cancer stem cell research program

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Stem Cell & Cancer Biology

Program overview.

This program brings together investigators who study various solid tumors and blood cancers with a focus on the molecular and cellular mechanisms that drive pre-cancer and cancer stem cells and make these cells resistant to therapy.

Healthy adult stem cells ensure that specialized cells in tissues, such as the blood and skin, are constantly replenished. In contrast, cancer stem cells, highly potent cancer cells that share many functional and molecular hallmarks of healthy adult stem cells, are responsible for cancer.

For several cancer types, such as some blood cancers, healthy stem cells become corrupted over time, and begin to churn out cancer cells instead of healthy tissue cells. In other cancers, stem cell-specific programs are activated in regular cells, endowing them with the ability proliferate unchecked, while disabling normal cell and tissue function. Cancer stem cells can drive disease progression, trigger relapse and metastatic disease, and can be highly resistant to standard therapy. Pre-cancer stem cells have acquired some but not all of the alterations that make them cancerous and often act as a critical reservoir for recurring disease.

Areas of Concentration

SCCB is a critical resource for basic science efforts allowing for fundamental discoveries. At the same time, the program interacts with translational and applied cancer research at Montefiore Einstein Comprehensive Cancer Center. SCCB investigators focus on: 

• How healthy stem cells function and prevent the formation of cancer
• The biochemical and molecular mechanisms behind gene regulation, RNA translation, and splicing driving stem cell differentiation
• Characterizing healthy and cancerous stem cells in patients in order to develop novel diagnostics and treatments

Current Initiatives

SCCB researchers have made important discoveries of early molecular and genetic changes that drive the formation of cancer stem cells and of factors that promote cancer evolution and resistance to therapy. Several basic mechanistic and pre-clinical studies have been translated into clinical therapeutic trials at Montefiore Einstein Comprehensive Cancer Center, and other studies have identified novel targets for drug development that are being pursued by investigators in SCCB and the Cancer Therapeutics program. Current collaborations and research initiatives include: 

• The role of a critical negative regulator of the tumor suppressor gene p53 (MDMX) in driving the transformation of blood-forming stem cells into cancer stem cells
• Identifying and overcoming resistance of cancer stem cells to current therapies (such as hypomethylating agents, immunotherapies, and radiation therapies)
• Investigating how age-associated molecular changes (other than DNA mutations) contribute to the formation and maintenance of pre-cancerous and cancerous stem cells, with a particular focus on pathways governing the cellular “recycling” process, known as autophagy.
• Examining the role and contribution of gene regulatory alterations in driving cancer evolution and their potential use as biomarkers for early cancer-risk stratification.

Kira Gritsman, MD, PhD

Assistant Director of Correlative Clinical Research, Associate Professor, Department of Oncology (Medical Oncology), Associate Professor, Department of Medicine (Oncology & Hematology), Associate Professor, Department of Cell Biology

Britta Will, Ph.D.

Assistant Director of Preclinical Modeling, Associate Professor, Department of Oncology (Medical Oncology), Associate Professor, Department of Medicine (Oncology & Hematology), Associate Professor, Department of Cell Biology

Stem Cell & Cancer Biology (SCCB) Research Program Members

Led by Dr. Kara Gritsman and Dr. Britta Will, the SCCB Program consists of highly dedicated experts committed to advancing the science of cancer care with thoughtfulness and integrity.

• Meet the SCCB Program Members

Cancer Clinical Trials

• Blood & Bone Marrow Cancers
• Brain, Spine & Central Nervous System Cancers
• Breast Cancer
• Childhood Cancers
• Endocrine System Cancers
• Gastrointestinal (GI) Cancers
• Genitourinary (GU) & Urologic Cancers
• Gynecologic Cancers
• Head & Neck Cancers
• Kaposi Sarcoma & AIDS-Related Cancers
• Lung & Chest Cancers
• Prostate Cancer
• Skin Cancer

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• Stem Cells Int
• v.2022; 2022

Cancer Stem Cells: From an Insight into the Basics to Recent Advances and Therapeutic Targeting

Shweta bisht.

1 Department of Biochemistry, Hemvati Nandan Bahuguna Garhwal University, Srinagar Garhwal, 246 174 Uttarakhand, India

Manisha Nigam

Shyam s. kunjwal.

2 Department of Zoology, School of Sciences, Uttarakhand Open University, Haldwani, 263139 Uttarakhand, India

Plygun Sergey

3 European Society of Clinical Microbiology and Infectious Diseases, Basel 4051, Switzerland

4 Laboratory of Biocontrol and Antimicrobial Resistance, Orel State University Named after I.S. Turgenev, 302026 Orel, Russia

Abhay Prakash Mishra

5 Department of Pharmacology, Faculty of Health Science, University of Free State, Bloemfontein 9300, South Africa

Javad Sharifi-Rad

6 Facultad de Medicina, Universidad del Azuay, Cuenca, Ecuador

Associated Data

The data used to support the findings of this study are available from the corresponding authors upon request.

Cancer is characterized by an abnormal growth of the cells in an uncontrolled manner. These cells have the potential to invade and can eventually turn into malignancy, leading to highly fatal forms of tumor. Small subpopulations of cancer cells that are long-lived with the potential of excessive self-renewal and tumor formation are called cancer stem cells (CSCs) or cancer-initiating cells or tumor stem cells. CSCs can be found in tissues, such as breast, brain, lung, liver, ovary, and testis; however, their origin is still a matter of debate. These cells can differentiate and possess self-renewal capacity maintained by numerous intracellular signal transduction pathways, such as the Wnt/ β -catenin signaling, Notch signaling, transforming growth factor- β signaling, and Hedgehog signaling. They can also contribute to numerous malignancies and are an important reason for tumor recurrence and metastasis because they are resistant to the known therapeutic strategies that mainly target the bulk of the tumor cells. This review contains collected and compiled information after analyzing published works of the last three decades. The goal was to gather information of recent breakthroughs related to CSCs, strategies to target CSCs' niche (e.g., nanotechnology with tumor biology), and their signaling pathways for cancer therapy. Moreover, the role of metformin, an antidiabetic drug, acting as a chemotherapeutic agent on CSCs by inhibiting cellular transformation and its selective killing is also addressed.

1. Introduction

Cancer stem cells (CSCs) represent specific type of rare cells found in the broad majority of tumors, possessing self-renewal and differentiation capacities. They contribute to the heterogeneous lineages of the cancer cells that form the tumor and share some of the common characteristics with stem cells. The concept of CSCs has been traced back to early 1900s when Julius Friedrich Cohnheim observed the similarity of the tissues of teratocarcinomas with the embryonic tissue. Cohnheim supported the theory of “embryonic rests” using the resemblance. In 1964, G. Barry Pierce showed that embryonal carcinoma (EC) cells that are derived from embryonal teratocarcinoma and carcinomas could generate multiple differentiated tissues and cause embryonal carcinoma upon transplantation into the mice. After this finding, he interpreted his observation supporting the theory of CSCs and stated that EC cells are multipotent. In the 1980s, the impacts of oncogenes on proliferation and genomic stability were given more attention than tumor differentiation issues. It continued till 1990s, because many researchers considered teratocarcinomas, which provided as a model for investigating differentiation phenomenon, as a unique case with little importance to the study of other cancers. The study of acute myelogenous leukemia (AML) led to the increased interest to explore more about the concept of CSCs [ 1 ].

In 1997, Dominique Bonnet and John E. Dick [ 2 ] were the first to identify CSCs from the mononuclear cells of the blood of human acute myeloid leukemia (also known as AML). They explained that these cells share similar characteristics (the ability of self-renewal and cellular differentiation into another cell type) with the normal stem cells (NSCs) and possess an exclusive phenotype of CD34 + and CD38 − surface markers and can be differentiated into leukemic blasts [ 2 ]. However, normal stem cells are noticeable for the diligence with which they control their proliferation and the ability with which they maintain their genomic integrity. Three distinctive properties of CSCs (self-renewal, the ability to develop into multiple lineages, and the potential to multiply quickly) are somewhat related to various forms of cancer [ 3 ]. CSCs also possess the property of plasticity via reversibly switching between the stem and nonstem cell states. They can escape apoptosis and can metastasize, though they may remain dormant for long duration. CSCs have the potential for self-renewal, can develop to all types, and are found in a specific sample of cancer having the capacity of continuous expansion into the population of malignant cells [ 2 , 4 , 5 ]. Due to their ability of tumor initiation, these cells are believed to play a crucial role in tumorigenic processes, such as oncogenesis, metastasis, and cancer recurrence [ 6 ]. Tumorigenesis and the behavior of the cell depend upon the microenvironment of tumor, and their identification depends on the surface markers along with their potency of self-renewal and propagation. CSCs contribute to the tumor heterogeneity, managing the vital malignant behaviors of processes such as invasion, metastasis, and therapy resistance, which is caused by epigenetic and genetic pathways. Many intracellular as well as extracellular factors that can be used for targeting drugs to treat cancers are responsible for controlling the activities of CSCs [ 7 ].

CSCs do not easily get destroyed via chemotherapy and radiotherapy, which means that following the effective destruction of the bulk of tumor by various therapies, a subset of residual CSCs may survive and facilitate cancer relapse leading to invasiveness and therapy resistance, thereby playing a critical role in the progression of cancer and therapy resistance. To prevent this condition, a deep understanding of the biology of CSCs is essential to develop effective therapies. Various CSC-targeted specific therapies are developing across the globe, for improving the survival rate and quality life of cancer patients, specifically for those with metastatic disease [ 8 ].

This review includes findings on CSCs from the recent reviews and research papers searched on PubMed database, using keywords such as “cancer,” “self-renewal,” “stemness,” “tumorigenesis,” and “signaling.” The goal was to summarize a detailed and conclusive understanding of the biological characteristic properties of CSCs, hypotheses of their origin, identification and regulating signaling pathways. Furthermore, recent breakthroughs in therapeutic strategies to target CSCs for cancer therapy are also discussed, with a focus on the role of metformin, an antidiabetic drug that acts as a chemotherapeutic agent against CSCs. Nevertheless, combining nanotechnology with tumor biology is considered to be critical, since nanosized materials may be exploited for CSC-driven anticancer therapy.

2. Theories of Origin and Niches of Cancer Stem Cells

2.1. theories of origin.

The theory of cancer stem cells states that a given subgroup of cancer cells drives tumor spread and growth which causes the progeny of cancer cells to be highly differentiated and destined to cease the proliferation because they have restricted mitotic divisions. The theory of CSCs ( Figure 1 ), therefore, shows that certain features of the cellular hierarchy which are observed in normal tissues can be seen in many tumors ( Table 1 and Table 2 ). It was earlier suggested that CSCs are born from NSCs ( Table 3 ), by observing the capability of differentiation of the leukemic cells into multiple mature lineages and allocating the expression of few markers with the NSCs [ 4 ].

Theories of origin of cancer stem cells. There are three possible theories: (i) CSCs could have possibly originated either from normal stem cells when they underwent mutation or oncogenic transformation, (ii) from progenitor cells who also undergone mutations, and (iii) from the fully differentiated cells that undergone several mutations via dedifferentiation (curved black arrows indicate self-renewal, while the straight arrows indicate the expression promotion).

Similarities between NSCs and CSCs [ 5 , 7 , 9 , 10 ].

Theories suggesting origin of CSCs [ 11 , 12 ].

Dissimilarities between NSCs and CSCs [ 5 , 7 , 9 , 10 ].

Based upon the application of stem cell concepts, which are known to be derived from embryogenesis to understand the process of tumorigenesis, some of the key features of CSC hypothesis are as follows: (i) When cancer cells were transplanted into immunodeficient mice, the cancer cells having tumorigenic potential were present in a small fraction [ 7 ]. (ii) By using distinctive surface markers, the CSC subpopulation can be distinguished from the other cancer cells [ 7 ]. (iii) Tumorigenic and nontumorigenic cells of the original tumor are present as a mixture in the tumor which is the result of CSCs [ 7 ]. (iv) The subpopulation of CSCs can be transplanted in series through several generations, indicating that it is a self-renewing population [ 7 ].

The progenitor cells arise from various types of stem cells possess the ability to divide further into differentiated or specialized cells for performing specific functions of the body. The origin of CSCs is still a controversy that whether they arise from stem cells, progenitor cells, or differentiated cells those present in adult tissues [ 13 ].

2.2. Niches of Cancer Stem Cells

Similar to normal stem cells, CSCs also reside in niches which are the specialized microenvironment known for regulating the normal stem cell fate via providing signals either by some secreted factors or through cell-cell contacts. Niches for mammalian stem cells have been identified in various epithelial tissues, such as the intestine, neural, epidermal, and hematopoietic systems. The components of normal niches are fibroblastic, endothelial, and perivascular cells or their progenitors, immune cells, extracellular matrix (ECM) components, networks of cytokines, and growth factors [ 12 ].

Cancer consists of malignant cells along with inflammatory cells, associated hematopoietic cells, stroma, and vasculature. So, the effect of niche may be inductive or selective depending upon subtype of every tumor. In the case of glioblastomas, there is a bidirectional relationship between the CSCs and the local environment as the niche can alter the cellular fate of cancer cells and can modify their microenvironment [ 14 ]. The CSC niche itself is isolated from the tumor microenvironment (TME), which is a collective term for neighboring stroma together with normal counterparts of tumorigenic cells [ 15 ]. The cells present in CSC niche produce some factors which further help in stimulating the self-renewal property of CSCs and induce angiogenesis ( Figure 2 ) [ 15 ]. Some additional factors, secreted by immune cells and other stromal cells that are recruited by cells residing in CSC niches, promote tumor cell invasion and metastasis via transdifferentiation into the vascular cells. CSCs which are present in glioblastomas contribute to the microvasculature [ 14 ], highlighting the close relationship between brain CSCs and their niche. In the case of cutaneous squamous cell carcinomas, the perivascular niche plays a very crucial role. Signals are provided by cellular and noncellular components of the niche which in turn regulate the proliferative and self-renewal signals in order to help CSCs so that they can maintain their quiescent state [ 15 ].

Crosstalk between CSCs and their niches. Cells present in the CSC niche produce some factors that stimulate self-renewal and angiogenesis and secrete factors involved in tumor cell invasion and metastasis. MSCs secrete CXCL12, IL-6, and IL-8 (the black arrows indicate the expression promotion) which promote CSC stemness via upregulating NF- κ B. To attract more MSCs towards CSCs, the latter also secretes IL-6. Gremlin 1 is an antagonist produced by MSCs to boost up the undifferentiated state. The tumor cells present around the CSC produce IL-4 which stimulates T H 2 and further produce TNF- α for upregulating the NF- κ B signaling. GM-CSF, G-CSF, and M-CSF are also produced by the same tumor cell to induce the expansion of some immune cells such as TAMs, TANs, MDSCs, and DCs. To enhance the plasticity of CSCs, TNF- α and TGF- β are produced by TAM to promote the NF- κ B-dependent or TGF- β -dependent EMT. TGF- β is also being produced by TAMs to stimulate the T reg cells. TAM, T reg , and hypoxic microenvironment also inhibit CD8 + T cell, NK cell cytotoxicity, and phagocytosis of macrophages thus inhibiting immunosurveillance (red arrows depicting inhibition). Hypoxic microenvironment increases the concentration of ROS, promotes cell survival, and induces EMT via the TGF- β signaling pathway. The downregulated c-Myc expression inhibits cell proliferation under hypoxia and enhances stemness. CXCL12 is produced by CAF to promote angiogenesis. Under hypoxic microenvironment, CSCs and ECs produce VEGF, which further induces angiogenesis. Nitric oxide production via the Notch signaling pathways leads to the self-renewal of CSCs. CAFs also produce TNC, HGF, and MMP2/3/9, which help in the enhancement of the Wnt and Notch signaling. It also produces MMP10 which promotes ECM degradation and remodeling thus enhances the CSC's stemness.

Mesenchymal stem cells (MSCs), being the multipotent stromal cells, secrete CXCL12, interleukin-6 (IL-6), and IL-8 which promote CSC stemness via upregulating NF- κ B. Gremlin 1, an antagonist, is also produced by MSCs to boost up the undifferentiated state [ 15 ].

The tumor cells surrounding CSC produce IL-4, which stimulates T H 2 to further produce TNF- α for upregulating the NF- κ B signaling pathway. Along with IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, and M-CSF are also produced by the same tumor cell for the expansion of some immune cells such as tumor-associated macrophages (TAMs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), and dendritic cells (DCs). T regulatory cells (T reg ) participate in immunosuppression recruited by TGF- β , which is secreted by TAMs [ 15 ].

Myofibroblast cells present in the tumor-associated stroma secrete HGF, which helps to maintain the function of CSCs by activating the Wnt pathway in the colorectal cancer, and they are present adjacent to stromal myofibroblasts thus induce some of the CSC features and tumorigenic capacity in differentiated cancer cells having the limited tumorigenic capacity [ 15 ]. Endothelial cells in the reverse direction secrete nitric oxide which results in the induction of the Notch signaling in glioma cells. Providing nutrients and oxygen to the cells, endothelial cells also secrete some factors that play a crucial role in promoting the self-renewal property as well as help in the survival of the head and neck CSCs [ 13 ].

Many cells surrounding the CSCs such as MSCs, cancer-associated fibroblasts (CAFs), TAMs, and some nonstem cancer cells also play a very important role in maintaining the CSC stemness.

2.2.1. Role of Exosomes in Tumor Microenvoirnment (TME) as well as in CSCs

Exosomes are the membrane-bound extracellular nanovesicles that are derived from the endosomes and possess a large number of functional proteins, RNA, microRNAs, and some DNA fragments [ 16 ]. They can be found in peripheral blood, breast milk, saliva, urine, and in many other body fluids. In comparison to normal cells, tumor cells secrete 10 times more vesicles, which is the most effective route for the tumor and metastatic information to reach both normal and tumor cells, as the transfer of tumor derived exosome (TDE) contents to nonmalignant cells has been shown to activate the tumor phenotype and metastatic properties [ 17 ]. The major role of exosomes when secreted in high concentration from the cancer cell is to induce differentiation of tumor-related fibroblasts, to promote angiogenesis, to regulate the microenvironment before metastasis, and to participate in the immune regulation of TME [ 16 ].

The interaction between CSCs and NSCs can be mediated by the exosome signaling which further regulates the development of tumors as well as the process of oncogenesis. By targeting some specific signaling pathways (such as Wnt, Notch, Hh, and NF- κ B), exosomes can regulate the growth of CSCs [ 18 ]. In the case of colorectal cancer, exosomes derived from fibroblast provide chemoresistance by promoting the growth of CSCs, while exosomes derived from CAF promote sphere formation by activating the Wnt pathway, thus increasing the number of CSCs [ 19 ]. Exosomes, when released from CSCs, increase the stemness of breast cancer cells and also affect the signal transduction in nearby cells [ 20 ]. They are considered as an ideal drug carrier for cancer therapy because of their easy storage, high drug loading capacity, long life, easy production, biocompatibility, and low immunogenicity [ 17 , 18 ].

2.2.2. Role of Hypoxic Microenvironment on Cancer Stem Cells

Hypoxia is an oxygen-deprived condition of the body resulting in the insufficient supply of the oxygen at the tissue level. This deprived state can promote genetic instability, metastasis, and invasiveness of tumor cells, resulting in the expression of HIFs by CSCs, where TGF- β is responsible for their regulation and stabilization. In order to adapt in this state, there is a phenotypic shift in the expression of genes that regulates the various cellular processes [ 21 ].

Hypoxia-inducible factors (HIFs), central to this shift, act as mediators under the hypoxic microenvironment for monitoring cellular responses to the oxygen level [ 21 ]. HIFs being heterodimeric are those helix-loop-helix transcription factors having an α and a β subunit (HIF- α and HIF- β ), as shown in Figure 3 [ 22 ]. Under the hypoxic condition in order to self-sustain, the role of HIFs in CSCs is to promote stemness and regulate tumor growth and cell survival by activating HIF-1 and HIF-2 as summarized in Figure 4 [ 23 ]. It was concluded from the knockdown experiments performed in vivo that those CSCs having low HIF activity are unstable in tumor propagation and cell survival. Furthermore, hypoxia increases the expression of Sox2 and Oct4 genes, both of which are involved in stem cell function. Sox2, along with Sox4, has been revealed to play a critical role in the preservation of stemness in CSCs [ 22 ]. Notably, some genes linked with the hypoxia response in normal cells, such as Glut1, Serpin B9, and VEGF, are elevated in CSCs [ 21 ]. In the case of solid tumors, the activity of oncogene can be regulated by HIF-1 α via various pathways such as Akt and epidermal growth factor receptor (EGFR) [ 21 ]. The specificity of HIF- α and HIF-2 α is essential for the survival and propagation of tumor. HIF-1 α when activated in hypoxia leads to the expansion of the subpopulation of the cells, which are positive for CSC marker, CD133. The level of CD44, a marker that is associated with stem-like phenotype, is also increased [ 21 ]. The expression of HIF-2 α stimulates expression of Oct-4 and promotes the activity of c-Myc, which ultimately ensures the property of undifferentiation in CSCs [ 22 ].

Structural and functional organization of hypoxic inducible factors (HIFs). HIF is a heterodimeric complex composed of an oxygen-dependent α -subunit (HIF- α ) and an oxygen-insensitive β -subunit (HIF- β ). The HIF- α has three subunits (HIF-1 α , HIF-2 α , and HIF-3 α ). The regulation of HIF-1 α and HIF-2 α is done by oxygen tension and is ubiquitously expressed in the normal tissue, whereas the HIF-1 β is the subunit of HIF- β . The carboxy-terminal domain (CTD) of HIF-1 α and HIF-2 α based on their regulation is divided (indicated by black arrows) into two domains: ODD (regulates stability) and TAD (regulates transcriptional activity via two transactivation domains (TADs))—(i) N-TAD and (ii) C-TAD. Some nuclear localization signals (NLS) are present in both the C and N-termini of the α -subunits such as N-NLS and C-NLS that give it the direction towards the nucleus.

Role of HIFs in CSCs. Under hypoxia, HIF-1 is formed after the dimerization of HIF-1 α with HIF-1 β and binds to the HRE (HIF-responsive element) present at the DNA. This binding results in the transcription of the targeted genes (HK1, PGK1, TP11, and BNIP3) (depicted in pink boxes) and regulates the various cellular processes like survival, angiogenesis, apoptosis, invasion, metastasis, metabolism, therapeutic resistance, and DNA repair.

Interestingly, there is a dynamic heterogeneous cellularity scattered within the edge and core of the tumor for producing adaptive mechanisms in response to various damaging situations. Edge cells are more quiescent, invasive, and resistant to infection than core cells, which multiply faster and have a higher cellular density. A rise in the number of resistant cells is an expected phenomenon in advanced malignancies and is partly due to core-to-edge cellular shifting, which expands the tumor's edge area, and hypoxia is a major contributor to this cellular dispersal [ 24 ].

The Hippo signaling pathway, in breast CSCs, is induced via HIF-1 α , by directly targeting the Hippo pathway effector, TAZ, which is a key regulator of breast cancer stem cell (BCSC) activity [ 25 ]. In patients with glioblastoma, HIF-1 α or HIF-2 α are targeted in CD133 + cells by short hairpin RNA that inhibits its proliferation and capability of neurosphere formation and induces caspase-dependent apoptotic effect in vivo and in vitro alters their tumor-initiating potential [ 26 ]. In AML, HIF-1 is overexpressed and preferentially activated in CD34 + CD38 subgroups [ 26 ], which enhances the stem-like phenotype and further results in increase in the number of stem cells of leukemia. Hypoxia also regulates various signaling pathways, such as Wnt and Notch, which induce EMT, further increase the invasiveness and stemness of CSCs, and also provide resistance to radiotherapy and chemotherapy [ 23 ].

3. Identification of Cancer Stem Cells

CSCs, first identified in AML, were confirmed with the subpopulations of CD34 + and CD38 − , similar to normal hematopoietic stem cells (HSCs) [ 2 ]. When these were transplanted into immune-deficient mice, then their tumor-initiating capacity was proved [ 27 ].

Some glycoproteins or proteinaceous markers can upregulate, downregulate, mutate, or silent the properties of CSCs. In order to detect CSCs, localization of these markers is crucial. Some of these markers are found in the cytoplasm, while others are present as the cell surface antigens [ 28 ]. In fact, breast cancer stem cells (BCSCs) were first identified in 2003 on the basis of the expression levels of cell surface antigens, CD44 + and CD24 − , that account for high capacity of invasiveness, migration, and proliferation [ 29 ]. CD44, which is a transmembrane glycoprotein receptor, acts as a crucial signal molecule that interacts with the cytoskeletal proteins or modulates gene expression thus eventually alters cell behavior. It is also involved in cell adhesion and migration [ 28 ]. According to scientists, CD44 and its isoform are reliable cancer stem cell markers and can be used alone as well as in a combination with other surface markers in order to identify CSCs. ALDH1, CD133, and CD61 are the examples of other markers that are correlated with BCSCs [ 29 ].

Although the most popular method which is being used to identify specific markers for CSCs is by observing the expression of cell surface antigen, however, those markers which are used to identify stem cells from one organ are not useful for identifying stem cells in other tissues. For example, Sca-1, a marker protein used to identify murine blood stem cells, cannot consistently be expressed by murine mammary duct stem cells [ 30 ]. Different biomarkers used for the identification of CSCs in various human cancers are depicted in Table 4 .

Markers used for identification of CSCs in tumors [ 19 ].

4. CSC Studies in Solid Tumors

An abnormal mass of tissue that does not contain any cysts or a liquid portion and can be benign (noncancerous) or malignant (cancerous) is referred to as solid tumors. The first solid tumor was studied in human breast cancer [ 5 ]. For the expression of CD44 and CD24, the samples of human breast tumors were analyzed and were found to be heterogeneous in the expression of surface antigen similar to the case of AML [ 27 ]. Human breast cancer cells were separated into different populations on the basis of the differences in the expression of surface antigen through flow cytometry. Upon injecting different populations into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice only those cells of human breast cancer formed tumors that can express a CD44 + CD24 low / − . Those human breast tumor cells, that are able to develop tumor when injected into NOD/SCID mice, specifically refer to “breast cancer stem cells” [ 92 ].

Using immunodeficient mice (SCID or NOD/SCID) as recipients for xenografts of the tumor, various other human solid tumors have been studied. For example, CD133 + and CD133 − were found in human brain tumors. When implanted into immunodeficient mice, only cancer cells expressing CD133 + , named “brain tumor-initiating cells”, were capable of forming tumors in the majority of patients [ 27 ].

Flow cytometry analysis of colon cancer tissues revealed heterogeneous groups of cells inside a tumor. The recent findings showed that in many patients having human colon cancer, only the CD133 + subsets of tumor cells known as “colon cancer-initiating cells” were capable of creating xenografted tumors in mice [ 93 ]. In the case of human colon cancer, the tumor-forming subsets in a patient's tumor were defined using CD44, epithelial cell adhesion molecule (EpCAM also known as an epithelial-specific antigen or ESA), and CD166. Only cells that were positive for these markers were able to produce tumors in mice whether utilized in pairs or all three together (ESA hi CD44 + , CD44 + , CD166 + , ESA hi CD166 + , or ESA hi CD44 + CD166 + ) [ 93 ]. CD44 + plays a major role in studying head and neck squamous cell carcinomas (HNSCC). Tumorigenic cancer stem cells in HNSCC possess the ability to propagate tumor formation in mice. To study HNSCCs, both NOD/SCID and Rag2/cytokine receptor common γ -chain double knockout (Rag2 γ DKO) mice were used as an immunodeficient mouse test model [ 92 ]. In human pancreatic cancers, cells having similar tumor propagation abilities were also found. They neither express CD44 nor CD24. Those cells expressing CD44 + , CD24 + , and ESA (named pancreatic cancer stem cells) were responsible for tumor formation [ 80 ].

5. Signaling Pathways Involved in CSCs

In the case of normal stem cells, various highly regulated molecular signaling pathways contribute to the different properties like self-renewal, survival, proliferation, and differentiation. On the contrary, in tumorigenesis or cancer stem cells, these signaling pathways are either repressed or abnormally activated. Moreover, these complex pathways are regulated by various extrinsic and intrinsic molecular signals, endogenous and exogenous genes, some regulatory elements, and microRNAs. Instead of being linear, these pathways are interwoven networks of signaling mediators that regulate and support the function of CSCs [ 19 , 94 ]. There are nine signaling pathways that are known to be involved in embryonic development as well as in cancer so far. Out of these nine, only seven pathways (such as the JAK/STAT pathway, MAP-kinase/ErK pathway, NOTCH pathway, NF- κ B pathway, P13K/Akt pathway, TGF- β pathway, and Wnt pathway) are common in both cancer and stem cells [ 19 ].

5.1. Hedgehog (Hh) Signaling Pathway in CSCs

It is a complex signaling network which consists of extracellular Hh ligands, transmembrane protein receptor (PTCH), a transmembrane protein (SMO), an intermediate transduction molecule, and the downstream molecule GLI. The role of SMO protein is to regulate the pathway positively, where PTCH plays a negative role in regulation [ 19 ]. The subtypes of GLI have different roles, where Gli1 acts in the activation of the transcription. Gli2 acts as both an activator and an inhibitor of transcription, but mainly as an activator. Gli3 inhibits transcription [ 95 ]. This signaling pathway plays an important role in the formation of the nervous system, skeleton, limbs, lungs, heart, gut, and embryonic development. In the absence of the Hh ligand, PTCH, which is present on the target cell membrane, binds to the SMO thus inhibits its activity and ultimately halts the signaling [ 96 ]. In the presence of Hh ligand, there is the spatial conformational change in the PTCH which activates the transcription factor Gli by eliminating the inhibition of SMO ( Figure 5 ). Gli upon translocation into the nucleus regulates growth, proliferation, and differentiation of the cell [ 19 ].

Signaling pathways involved in cancer stem cells. (a) The JAK/STAT pathway (extreme left): JAKs get activated when ligands bind to its receptor; JAK1 and JAK2 auto and transphosphorylate each other and also phosphorylate the tyrosine residues present in the cytoplasmic domain of the receptor. STATs upon phosphorylation by JAKs form dimers and are then translocated into the nucleus to initiate the transcription of the targeted genes. (b) The Hedgehog pathway (left): Hh, when secreted from the other cells, binds to PTCH and allows the activation (indicated by black arrows) of SMO. SMO protein complex secretes Gli1/2 and translocates it into the nucleus, leading to the transcription of Hh-associated genes (depicted by purple arrows). (c) The Notch pathway (right): the binding of the delta ligand to the other cell; two different enzymes responsible for two different cleavages are ADAM10 or TACE and a metalloprotease that catalyzes the S2 cleavage and hence producing a substrate for S3 cleavage via the γ -secretase complex. Due to proteolysis, it mediates the release of NCID, which upon translocation into the nucleus starts interacting with the DNA-binding CSL protein and MAML which further activate the transcription process of the targeted genes. (d) The Wnt pathway (extreme right): Wnt ligand binds to Fz, a receptor, and induces the phosphorylation of the coreceptors, LRP5/6, which further forms the docking site for AXIN. The binding of the ligand to the receptor signals Dvl to recruit AXIN 1 along with the other kinases CK1 α and GSK3 β to the membrane, which interrupts the destruction complex leading to impairment of the phosphorylation of β -catenin and results in its destruction. Accumulated β -catenin then translocates from the cytoplasm to the nucleus and functions as an activator of TCF/LEF-mediated transcription of Wnt target genes.

Self-renewal capacity and metastasis of CSCs can also be promoted by the Hh signaling via upregulating the expression of related downstream markers of CSCs (e.g., ALDH1, Bmi-1, CCND1, CD44, C-MYC, Jagged1, Nanong, Oct4, PDGFR α , Snail, Twist1, and Wnt2). Directly or indirectly, some protooncogenes and suppressor genes regulate the Hh signaling in the proliferation and migration of CSCs [ 19 ]. In the case of medulloblastoma stem cells, a transcriptional repressor, BCL6, and lymphoma oncoprotein directly repress the Sonic Hh effectors Gli1 and Gli2. It involves the degradation of Gli1, due to its increased physical interaction with the β -catenin [ 96 ]. In the case of lung CSCs, miR-122 directly targets the Shh and Gli1 [ 97 ]. Thus, it can be suggested that the amplified Hh signaling is important for self-renewal, growth, and metastasis of CSCs.

5.2. JAK-STAT Signaling Pathway in CSCs

Cytokines are responsible for the stimulation of the Janus kinase/signal transducers and activators of transcription (JAK-STAT). Interleukin 2-7, granulocytes/macrophage colony-stimulating factor, growth hormone, EGF, PDGF, and interferon are the examples of some cytokines and growth factors that are responsible for transmitting the signal via this pathway. Many vital biological processes such as apoptosis, cell proliferation, differentiation, and immune regulation involve the presence of the JAK-STAT signaling pathway. The tyrosine kinase-related receptor, the tyrosine kinase JAK, and the transcription factor STAT are the three main components of this pathway. The binding site for the tyrosine kinase JAK is present in the cells. Tyrosine residues of various target proteins after binding with the ligands get phosphorylated via JAK activation, in order to achieve signaling from the extracellular to the intracellular space. The four members of JAK protein family are JAK1, JAK2, JAK3, and Tyk2, while STAT has seven members in the family (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6) that play an important role in signal transduction and transcriptional activation [ 19 ].

When JAK receives the signal from the upstream receptor molecule, it activates the tyrosine kinase-related receptor and gets activated to catalyze tyrosine phosphorylation of the receptor. The phosphorylated tyrosine present on the receptor molecule acts as a signal molecule and binds to the SH2 site of STAT. Upon binding to the receptor, the tyrosine phosphorylation of STAT also occurs, resulting in the formation of a dimer that enters the nucleus and affects the expression of the targeted genes (summarized in Figure 5 ) and ultimately proliferates and differentiates the target cells [ 98 ].

Constitutive and abnormal activation of STAT3 and mutation in JAK2 are observed in many tumors. The self-renewal of glioma stem-like cells is promoted by HIF-1 α via the JAK1/STAT3 pathway [ 99 ]. This pathway is activated in ALDH high and CD126 + endometrial CSCs by IL-6, which also converts the nonstem cancer cells into cancer stem cells by activating the downstream Oct4 gene, in the case of breast CSCs [ 36 ]. A scaffold protein, AJUBA, which plays an important role in cell adhesion, differentiation, proliferation, and migration, is also responsible to promote colorectal CSC survival and proliferation through the JAK1/STAT1 pathway [ 100 ]. In the case of lung CSCs, the gene expression of JAK3 and IL-6 receptor is negatively regulated by miR-218 because microRNAs activate the JAK/STAT signaling by inhibiting the negative regulatory factor of JAK2/STAT3 [ 19 ]. Thus, some recent studies on this pathway suggest that the JAK/STAT signaling pathways play an important role in the survival, self-renewal, and metastasis of CSCs.

5.3. NF- κ B Signaling Pathway in CSCs

NF- κ B comprising five different proteins (mainly p65, RelB, c-Rel, NF- κ B1, and NF- κ B2) is the rapidly inducible transcription factor [ 36 ]. Two major pathways regulate its activity. Those pathways are the canonical NF- κ B and noncanonical NF- κ B signaling pathways. The canonical NF- κ B pathways activate when the ligand (e.g., cell components of bacteria, IL-1 β , TNF- α , or lipopolysaccharides) binds to the receptor (such as Toll-like receptor, TNF receptor, IL-1 receptor, and antigen receptor). These receptors upon stimulation further phosphorylate and activate I κ B kinase (IKK protein). IKK1 proteins also get phosphorylated and activated in the noncanonical pathway by inducing the kinase (NIK) which further activates NF- κ B as shown in Figure 6 . The IKK enzyme activity induces the production of p52 by stimulating the phosphorylation of p100 [ 101 ].

Signaling pathways involved in cancer stem cells. (a) The NF- κ B pathway (left): TNF- α , the proinflammatory cytokine, binds to the TNF receptor and induces the formation of IKK complex, which phosphorylates I κ B-a. Phosphorylation of I κ B-a results in its degradation via proteasome which leads to the accumulation of p65-p50 (acting as an NF- κ B) dimer into the nucleus and regulates the transcription of the targeted genes. (b) The TGF- β pathway (middle): TGF β 1 ligand upon binding to the TGF-beta receptor type-2 (TGF β R2) promotes (indicated by black arrows) the dimerization of TGF β R2 with TGF β R1, resulting in the transphosphorylation of TGF β R1. The activated TGF β R1 further activates R-SMADs (SMAD2 and SMAD3) by phosphorylation. SMAD2/3 trimerizes with a co-SMAD (SMAD4). The SMAD trimer upon localization into the nucleus activates (indicated by purple arrows) the gene transcription and promotes cell growth and survival. (c) The PI3K pathway (right): the binding of the ligand to the RTK results in the phosphorylation of the membrane lipid PIP 2 via intracellular PI3K and it then converts to PIP 3 . PKB bounds to its docking site in PIP 3 , and upon phosphorylation, it gets activated by various kinases involving mTOR and DNA-dependent protein kinases, which further enhances PKB-mediated phosphorylation and the activation or repression of downstream mediators. PTEN, a phosphatase that is a negative regulator (inhibition is indicated by red arrows) of this process, helps in the dephosphorylation of PIP 3 to PIP 2 (the black arrows are depicting pathway activation/signal propagation).

In order to activate the NF- κ B signaling pathway, the process of tumor development and progression produces some cytokines, proteases, and some factors responsible for growth and angiogenesis. In many cancers, overactivation of the NF- κ B signaling has been reported [ 19 ]. In the case of ovarian CSCs, CD44 + cells promote self-renewal, metastasis, and maintenance of CSCs by increasing the expression of RelA, RelB, and IKK α and mediate nuclear activation of p50/RelA (p50/p65) dimer [ 19 ]. The inflammatory mediator prostaglandin E2 (PGE2) activates this signaling by the EP4-PI3K and EP4-MAPK pathways which contribute to tumor formation and metastasis in colorectal CSCs [ 102 ]. miR-221/222 inhibits the expression of PTEN thereby inducing the phosphorylation of Akt which results in the increased level of p65, p-p65, and COX2 and promotes self-renewal, migration, and invasion in breast CSCs [ 103 ]. Thus, it can be determined that the elevated NF- κ B signaling is crucial for regulating apoptosis, proliferation, and metastasis in CSCs.

5.4. Notch Signaling Pathway in CSCs

The Notch signaling pathway is a highly conserved pathway involving four Notch receptors (mainly Notch1, Notch2, Notch3, and Notch4) and five structurally similar Notch ligands (Delta-like1, Delta-like3, Delta-like4, Jagged1, and Jagged2). Under some physiological conditions, binding of the delta ligand to the Notch receptor results in the expression on the neighboring cells in a juxtacrine manner. The proteolytic cleavages of the intracellular domain (ICD) of Notch by two different enzymes responsible for two different cleavages are ADAM10 or TACE (TNF- α -converting enzyme, also known as ADAM17) and a metalloprotease that catalyzes the S2 cleavage and hence producing a substrate for S3 cleavage via the γ -secretase complex ( Figure 5 ). Due to proteolysis, it mediates the release of NCID and hence is translocated into the nucleus where it binds to the transcription factor CSL. This binding leads to the formation of transcriptional activation complex NICD/CSL that activates the targeted genes of the BHLH transcription inhibitor family [ 19 ].

Depending upon the microenvironment, Notch can act either as an enzyme, oncogene, or tumor suppressor gene. When the Notch is activated, it promotes self-renewal, metastasis, and cell survival but inhibits apoptosis. In gastric CSCs, an abundance of Delta-like ligand 4 (DLL-4) promotes tumor angiogenesis and metastasis [ 104 ]. In cervical CSCs, the Notch pathway gets activated when MAP17 (DD96, PDZKIP1), a nonglycosylated membrane-associated protein present on the Golgi apparatus and the plasma membrane, interacts with NUMB via the PDZ-binding domain. TACE/ADAM17 is activated to regulate the Notch1 signaling. They are being activated by inducible nitric oxide synthase which promotes the self-renewal capacity of CD24 + /CD133 + liver cells [ 105 ]. In oral squamous cell carcinoma, the Notch1 signaling is activated to enhance the CSC-like phenotype by TNF- α . The migration and invasion of ovarian CSCs are induced by Notch1 when there is no sign of hypoxia [ 19 ]. Thus, these recent studies emphasize the important role played by the Notch signaling in metastasis, self-renewal, and growth of CSCs.

5.5. PI3K/Akt Signaling Pathway in CSCs

PI3K is an intracellular enzyme mainly phosphatidylinositol kinase that possesses a regulatory and a catalytic subunit. p85 is the regulatory subunit, and p110 is the catalytic subunit which has the activities of both the kinases serine/threonine (Ser/Thr) kinase and phosphatidylinositol kinase. Serine/threonine kinase is present as AKT which further has three isoforms (AKT1, AKT2, and AKT3), and their proteins are important effectors of PI3K because they can be directly activated after getting a response from PI3K. When the ligand binds to receptor tyrosine kinase (RTK) which phosphorylates the membrane lipid, phosphatidylinositol (3,4)-bis-phosphate (PIP 2 ) via intracellular PI3K, it then converts to phosphatidylinositol (3,4,5)-tris-phosphate (PIP 3 ). Protein kinase B (PKB) bounds to its docking site in PIP 3 , and upon phosphorylation, it gets activated by various kinases involving mTOR and DNA-dependent protein kinases that further enhance PKB-mediated phosphorylation and the activation or repression of downstream mediators. A conserved serine/threonine kinase, the mammalian target of rapamycin (mTOR) complex, is one of the important downstream target genes of Akt. mTOR is found to be in two different multiprotein complexes, mTORC1 (consists of mTOR, raptor, mLST8, and two negative regulators PPRAS40 and DEPTOR) and mTORC2. The role of mTORC2 is to phosphorylate Akt at serine/threonine 473 for the activation of Akt ( Figure 6 ) [ 19 ].

The PI3K/Akt signaling is involved in cell proliferation in ovarian cancer and also in EMT [ 106 ]. Upon activation, this signaling enhances the properties of migration and invasiveness in the prostate and pancreatic cancer [ 107 ]. In the case of head and neck squamous CSCs, the activation of PI3K increases cell proliferation, migration, and invasion in ALDH + and CD44 high cells [ 108 ]. In colorectal cancer, the expression of ALDH1 increases due to the activation of mTORC1. The expression of hepatic CSC marker EpCAM and tumorigenicity, in the hepatocellular CSCs, is upregulated upon activation of mTORC2 [ 36 ]. An inhibitor of mTOR, matcha green tea (MGT), inhibits the proliferation of breast CSCs by targeting the mitochondrial metabolism, glycolysis, and multiple signal transduction pathways [ 109 ].

5.6. TGF- β Signaling Pathway in CSCs

A TGF- β signaling pathway is structurally simple and regulates numerous cellular processes, such as cell proliferation, differentiation, apoptosis, and homeostasis. The TGF- β superfamily ligands are divided into two groups: (i) TGF- β /activin that consists of TGF- β , activin, and Nodal and (ii) BMP/GDP which includes BMP, GDF, and AMH ligands. SMAD proteins on the basis of their structure are divided into three subfamilies: R-SMADs (receptor-activated or pathway-restricted SMAD), co-SMAD, and inhibitory SMADs (I-SMADs). The ligand of TGF- β superfamily binds to the type II receptor and phosphorylates it. Type I receptor binds to a common pathway SMAD (co-SMAD) by phosphorylating receptor-regulated SMADs (R-SMAD). The complex of R-SMAD/co-SMAD acts as a transcription factor by accumulating into the nucleus and regulating the expression of the targeted genes [ 19 ]. The process is summarized in Figure 6 .

The activated TGF/SMAD signaling is also found in human cancers. In the case of lung cancer, cellular transformation and stemness are mediated via nuclear NPM1 protein, while the TGF- β signaling is promoted by cancer upregulated gene 2 [ 110 ]. The role of TGF/SMAD is to proliferate CSCs; for example, in order to regulate the self-renewal of liver CSCs, the cyclin D1-SMAD2/3-SMAD4 signaling is promoted after the activation of SMAD2/3 and SMAD4 by their interaction with cyclin D1 [ 19 ]. The expression of p-SMAD2/3, SMAD4, and CD133 is induced by the upregulation of TGF- β in liver CSCs. In order to regulate glycolysis in glioma stem cells, the expression of PFKFB3 is upregulated by TGF- β 1 via activating the p38 MAPK and PI3K/AKT signaling pathways [ 111 ]. SMAD7, a target gene of miR-106b, acts as an inhibitor of the TGF- β /SMAD signaling and inhibits the sphere formation of gastric CSCs [ 112 ]. Although there are limited studies on the TGF- β /SMAD signaling pathway in CSCs, this can be concluded from the earlier studies that it plays a vital role in cellular processes in CSCs.

TGF- β has recently been proposed as a mediator of immaturity that contributes to tumor development and relapse. It acts not only on tumor cells but also on immune system cells like dendritic cells and natural killer cells, potentiating TME's immunosuppressive property [ 113 ], whose inhibition normalises tumor stroma and makes tumors more sensitive to therapy [ 114 ], including immune checkpoint inhibitor therapy [ 115 ].

5.7. Wnt Signaling Pathway in CSCs

The abnormal canonical and noncanonical WNT signaling is involved in CSC survival, bulk-tumor expansion, and invasion/metastasis in a variety of human cancers [ 116 ]. On the basis of the transcriptional regulator β -catenin acting as a mediator, the Wnt pathway can be divided into two signaling pathways, namely, the canonical and noncanonical Wnt signaling pathways [ 116 ].

By forming the stem cell signaling network, the WNT signaling, along with other signaling cascades such as FGF, Notch, Hedgehog, and TGF/BMP, regulates the expression of functional CSC markers [ 116 ].

The canonical WNT/-catenin signaling cascade is involved in stem cell self-renewal and progenitor cell proliferation or differentiation [ 53 , 117 ], whereas the noncanonical WNT signaling cascades are involved in stem cell maintenance, directional cell movement, or suppression of the canonical WNT signaling pathway [ 118 , 119 ]. WNT signaling cascades, both canonical and noncanonical, play a significant role in the development and evolvement of CSCs. WNT2B, WNT3, and other canonical WNT ligands derived from cancer supporting cells or stromal cells, as well as genetic alterations in the canonical WNT/-catenin signaling components, activate the canonical WNT signaling in CSCs [ 120 ]. LGR5, which encodes an R-spondin (RSPO) receptor, is a target gene of the WNT/-catenin signaling cascade in both quiescent and cycling stem cells. Canonical WNT signals stimulate the LGR5 receptor on CSCs, allowing them to remain canonical WNT responsive and directly promote CSC proliferation by upregulating the proteins CCND1, FOXM1, MYC, and YAP/TAZ [ 121 , 122 ].

WNT5A, WNT11, and other noncanonical WNT ligands secreted by cancer cells or stromal/immune cells, as well as genetic alterations that transactivate noncanonical WNT signaling cascades, activate the noncanonical WNT signaling in CSCs [ 116 ]. Through PI3K-AKT signaling activation and YAP/TAZ-mediated transcriptional activation, the noncanonical WNT signaling promotes CSC survival and therapeutic resistance [ 116 ].

In contrast, invasion and metastasis are driven by both canonical and noncanonical WNT signaling cascades. Canonical WNT/-catenin and WNT/STOP (stabilization of proteins) signaling cascades, for example, upregulate SNAI1 to repress epithelial genes, such as CDH1 (E-cadherin), for the initiation of CSC EMT, whereas noncanonical WNT signals promote CSC invasion, survival, and metastasis [ 116 , 122 , 123 ].

These findings indicate that the canonical WNT/-catenin signaling, as well as other WNT signaling cascades, plays a critical role in CSC malignancy.

WNT signaling cascades are the primary cause of many types of human cancers [ 116 , 122 , 123 ], but the development of many WNT signaling-targeted therapeutics is ground to a halt due to the complexity of WNT signaling cascades and genetic alterations in nonenzymatic signaling components. WNT signaling-targeted therapeutics in clinical trials or preclinical studies encompass anti-FZD mAb, anti-ROR1 mAb, anti-RSPO3 mAb, PORCN inhibitors, and β -catenin inhibitors [ 120 ].

6. Role of Cancer Stem Cells in Tumorigenic Processes

6.1. cancer stem cells in oncogenesis.

The hypothesis of CSCs could be compared with the working theory for oncogenesis, that due to the accumulation of mutations in the protooncogenes or tumor suppressor genes, differentiated cells get converted into tumorigenic. These genes regulate cell growth by regulating some growth-related factors, as the overactivation of these genes can lead to uncontrolled growth and even develop cancer [ 124 ].

In order to initiate cancer, many adult stem cells, their derivative progenitor cells, or many differentiated cells can convert into CSCs. Adult stem cells are virtually present in all tissues, and due to their long-lived nature, they are prone to develop the numerous mutations than any other cells. These mutations then further lead to cancer [ 3 ]. Markers such as CD133 and ALDH1 [ 125 ] which are associated with adult stem cells are also being expressed by CSCs. It also appears that CSCs and NSCs share some similar epigenetic and genetic profiles and activated signaling pathways, such as Hedgehog, Notch, and Wnt. In the case of AML, progenitor cells can be mutated to become CSCs since they have a phenotype similar to that of progenitor cells. Due to mutation in the differentiated cells, they might acquire the properties of progenitor or stem cells and can give rise to CSCs. The first genetic or epigenetic modifications might be possible to occur in adult stem cells, and subsequent mutations may accumulate in a progenitor or more differentiated daughter cell [ 126 ]. Cell surface marker proteins found on CSCs of different tissue types such as CD133 and CD44 are most likely to be the true markers of CSCs which are involved in oncogenesis because their existence on CSCs is reproducible. However, these markers may represent some cell's ability to survive purification procedures or trigger tumor growth in mice [ 29 ].

6.2. Cancer Stem Cells in Tumor Growth

CSCs are suggested to participate in tumor growth, but the number of different types of CSCs involved in this process and the importance of different percentages of CSCs contained in tumors are unclear [ 127 ].

Due to the constantly evolving nature of cancer cells via genetic/epigenetic changes along with the impact of their microenvironment, any of them could become CSCs [ 128 ]. A mutation pattern found in different regions of a single tumor suggests the multiple clonal cell populations, some of which may be cancer stem cells. Moreover, various markers used to isolate CSCs can reflect the diversity of these cells [ 5 ]. Some tumor cell populations are likely to be overlooked due to the detection of CSCs, including the fact that tumor tissue samples may not be representative of the entire population.

Tumors tend to differ in the percentage of CSCs with the recorded values between 0.03% and almost 100%. This percentage is probably determined by the specific characteristics of the CSCs that initiated the tumor and by the microenvironment by controlling the frequency with which additional CSCs are produced [ 127 ]. The degree of CSC expression tends to correlate with patient prognosis since the percentage of CSCs in a tumor may reflect tumor subtype or progression level, with more CSCs generally leading to poor clinical outcome. The presence of large population of CSCs indicates the rapid proliferation rate of the tumor cells. Tumor cells are more genetically unstable as they lack the property of differentiation, and hence, it is not possible to generate differentiated progeny thus enhancing the selective advantage in context to cancer therapy [ 128 ].

6.3. Cancer Stem Cells in Metastasis

The term “metastasis” can be defined as the dispersion of the cancer cells via the lymphatic system or bloodstream, from the tumor it has been originated (the primary tumor) to the tissues or organs surrounding the tumor. CSCs participate in the metastasis in two ways: firstly, as the original CSCs that started primary tumor, and secondly, as CSCs derived from first one or another cell in the tumor that acquired metastatic traits. The second type of cells is more invasive than the first one and therefore more likely to metastasize due to additional genetic and epigenetic alterations.

The CSC model can be used to describe the biology of metastases and to explain the similarities between primary tumors and autologous lymph node. These similarities are in contrast to traditional cancer models which reveal that metastasis originates from monoclonal expansions of specific individual tumor subclones having specific genotypic and phenotypic features; thus, they are different from primary tumors [ 129 ]. The genetic changes that are acquired at the initial stages of tumor development are the reason for the predetermining the metastasis capacity. Because of the metastatic potential of solid tumors, the poor prognosis issue of the patients can be successfully determined using various genomic approaches and molecular signatures. Therefore, it can be suggested that the majority of cancer stem cells found in primary tumors have a metastasis gene program [ 130 ]

A subset of CSCs, which are essential for metastasis, can be found at the invasive edge of pancreatic carcinomas. CSCs are likely to aid the migration of tumor cells away from the primary tumor which is one of the prime steps in the metastatic cascade to form secondary tumors in distant organs. Thus, it can be suggested that CSCs may metastasize together along with another type of cancer cells [ 14 ]. Various genetic signatures present in CSCs might predict the recurrence and metastasis of tumor. The biomarkers such as CD133, CD44, and CD166 when combined together easily identify the risk of recurrence and metastasis in the patients suffering from colorectal cancer. The putative stem cells CD44 + and CD24 -/low can be detectable in the metastatic pleural fluid found in breast cancer and are mainly responsible for generating primary tumors in an orthotopic site and cause lung metastasis [ 130 ]. Thus, it can be concluded that CSCs undergo neoplastic growth in the new location when they metastasize [ 14 ].

6.4. Cancer Stem Cells in Cancer Recurrence

The role of CSCs in cancer recurrence can be found due to their tumorigenic properties and their tendency to resist many therapies like chemotherapy and radiotherapy [ 131 ]. CSCs found in breast cancer when grown in the culture are found to be resistant to the chemotherapeutic agents. Thus, after chemotherapy in breast cancer patients, there are high percentages of cells with breast CSC properties. This indicates that the treatment may have been less effective in destroying CSCs rather than removing other cancer cells. This study observed the biopsies of patients by measuring cell surface proteins. However, the fact that whether the location of these measured proteins was actually on clonally related cancer cell population, or whether there was some change in their pattern by the treatment, or whether the sensitivity of the treated cells was different in those patients who responded therapy was ambiguous. The drug-resistance mechanism of CSCs is not completely known, but it could be possible that the overexpression of antiapoptotic proteins or drug-metabolizing proteins on them could pump out the drugs out of the cell [ 6 ].

Daunorubicin and Ara-C are the examples of some chemotherapeutic drugs, to which CSCs of leukemia are resistant [ 132 ]. CSCs of various other cancers, such as pancreas or colon cancer, were also found to be resistant to chemotherapy, while some CSCs are resistant to radiation also [ 133 , 134 ]. Interestingly, parthenolide and rapamycin are the drugs that kill the CSCs of AML but do not work against normal hematopoietic stem cells. A decrease in tumorigenicity was reported by temozolomide and bevacizumab when treated against CSCs in glioblastoma [ 135 ].

The use of the HER1/HER2 inhibitor lapatinib in HER2-positive breast cancer, responsible for the amplification of the HER2 gene, is the best example for eliminating CSCs by targeting a particular cancer-specific genetic alteration [ 131 ].

Most of the cases studied had revealed that CSCs could be effectively destroyed by the introduction or inhibition of specific genetic alterations. This demonstrated that an effective therapeutic approach can be achieved by the targeting of a mutation in all tumor cells and further added that in cancer recurrence, there is an involvement of both CSCs and differentiated cells [ 6 ].

7. Cancer Stem Cells in Evading Programmed Cell Death

Several existing therapeutics fail to eliminate tumors owing to CSCs' capacity to evade various programmed cell deaths which are all dysregulated in CSCs. As a result, establishing CSC-selective and programmed death-inducing therapeutic approaches seems to be essential.

7.1. Apoptosis

The proper balance of life and death signals is critical as the breakdown in control can lead to tumor growth [ 136 ]. In this aspect, apoptosis is critical in the prevention of cancer development. Many studies have demonstrated that genetic abnormalities turn normal stem cells into CSCs, permitting them to escape apoptosis and hence develop tumors [ 136 ]. Dedifferentiation and reprogramming are two alternative ways for forming apoptosis-resistant CSCs. Unfortunately, there are no therapeutics that specifically target CSCs. In addition to PI3K/Akt, NOTCH1, and Wnt/ β -catenin, CSCs overexpress antiapoptotic proteins and possess fast DNA repair, resulting in apoptosis resilience [ 136 ] and eventually resistance to several chemotherapeutic treatments [ 136 , 137 ]. Thus, while traditional anticancer medicines can reduce or eliminate cancer cells, CSCs can endure, resulting in relapses in many types of cancer or metastases by migrating beyond the initial location of the tumor [ 137 ].

7.1.1. Mechanisms of CSCs to Evade Apoptosis

CSCs exhibit inherent resistance to apoptosis via a variety of mechanisms, including the overexpression of multidrug resistance transporters such as the ATP binding cassette (ABC) transporter family. ABC transporter overexpression has been documented in numerous malignancies, most noticeably in CSCs [ 138 ]. CSCs have been demonstrated to be enhanced in a variety of malignancies, leading to treatment resistance [ 139 ]. The PI3K/AKT/mTOR signaling is another route implicated in the evasion strategy of CSCs. This route is essential for cell proliferation, metabolism, invasion, survival, and thus for tumor formation and CSC maintenance [ 19 ]. Furthermore, a modulation in the ratio of apoptotic to antiapoptotic proteins is needed for the development of many cancers and contributes to the survival of CSCs. However, their role to drug resistance is not fully understood. BCL2 family proteins, which include proapoptotic proteins Bax, Bak, Bid, Bim, Bik, Noxa, and Puma as well as antiapoptotic molecules Bcl-2, Bcl-XL, and Mcl-1, were shown to be overexpressed in CSCs [ 140 ]. The altered interaction of pro- and antiapoptotic proteins is linked to CSC resistance to apoptosis and anticancer therapeutics.

Furthermore, there is a significant rise in the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) in CSCs which is a redox-sensing transcription factor that supports and enhances CSC survival.

TRADD, a factor implicated in multiple receptor signaling pathways of cell survival and death, is required for NF-B activation in CSCs that in turn promotes the development of a range of inflammatory cytokines and apoptosis inhibitory protein [ 141 ]. Furthermore, lowering NF-B activation by TRADD silencing reduced cell viability, demonstrating TRADD's function in CSC survival.

XIAP, a member of the inhibitor of apoptosis (IAP) family of proteins, regulates apoptosis in CSCs and is expressed at greater levels in glioblastoma and nasopharyngeal cancer stem cells [ 142 ]. FLICE-inhibitory protein (c-FLIP), the major antiapoptotic protein responsible for resistance to chemotherapy-induced apoptosis, is overexpressed in a variety of malignancies. Its levels in CSCs, on the other hand, are substantially greater than in normal cancer cells [ 143 ], conferring resistance to TRAIL-induced apoptosis. Moreover, inhibiting cFLIP makes CSCs susceptible to TRAIL-induced apoptosis, indicating a function for cFLIP in death resistance [ 143 ].

7.2. Autophagy

Autophagy is required for preservation of pluripotency or the ability to self-renew and remain undifferentiated which is an essential feature of CSCs [ 144 ]. In fact, it has been discovered that CSCs from various malignancies retain a strong autophagic flux [ 138 ], and along with hypoxia, it is required for the preservation of the stem cell niche. Interestingly, Zhu et al. established that autophagy is HIF-1-dependent and essential for maintaining the balance between pancreatic CSCs and normal cancer cells [ 145 ]. As a result, autophagy is an adaptive process required for CSC maintenance.

7.2.1. Mechanisms of CSCs to Evade Autophagy

Autophagy and autophagy proteins are elevated in breast CSCs [ 146 ]. Its failure has a deleterious impact on the expression of staminal markers and, hence, the ability to self-renew in a variety of CSCs.

The basic pathways of autophagy-dependent CSC maintenance have been [ 147 ] demonstrated to occur via the EGFR/Stat3 and TGF/Smad pathways in breast cancer stem-like cells. Inhibiting autophagy in triple-negative breast CSCs inhibited the STAT3/JAK2 pathway-mediated production of IL-6, a cytokine critical for CSC survival and required to produce the CD44 + /CD24 low phenotype in breast cancer cell lines [ 148 ]. As a result, the IL-6-JAK2-STAT3 pathway appears to play an important role in the conversion of non-CSCs to CSCs.

FOXO proteins that govern autophagy affect cancer development and metastasis as well as the fate of CSCs [ 149 ], which needs to be explored. FOXO3 inhibition resulted in long-term CSC self-renewal in many malignancies [ 150 ]. However, further research is needed to understand how FOXO-dependent control of stemness and autophagy pathways are linked in carcinogenesis. The study on ovarian CSCs showed a link between autophagy and stemness [ 151 ]. Forkhead Box A2 (FOXA2) has been reported to be overexpressed in ovarian CSCs and is regulated by autophagy. Moreover, inhibition of autophagy by pharmacological and genetic techniques results in FOXA2 decrease and, as a result, loss of self-renewal potential. Several studies have shown that autophagy plays a function in chromosomal stability management; hence, CSCs may activate autophagy to avoid additional DNA damage and thus maintain their survival [ 152 ].

8. Therapeutic Strategies to Target CSCs

CSCs have been found to influence the tumor metastasis and drug resistance. By targeting CSCs, the problem of the poor prognosis of the patients can be overcome leading to increased survival rates of the patient. There are several ways or approaches to target CSCs; some of them are explained in the following section.

8.1. Targeting CSC Niche

The main properties of the CSC microenvironment are inflammatory cytokines, hypoxia, and the regulation of the potency of self-renewal, proliferation, and differentiation via the perivascular niche. Several inflammatory cytokines (e.g., IL-1 β , IL-6, and IL-8) are responsible for the activation of various pathways such as STAT3 and NF- κ B in tumors as well as stromal cells. The process of angiogenesis, metastasis, and self-renewal is promoted by the secretion of cytokines via the aforementioned pathway in a positive feedback loop. It has been observed that blocking of the IL-6/IL-8 cytokine signaling leads to decrease in tumor growth [ 153 ]. Repertaxin which is known to be a noncompetitive inhibitor of the IL-8 and CXCR1 signaling is responsible for reducing tumor size and increasing the efficacy of chemotherapy. Plerixafor (AMD3100), a drug that targets CXCR4, is an effective mobilizer for HSC and used in the treatment of the patients suffering from multiple myeloma and non-Hodgkin lymphoma (NHL) [ 154 ].

Hypoxic microenvironment results in the activation of HIFs which resist cellular differentiation and chemotherapy/radiotherapy. Moreover, it also modulates angiogenesis and apoptosis [ 155 ]. Few small molecules acting as inhibitors of the HIF pathway approved by the Food and Drug Administration (FDA) are bortezomib (Velcade®, PS-341) approved in 2003 against multiple myeloma and temsirolimus (Torisel®, CCI-779) approved in 2007 against renal cell carcinoma. Bevacizumab (Avastin®), cediranib (AZD2171), sunitinib, and vandetanib are examples of some other drugs which inhibit angiogenesis by blocking the vascular endothelial growth factor (VGEF) that accounts for the migration of the endothelial cells. These drugs also suppress the self-renewal capacity of CSCs and thus inhibiting tumor propagation and metastasis [ 156 ].

8.2. Targeting CSC Signaling Pathway for Cancer Therapy

The targeting CSC signaling pathways involved in self-renewal, proliferation, and differentiation, in order to maintain the stem cell properties, offers new avenues for cancer treatments. Therefore, targeting the essential pathways, such as Notch, Wnt, and Hedgehog (Hh), can block the self-renewal potency of CSCs [ 19 ]. Disulfiram, an antialcoholism drug in the case of breast CSCs, inhibits TGF- β -induced metastasis by the ERK/NF- κ B/Snail pathway. Vismodegib, approved by European Medicines Agency (EMA) in 2013 and US FDA in 2012, is a drug that targets the Hh pathway and is used against the therapy of those metastatic basal cell carcinoma (BCC) patients whose surgery and radiotherapy could not be done [ 157 ]. BMS-833923, saridegib (IPI-926), sonidegib/erismodegib (LDE225), PF-04449913, LY2940680, LEQ 506, and TAK-441 were used as monotherapy. Vantictumab (OMP-18R5) is a mAb that blocks the Fz receptors (such as Fz1, Fz2, Fz5, Fz7, and Fz8) [ 57 ] and reduces proliferation of tumor cell and tumor-initiating cell number in tumors of the lung, breast, colon, and pancreas.

Identification of molecular mechanisms and signaling pathways characteristics for CSCs in solid and hematologic malignancies has been the focus of numerous studies. Notch, Hedgehog, Wnt, and NF- κ B cascades have been reported to be dysregulated in cancers and are linked with high proliferative multidrug resistance. These associations serve as potential targets for CSC specific eradications. However, further studies are required to determine the safety of these targeted therapies because these signaling pathways are also crucial for normal stem cell maintenance [ 19 ].

8.3. Targeting CSCs for Immunotherapy

Various experiments have been conducted, focusing on how the immune system plays a role in preventing tumor growth. However, cancer cells' escape mechanisms from the immune system have also been studied. Along with chemotherapy and radiotherapy, CSCs are found to be resistant to immune therapy also. The evidence can be found in many cases: the absence of the expression of major histocompatibility complex class I leads to easy escape from T lymphocytes [ 158 ].

Cancer immunotherapy mainly targets the growth and expansion of cancer cells by recognizing them via the immune system. Some cell surface molecules which are being expressed on CSCs play a major role in identifying particular antigen and detecting specific targets for CSC immunotherapies [ 19 ], e.g., ALDH, CD44, CD133, EpCAM, and HER2. Immune checkpoints act as the endogenous regulators of the immune response. They also play a role to limit the autoimmunity as they mediate coinhibitory signaling pathways. An example of such immunoinhibitory pathways is CTL antigen 4 (CTLA-4/B7) or the programmed cell death-1 (PD1/PDL1). These are the negative immune regulatory pathways that have been identified as protecting cancer cells from being killed by immune cells [ 153 ]. Recently, several novel anti-CSC immunotherapy strategies have been developed, such as immunological checkpoint blocking or chimeric antigen receptor-T (CAR-T) therapies [ 159 ]. CAR-T cells are engineered T cells possessing an artificial receptor specific for tumor-associated antigens (TAAs) through which they perfectly target and eradicate cancer cells.

In preclinical studies, CSC markers used in CAR-T cell therapies include CD20, CD44, c-Met CD133, CD166, CD38, CLL-1, CD123, EpCAM, CD171, ROR1, CD47, and CD117. Moreover, many of them have recently undergone clinical trials, resulting in substantial cancer regression [ 159 ].

Some drugs are approved by the FDA that target checkpoint receptors of the immune system and have shown effectiveness in the cancer patients, e.g., nivolumab, pembrolizumab, cemiplimab (CTLA-4, PD-1), avelumab, durvalumab, and atezolizumab (PD-L1) [ 19 ].

In order to enhance the efficacy for treating cancer, many effectors capable of recognizing and killing CSCs are also targeted for immunotherapy, such as cells involved in innate immunity (e.g., natural killer (NK) cells and γδ T cells), antibodies involved in acquired humoral immunity, and cells in acquired cellular immunity (e.g., CSC-based dendritic cells and CSC-primed cytotoxic T lymphocytes) [ 137 ].

8.4. Metformin: An Antidiabetic Drug Targeting CSCs

Metformin ( N ′, N ′ − dimethylbiguanide), a well-known antidiabetic drug used for treating the patients of type 2 diabetes mellitus (DM), is an oral hypoglycemic agent. It acts by downregulating hepatic gluconeogenesis thus reduces blood glucose level and upregulates the uptake of glucose in the peripheral tissues [ 160 ].

It has been observed that metformin possesses antitumor effects as its consumption reduces the chances of breast and pancreatic cancers in the patients suffering from DM, but the mechanism of drug action is still unknown. The increase in insulin sensitivity caused by metformin inhibits cancer cell growth by activating AMP kinase (AMPK), which then inhibits the PI3K/Akt/mTOR signaling pathway through mTOR phosphorylation resulting in rapid inhibition of protein synthesis and cell growth [ 161 ]. By regulating various processes, such as expression of cyclin D1-mediated cell cycle, p53, and phosphorylation in breast cancer and pancreatic cancers, metformin can directly suppress the tumor growth and cell proliferation. By inactivating the NF- κ B pathway and HIF-1 α , metformin can reduce the production of inflammatory cytokines (e.g., IL-6, TNF- α , and VEGF). Metformin's antitumor activity in vitro and in vivo may also be associated with inhibition of the insulin/IGF-1 pathway via AMPK activation by inactivation of breast CD44 + /CD24 CSCs and the EMT phenotype, or even with inhibiting cellular proliferation, clonogenic potential, migration/invasion, and CSC self-renewal capacity in gemcitabine-resistant pancreatic cancer cells [ 162 ]. Metformin also functions as an immunomodulator by activating AMPK and blocking the HIF-1 pathway, which reduces CD39/CD73 expression-dependent MDSC immunosuppressive activity in ovarian cancer patients and boosts antitumor T cell immunological responses [ 163 ].

Metformin can considerably reduce microvascular density (MVD), improve vascular normalization, and suppress tumor angiogenesis in metastatic breast cancer models, with downregulation of platelet-derived growth factor B (PDGF-B) playing a key role in this process [ 164 ]. Moreover, restoration of normalization in the tumor vasculature further increases the sensitivity of CSCs into therapy [ 165 ]. Recently, Seo et al. [ 166 ] evaluated the effect of the mevalonate (MVA) pathway on the metformin-induced tumor suppression as this pathway results in the synthesis of sterols and protein prenylation, both of which play a pivotal role in tumor growth. They found that in the case of colorectal cancer (CRC), metformin acts as a negative regulator of the mevalonate pathway and shows inhibitory effects on CSCs. The increased expression of the MVA pathway enzymes (e.g., FDPS, GGPS1, HMGCR, and SQLE) was reversed upon adding mevalonate. Metformin also suppressed CSCs by inhibiting the processes of protein prenylation via geranylgeranylation and farnesylation that occurs via the MVA pathway.

The expressions of CSC surface marker CD44 and EpCAM; CSC genes such as EZH2, Notch1, Nanong, and Oct4; and miRNA of let-7 and miRNA-200 family in the CSC-like sphere cells of gemcitabine-resistant cells have also been found to be inhibited by metformin [ 167 ]. Overall, these data can suggest that metformin possesses antitumor effects and may commit to targeting CSCs.

8.5. Targeting CSCs via Nanotechnology

It is reported that the CSCs promote tumor growth and are highly resistant to traditional therapies, such as chemotherapy and radiotherapy, leading to tumor relapse and metastasis. Therefore, the use of nanotechnology along with tumor biology is of great importance, such that nanosized materials may be used for anticancer therapies driven by CSC [ 168 ].

Nanotechnology is a branch of science concerned with the study of devices with dimensions ranging from 1 to 1000 nanometers. Recently, several nanostructures from organic as well as inorganic materials have been used in either passive or active targeting of tumors for cancer therapy and diagnosis. A novel conception of nanovesicles, polymeric micelles, liposomes, dendrimers, and polymeric nanoparticles (NPs) can access a solid tumor tissue via the porous structure of a tumor vascular system and selectively deliver therapeutic agents to the targeted sites [ 8 ]. The surface of the nanoparticles has been developed to target CSCs accurately and effectively. Due to their magnetic property, the nanoparticle may be concentrated in the tumor region and the particular drug or monoclonal antibody can be targeted and released directly at the tumor site without affecting any other parts of the body [ 8 ].

Numerous nanoparticles with different sizes can be easily prepared by modifying their surfaces to target CSCs, such as allotropes of carbon (e.g., nanodiamond, graphene, and carbon nanotubes), noble metal (e.g., gold nanoparticles), organic polymers, and liposome nanoparticles. Graphene oxide (GO) is a graphene derivative with carbon atoms linked to oxygen functional groups, giving it exceptional chemical versatility. As a result, graphene's surface can be easily modified with various biochemical molecules and agents of interest, making graphene an excellent carrier of drugs or nucleic acids for the targeted cancer therapies. GO specifically targets a global phenotypic property of CSCs, and it has the potential to reduce the number of genuine CSCs by inducing differentiation and inhibiting proliferation. Carbon nanotubes on the other hand are the cylindrical nanostructure of graphene that are mainly used in carbon nanotube-mediated thermal treatments for the elimination of both the differentiated cells that are responsible for creating the bulk of tumor and the breast cancer stem cells that promote growth and recurrence of tumors. Nanodiamonds are the truncated semioctahedral carbon structure that are found to be the very efficient nanomedicine-based approach for overcoming chemoresistance in hepatic CSCs after forming a nanodiamond drug complex by the process of physical adsorption of epirubicin on nanodiamonds [ 169 ]. Gold nanoparticles are used as nanovectors for translational purposes, as they are biocompatible as well as nontoxic. To target glioblastoma CSCs, gold NPs are coupled with a peptide recognizing CD133. PEGylated gold NP functions efficiently with an anti-CD44 antibody to target breast or gastric CSCs. To target CSCs, organic nanoparticles such as liposomes and polymeric nanoparticles are also used [ 169 ]. Disulfiram, an antialcoholism drug as well as an NF- κ B inhibitor, when combined with copper in vivo , is employed in a liposome to target CSCs and reversing the process of chemoresistance [ 170 ].

Recently, nanotech-based drugs have been the interest of many researchers because of their effectiveness in developing anticancer therapies and targeting CSCs. The examples of some clinically approved nanomedicines are albumin-bound paclitaxel particles (Abraxane), iron oxide nanoparticles (nanotherm), methoxy-PEG-poly(d,l-lactide)-paclitaxel micelle (gene-xol-PM), PEG-1 asparaginase (Oncaspar), PEGylated liposome (Doxil), and SMANCS (zinostatin) [ 171 ]. The nanotech-based anticancer drugs could help in the treatment and prevention of various types of cancers because of their excellent diffusion capacity and effectiveness against various tumors and CSCs. Furthermore, nanomedicines used to target CSCs have several advantages, which include enhanced cell absorption, greater systemic circulation, improved biodistribution profiles, and the ability to address the problems of low stability and solubility with minimal side effects [ 8 ].

9. Conclusions

A subset of a distinct group of CSCs is related to the tumor phenotype characterized by the enhanced cell survival rate, invasive and metastatic ability, resistance to treatment, and recurrence of tumors which can lead to poor prognosis. As the environmental factors affecting CSC niche are not well understood, therefore, there is still a lot of scope for improving the current methods used for isolating, identifying, and targeting CSCs. Numerous novel anti-CSC immunotherapy strategies, such as chimeric antigen receptor-T cell therapeutics, are increasingly being optimized for improving the specificity and clinical outcomes leading to the reduction of adverse side effects in cancer patients [ 159 ]. Targeting tumor cells via chemotherapy results in the emergence of drug-resistant tumor cells which probably originate from CSCs. This is due to the CSC model, which states that CSCs divide symmetrically to replenish the CSC pool and asymmetrically to generate daughter cells (non-CSCs) with low tumorigenic potential. However, due to transcriptional, epigenetic, or environmental changes, non-CSCs can undergo a dedifferentiation process to acquire stem-like characteristics and get reprogrammed towards a more aggressive tumorigenic fate. Thus, the dedifferentiation of non-CSCs into CSCs can make latter resistant to various conventional therapies. To eliminate the chances of tumor recurrence, there is a need for precise diagnostic and screening methods that could detect and monitor the residual CSCs who have escaped the conventional therapy. The novel approach of combining small molecules and immunotherapies with traditional chemotherapeutic drugs that specifically target CSCs only could provide a better direction for the treatment of cancer patients.

As discussed in this review, both NSCs and CSCs contain a variety of biomarkers and signaling pathways. Therefore, all the regulatory factors cannot be used as therapeutic targets that contribute to CSCs. Effective targeting of CSCs without destroying the normal cells needs some of the novel approaches for identifying the realistic drug targets specific to CSCs only. Besides, factors responsible for the stemness of CSCs should also be considered. Still numerous challenges must be overcome to effectively target CSCs, such as exploration of tumor-specific characteristics of CSCs, lack of models that recapitulate the biological complexity of tumors, and obstacles in mimicking the CSC-specific niche. Moreover, most interesting question in the therapeutic use of cancer is whether CSCs should be triggered or hindered. As a matter of fact, various promising strategies for suppressing tumor relapse and metastasis in terms of targeting specific CSCs of a specific cancer must be investigated in order to achieve successful CSC-targeted therapies and thus improve cancer patient survival rates.

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Conflicts of interest.

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Authors' Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas, that is, revising or critically reviewing the article, giving final approval of the version to be published, agreeing on the journal to which the article has been submitted, and confirming to be accountable for all aspects of the work.

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• Review Article
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• Published: 07 February 2020

Targeting cancer stem cell pathways for cancer therapy

• Liqun Yang 1 , 2   na1 ,
• Pengfei Shi 1 , 2   na1 ,
• Gaichao Zhao 1 , 2 ,
• Jie Xu 1 , 2 ,
• Wen Peng 1 , 2 ,
• Jiayi Zhang 1 , 2 ,
• Guanghui Zhang 1 , 2 ,
• Xiaowen Wang 1 , 2 ,
• Zhen Dong 1 , 2 ,
• Fei Chen 3 &
• Hongjuan Cui 1 , 2  

Signal Transduction and Targeted Therapy volume  5 , Article number:  8 ( 2020 ) Cite this article

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• Cancer stem cells

Since cancer stem cells (CSCs) were first identified in leukemia in 1994, they have been considered promising therapeutic targets for cancer therapy. These cells have self-renewal capacity and differentiation potential and contribute to multiple tumor malignancies, such as recurrence, metastasis, heterogeneity, multidrug resistance, and radiation resistance. The biological activities of CSCs are regulated by several pluripotent transcription factors, such as OCT4, Sox2, Nanog, KLF4, and MYC. In addition, many intracellular signaling pathways, such as Wnt, NF-κB (nuclear factor-κB), Notch, Hedgehog, JAK-STAT (Janus kinase/signal transducers and activators of transcription), PI3K/AKT/mTOR (phosphoinositide 3-kinase/AKT/mammalian target of rapamycin), TGF (transforming growth factor)/SMAD, and PPAR (peroxisome proliferator-activated receptor), as well as extracellular factors, such as vascular niches, hypoxia, tumor-associated macrophages, cancer-associated fibroblasts, cancer-associated mesenchymal stem cells, extracellular matrix, and exosomes, have been shown to be very important regulators of CSCs. Molecules, vaccines, antibodies, and CAR-T (chimeric antigen receptor T cell) cells have been developed to specifically target CSCs, and some of these factors are already undergoing clinical trials. This review summarizes the characterization and identification of CSCs, depicts major factors and pathways that regulate CSC development, and discusses potential targeted therapy for CSCs.

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Introduction.

Cancers are chronologic diseases that seriously threaten human life. Many strategies have been developed for cancer treatment, including surgery, radiotherapy, chemotherapy, and targeted therapy. Because of all these treatments, the incidence rate of cancer has been stable in women and has declined slightly in men in the past decade (2006–2015), and the cancer death rate (2007–2016) also declined. 1 However, traditional cancer treatment methods are effective only for some malignant tumors. 2 The main reasons for the failure of cancer treatment are metastasis, recurrence, heterogeneity, resistance to chemotherapy and radiotherapy, and avoidance of immunological surveillance. 3 All these failures could be explained by the characteristics of cancer stem cells (CSCs). 4 CSCs can cause cancer relapse, metastasis, multidrug resistance, and radiation resistance through their ability to arrest in the G0 phase, giving rise to new tumors. 5 Therefore, CSCs could be considered the most promising targets for cancer treatment.

CSCs were first identified in leukemia and then isolated via CD34 + and CD38 − surface marker expression in the 1990s. 6 , 7 CSCs expressing different surface markers, such as CD133, nestin, and CD44, have been subsequently found in many nonsolid and solid tumors, and these cells also form the bulk of the tumor. 8 , 9 CSCs can generate tumors via the self-renewal and differentiation into multiple cellular subtypes. 10 The activities of CSCs are controlled by many intracellular and extracellular factors, and these factors can be used as drug targets for cancer treatment. 11 To understand the nature of CSCs, we summarized their characteristics, methods for identification and isolation, regulation and current research on targeting CSCs for cancer therapy both in basic research and clinical studies.

The concept of CSCs

Biological characteristics of cscs.

With the deepening of tumor biology research, clinical diagnosis and cancer treatment have significantly improved in recent years. However, the high recurrence rate and high mortality rate are still unresolved and are closely related to the biological characteristics of CSCs. With further understanding of CSC characteristics, research on tumor biology has entered a new era. Therefore, understanding the biological properties of CSCs is of great significance in the diagnosis and treatment of tumors.

CSCs have a strong self-renewal ability, which is the direct cause of tumorigenesis. 12 CSCs can symmetrically divide into two CSCs or into one CSC and one daughter cell. 13 CSCs expand in a symmetrical splitting manner to excessively increase cell growth, ultimately leading to tumor formation. 14 CSCs isolated from original tumor tissue that were transplanted into severe combined immunodeficiency disease (SCID) mice then formed new tumors. 15 CSCs and normal stem cells also share some of the same regulatory signaling pathways, such as the Wnt/β-catenin, 16 Sonic Hedgehog (Hh), 17 and Notch pathways, which are involved in the self-renewal process. 18 In addition, other signaling molecules, such as PTEN and the polycomb family, also play important roles in the regulation of CSC growth. 19 The regulation of CSC self-renewal is the key link to understanding tumorigenesis. These studies will provide a clear target for cancer treatment.

In addition to their self-renewal ability, CSCs also have the ability to differentiate into different cell types. Bonnet and Dick 7 demonstrated in 1997 that CD34 + /CD38 − leukemia stem cells (LSCs) have the ability to differentiate and proliferate in SCID mice. Brain CSCs isolated from patients are positive for the markers CD133 and nestin, which are the same markers as those of normal neuronal stem cells, but some cells lack surface markers for differentiation. 20 Generally, various signaling pathways regulate the self-renewal and differentiation of normal stem cells to promote their proliferation and differentiation in a relatively balanced manner. Once the regulatory balance is destroyed, uncontrolled CSCs ultimately lead to tumorigenesis. 21 CSCs also transdifferentiate into other multilineage cells to regulate tumorigenesis. 22 Bussolati et al. 23 found that renal CSCs differentiated into vascular endothelial cells (ECs) in the bulk of tumors formed in SCID mice after injection of human renal CSCs. Additionally, CSCs that differentiate into vascular ECs and promote angiogenesis have been found in a variety of cancers, such as glioblastoma 24 and liver cancer. 25

Metastasis refers to the process by which cancer cells travel from the primary site through lymphatic vessels, blood vessels, or the body cavity. 26 Since stromal cells (such as granulocytes and macrophages) secrete signaling molecules in the tumor microenvironment (TME), these cells stimulate epithelial–mesenchymal transformation (EMT) to promote the invasion of tumor cells, 27 which induce differentiated human mammary epithelial cells to form mammary glands. 28 Activation of the RAS/MAPK (mitogen-activated protein kinase) signaling pathway transforms nontumorigenic CD44 − /CD24 + breast cancer cells into tumorigenic CD44 + /CD24 − breast cancer cells. 29 A study showed that CSCs are closely related to EMT, and EMT is likely to be the basis for tumor invasion and metastasis. In addition, CD133 + /CXCR4 + pancreatic cancer cells 30 and CD44 + /α2β hi 1/CD133 + prostate cancer cells 31 are also tumorigenic. Therefore, these studies indicate that CSCs play a crucial role in tumor metastasis and development.

Furthermore, understanding the mechanism of CSC drug resistance is vital for cancer treatment and preventing recurrence. 32 CSCs efficiently express ATP-binding cassette (ABC) transporters (including MDR1 (ABCB1), MRP1 (ABCC1), and (ABCG2)), which are multidrug resistance proteins, and these proteins protect leukemia and some solid tumor cells from drug damage and induce drug resistance. 33 According to previous studies, aldehyde dehydrogenase (ALDH), a marker in many CSCs, 34 eliminates oxidative stress and enhances resistance to chemotherapeutic drugs, such as oxazolidine, taxanes, and platinum drugs. 35 ALDH also removes free radicals induced by radiation and stimulates resistance to radiation. 35 Inducing DNA damage and apoptosis through chemotherapy and radiotherapy are commonly used cancer treatments. However, CSCs can effectively protect cancer cells from apoptosis by activating DNA repair abilities. 36

It is currently believed that CSCs are the key "seeds" for tumor initiation and development, metastasis, and recurrence. 37 CSCs have evolved and are highly heterogeneous. 38 Breast CSCs have different expression patterns of surface biomarkers, such as CD44 + , CD24 − , SP, and ALDH +. 29 , 34 , 39 CD271 − or CD271 + melanoma stem cells can form tumors in SCID mice. 40 The heterogeneity of CSCs has also been found in other cancers, including glioblastoma, 41 prostate cancer, 42 and lung cancer. 43 The heterogeneity of CSCs is so complex that more effective biomarkers are needed to identify CSCs or distinguish the heterogeneity of CSCs.

Isolation and identification of CSCs

It is known that the proportion of CSCs in tumor tissues is very low and generally accounts for only 0.01–2% of the total tumor mass. In addition, CSCs and normal stem cells also share similar transcription factors and signaling pathways. Therefore, it is more challenging to isolate and identify CSCs. However, an increasing number of techniques and means have emerged.

CSCs have been identified through different biomarkers in human cancers (Table 1 ). CSCs can be separated by combining specific biomarkers that are mostly located on the cell surface. 3 The primary separation techniques are fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS). 44 , 45 Since Dick JE first screened CSCs from leukemia by using FACS technology, 7 FACS has become the most widely used technique for cell separation. It can perform multibiomarker sorting at one time and has high purity and strong specificity. MACS is a MACS technique. MACS separation is relatively simple, but the technique is cumbersome. Therefore, this method requires high activity of CSCs. 44 , 46 These two methods are effective in separating CSCs from large numbers of cells.

Additionally, there are other ways to separate CSCs from tumors. In 1996, Dr. Goodell observed that after adding Hoechst 33342 to a culture of bone marrow cells, a few cells did not accumulate dyes, and he claimed that these few cells were side population (SP) cells. Therefore, SP cells can be separated by fluorescence screening after the outflow of Hoechst 33342. Recently, SP cells have been identified in various normal tissues and tumor cells. SP cells have high homology, self-renewal and multidirectional differentiation potential. 47 , 48 Some reports have shown that ABCG2 is highly expressed in SP cells. 47 , 49 ABCG2 is highly related to the drug resistance of CSCs and is used as a phenotypic marker for CSCs, 50 , 51 including ovarian cancer, 52 AML, 53 breast cancer, 54 lung cancer, 55 nasopharyngeal carcinoma, 56 and hepatocellular carcinoma (HCC). 57 Montanaro et al. 58 explored the optimal concentration of Hoechst 33342 to reduce the toxic effect. The SP sorting method has universal applicability in the separation and identification of CSCs, especially CSCs with unknown cell surface markers, and is an effective method for CSC research.

The colony-forming ability of CSCs is also used for separation and identification. 59 After digestion of the tumor tissues into single cells, low-density cell culture can be conducted in serum-free medium containing epithelial growth factor (EGF) and basic fibroblast growth factor (FGF). 60 Under this condition, a single CSC will form a cell colony or sphere. Taylor et al. 61 successfully isolated CSCs from a variety of neurological tumors by using this colony formation assay. However, the cell purification rate is low, and the CSC specificity is poor in this assay. The in vivo limited dilution assay (LDA) can be used for assessing CSC activity. After low-density transplantation of immune-deficient mice with the limiting dilution method, CSCs can be identified by ELDA software analysis, and this method is affected by cell density and the microenvironment in mice. 62

Traditional chemotherapeutic drugs mainly affect cancer cells, but CSCs are mostly arrested in the G0 phase and are relatively static, thus evading the killing effect of chemotherapeutic drugs. 63 Hence, the drug-resistant characteristics of CSCs can be used to isolate and identify CSCs. 64 Previous studies have shown that radiotherapy combined with hypoxic culture can also be used to enrich CSCs. 65 In addition, the separation of CSCs can also be accomplished by physical methods. Hepatoma stem cells can be isolated from rat liver cancer tissue by Percoll density gradient centrifugation; a cell fraction with a high nuclear-to-cytoplasmic ratio is obtained. 66 Recently, Rahimi et al. 67 used the miR-302 host gene promoter to overexpress neomycin in cancer cells and selected and collected neomycin-resistant CSCs.

Factors regulating CSCs

CSCs can originate from at least four cell types, including normal stem cells, directed group progenitor cells, mature cells, and the fusion of stem cells and other mutant cells. 68 Therefore, transformed CSCs from normal cells require multiple gene mutations, epigenetic changes, uncontrolled signaling pathways, and continuous regulation of the microenvironment. It is currently believed that there are many similarities between CSCs and embryonic stem (ES) cells, especially regarding their ability to grow indefinitely and self-renew, signaling pathways and some transcription factors. In addition, CSCs exist in the supporting microenvironment, which is vital for their survival. Moreover, the complex interaction between CSCs and their microenvironment can further regulate CSC growth. This section will discuss the effects of transcription factors, signaling pathways, and the microenvironment on CSC survival, apoptosis, and metastasis.

Major transcription factors in CSCs

Generally, stem cells have at least two common characteristics: the ability to self-renew and the potential to differentiate into one or more specialized cell types. 69 Somatic cells can be reprogrammed to become induced pluripotent stem cells by transient ectopic overexpression of the transcription factors Oct4, Sox2, Nanog, KLF4, and MYC. 70 , 71 , 72 In addition, there are some similarities between CSCs and ES cells. It is reasonable that some embryonic transcription factors can be re-expressed or reactivated in CSCs. 69 Therefore, these transcription factors play a very important role in the regulation of CSC growth.

Oct4, a homeodomain transcription factor of the Pit-Oct-Unc family, is recognized as one of the most important transcription factors. 73 Recently, Oct4 has emerged as a master regulator that controls pluripotency, self-renewal, and maintenance of stem cells. 74 Some studies have reported that Oct4 is highly expressed in CSCs. 70 , 73 High expression of Oct4 is positively correlated with glioma grades 75 and promotes self-renewal, chemoresistance, and tumorigenicity of HCC stem cells. 76 High expression of Oct4 is also observed in breast CSC-like cells (CD44 + /CD24 − ). 77 Cisplatin, etoposide, adriamycin, and paclitaxel γ-irradiation upregulate the expression of Oct4 in lung cancer cells, and CD133 + cells are more resistant to drug treatments than CD133 − cells. 78 Data also show that Oct4 expression is associated with poor clinical outcome in hormone receptor-positive breast cancer. 79 Knockdown of Oct4 also reduces the stemness of germ cell tumors. 80 Hence, these studies have proven that Oct4 is a pluripotent factor in CSCs.

Sox2 belongs to the family of high-mobility group transcription factors and plays a significant function in the early development and maintenance of undifferentiated ESCs. It is also one of the key transcription factors in CSCs. Rodriguez-Pinilla et al. 81 found that increased expression of Sox2 in basal-like breast cancer may help to characterize poorly differentiated/stem cell phenotypes. 82 Hagerstrand et al. 82 also found that a high level of Sox2 can induce xenograft glioma. Further studies showed that knockout of Sox2 inhibits glioblastoma cell proliferation and tumorigenicity, which suggests that Sox2 is the basis for maintaining the self-renewal ability of tumor-initiating cells (TICs). 83 Sox2 also maintains the self-renewal of TICs in osteosarcomas, and downregulation of Sox2 drastically decreases its transformative characteristics and tumorigenesis ability in vitro. Furthermore, osteosarcoma cells that lose Sox2 cannot form osteospheres and differentiate into mature osteoblasts any longer. 84 Sox2 is found in invasive cutaneous squamous cell carcinoma (SCC) and promotes the metastasis of cancer cells. 85 These studies suggest that Sox2 promotes self-renewal and tumorigenesis and inhibits differentiation in CSCs.

Nanog, a differentiated homeobox (HOX) domain protein that was first discovered in ESCs, has typical self-renewal and multipotent transcriptional regulatory functions. 86 Although Nanog is silenced in normal somatic cells, abnormal expression has been reported in human cancers, such as breast cancer, cervical cancer, brain cancer, colon cancer, head and neck cancer, lung cancer, and gastric cancer. 86 , 87 , 88 , 89 , 90 Compared to levels in benign tissues, Nanog messenger RNA (mRNA) is elevated in malignant tumors. In a number of patients with colorectal cancer ( n  = 175), high Nanog protein is associated with lymph node positivity and Dukes grade. 91 Similarly, overexpression of Nanog in colorectal CSCs promotes colony formation and tumorigenicity in vivo. 92 In addition, gastric cancer patients with high Nanog levels have a lower 5-year survival rate. 88 The expression level of Nanog is increased in HCC cell lines and primary tumors and is associated with advanced diseases (tumor node metastasis (TNM) stage III/IV). 93 Through the study of prostatic cell lines, xenografts and primary tumors, it was found that Nanog short hairpin RNA inhibits the formation of primary prostate cancer cells (PCA) spheres, clonal growth, and tumorigenesis. 94 In 43 cases of pancreatic cancer tissue microarray analysis, Kaplan–Meier analysis showed that high expression of Nanog (and Oct4) predicted worse prognosis and was negatively correlated with patient survival. 95 These studies indicate that Nanog plays an important role in regulating the self-renewal and proliferation of CSCs.

KLF4 is expressed in many tissues and plays an important role in many different physiological processes. As a bifunctional transcription factor, KLF4 activates or inhibits transcription according to different target genes and utilizing different mechanisms. KLF4 can play an oncogenic or anticancer role, depending on the type of cancer involved. For example, KLF4 is an anticancer factor in the intestinal epithelium and gastric epithelium. 96 The expression of KLF4 is downregulated with hypermethylation and loss of heterozygosity in colorectal CSCs and gastric CSCs. 97 Downregulation of KLF4 is also found in other cancers, such as non-small-cell lung carcinoma, 98 liver cancer, 99 leukemia, 100 anaplastic meningioma, 101 bladder cancer, 102 and esophageal cancer. 103 Although these data clearly demonstrate that KLF4 plays an anticancer role in those cancers, KLF4 may also be an oncogene, which was demonstrated for the first time in nearly a decade. 104 Overexpression of KLF4 in transformed rat renal epithelial cells induces tumorigenesis of laryngeal SCC. 105 In addition, depletion of KLF4 inhibits melanoma xenograft growth in vivo. 106 High expression of KLF4, an oncogene in human breast CSCs, is correlated with an aggressive phenotype in canine mammary tumors. 107 These studies suggest that KLF4 has different functions in different CSCs.

MYC has three family members (C-Myc, N-Myc, and L-Myc, which are encoded by the proto-oncogene family and are essential transcription factors in the DNA-binding proteins of the basic helix–loop–helix (bHLH) superfamily). MYC regulates a large number of protein-coding and noncoding genes and coordinates various biological processes in stem cells, such as cell metabolism, self-renewal, differentiation, and growth. 108 , 109 Although the MYC gene is one of the most commonly activated oncogenes that is involved in the pathogenesis of human cancer, overexpression of MYC alone is surprisingly unable to induce the transformation of normal cells into tumor cells. The overexpression of MYC in normal human cells may be ineffective or highly destructive, resulting in stagnation of proliferation, aging, or apoptosis. 110 MYC is usually deregulated in human cancers, plays an important role in maintaining the number of invasive CSCs, 111 and is also one of the most effective oncogenes for detecting the cell transformation phenotype in vitro and in vivo. Previous studies have shown that deletion of the tumor suppressor gene p53 and MYC synergizes to induce hepatocyte proliferation and tumorigenesis. 112 In addition to p53 deletion, overexpression of Bcl-2 and Bmi-1 and loss of p19ARF also assist MYC in regulating the survival and proliferation of CSCs. 113 The expression of the three members of the MYC family is different in different tumors, such as C-MYC in leukemia and tongue SCC stem cells 114 , 115 and N-MYC in small-cell lung cancer, prostate cancer, neuroblastoma, and medulloblastoma. 116 , 117 L-MYC is expressed in hematopoietic malignancies. 118 In addition, inactivation of MYC results in HCC stem cells differentiating into hepatocytes and biliary duct cells to form bile duct structures, which might be associated with the loss of the tumor marker α-fetoprotein and increased expression of cytokeratin 8, hepatocyte markers, carcinoembryonic antigen, and the liver stem cell marker cytokeratin 19. 119 Studies have also shown that MYC is highly expressed in glioblastoma multiforme stem cells and induces cell proliferation and invasion and inhibits apoptosis. 111 Increased copy number of the MYC gene in human and mouse prostate CSCs has also been found. 120 These studies indicate that MYC induces tumorigenesis with the help of other factors.

Major signaling pathways in CSCs

Many signaling pathways that contribute to the survival, proliferation, self-renewal, and differentiation properties of normal stem cells are abnormally activated or repressed in tumorigenesis or CSCs. Many endogenous or exogenous genes and microRNAs regulate these complex pathways. These signaling pathways can also induce downstream gene expression, such as cytokines, growth factors, apoptosis genes, antiapoptotic genes, proliferation genes, and metastasis genes in CSCs. These signaling pathways are not a single regulator but interwoven networks of signaling mediators to regulate CSC growth. Therefore, this section will describe how signaling pathways regulate CSC growth.

Wnt signaling pathway in CSCs

Wnts include large protein ligands that affect diverse processes, such as the generation of cell polarity, and cells fate. 121 The Wnt pathway is highly complex and evolutionarily conserved and includes 19 Wnt ligands and more than 15 receptors. 122 The Wnt signaling pathway can be divided into canonical Wnt signaling (through the FZD-LRP5/6 receptor complex, leading to derepression of β-catenin) and noncanonical Wnt signaling (through FZD receptors and/or ROR1/ROR2/RYK coreceptors, activating PCP, RTK, or Ca 2+ signaling cascades). 123 In canonical Wnt signaling, in the absence of Wnt ligands (inactive Wnt signaling state, Fig. 1 , left), β-catenin is phosphorylated by glycogen synthase kinase 3β (GSK3β), which leads to β-catenin degradation via β-TrCP200 ubiquitination and inhibits translocation of β-catenin from the cytoplasm to the nucleus. 124 In contrast, in the presence of Wnt ligands (e.g., Wnt3a and Wnt1), the ligands combine with Fzd receptors and LRP coreceptors (active Wnt signaling, Fig. 1 , right). LRP receptors are phosphorylated by GSK3β and CK1α. 125 β-Catenin is released from the Axin complex to enter the nucleus. In addition, β-catenin combines with LEF/TCF and enhances the recruitment of histone-modifying coactivators, such as BCL9, Pygo, CBP/p300, and BRG1, to activate transcription. Noncanonical Wnt signaling does not involve β-catenin. During Wnt/PCP signaling, Dvl is activated through binding of Wnt ligands and the ROR-Frizzled receptor. 126 Dvl inhibits the binding of the small GTPase Rho and the cytoplasmic protein DAAM1. 127 The small GTPases Rac1 and Rho together trigger ROCK (Rho kinase) and JNK (c-Jun N-terminal kinase). This results in cytoskeletal rearrangement and/or transcriptional responses. 128 Wnt/Ca 2+ signaling is activated by G protein-triggered phospholipase C activity, which results in intracellular calcium flux and downstream calcium-dependent cytoskeletal and/or transcriptional responses. 129 , 130

Wnt/β-catenin pathway in cancer stem cells. The canonical Wnt/β-catenin pathway regulates the pluripotency of CSCs and determines the differentiation fate of CSCs. In the absence of Wnt signaling, β-catenin is bound to the Axin complex, which contains APC and GSK3β, and is phosphorylated, leading to ubiquitination and proteasomal degradation through the β-Trcp pathway. However, the complex (TAZ/YAP), the long noncoding RNA TIC1 and proteins (TRAP1 and TIAM1) regulate the β-Trcp pathway. In the presence of Wnt signaling, the binding of LRP5/6 and Fzd inhibits the activity of the Axin complex and the phosphorylation of β-catenin, which makes β-catenin enter the nucleus, and then bind to TEF/TCF to form a complex, which then recruits cofactors to initiate downstream gene expression. Some proteins (DKK2 (Dickkopf-related protein 2), DACT1, CDH11, GECG, PKM2, EZH2, CD44v6, MYC, and TERT), microRNAs (miR-1246, miR-9, miR-92a, miR-544a, and miR-483-5p), and long noncoding RNAs (lncR-β-catm and lncR-TCF7) regulate the activation of the Wnt/β-catenin pathway in CSCs

Aberrant Wnt signaling is found in many cancers, such as invasive ductal breast carcinomas, 131 colorectal cancer, 132 papillary thyroid cancer, 133 esophageal cancer, 134 and colorectal cancer. 135 The activation of Wnt signaling is different in different tumors. Some Wnt activation is caused by mutations in Wnt components, such as Axin mutation in gastrointestinal cancers, 136 APC mutation in colorectal cancer, 137 and β-catenin mutation in gastric cancer and liver cancer. 138 , 139 GSK3 genes are critical for β-catenin regulation; therefore, many researchers expect the occurrence of GSK3 mutations, but GSK3 mutations are not correlated with cancer occurrence. In addition, some genes (pyruvate kinase isozyme M2 (PKM2) in breast cancer 140 and telomerase reverse transcriptase (TERT) in prostate cancer 141 ) and microRNAs (miR-164a in colorectal cancer 142 and miR-582-3p in non-small-cell lung cancer 143 ) inhibit the activity of APC, Axin, and GSK3β to promote the accumulation of β-catenin in the cytoplasm.

Stem cell signaling pathways and transcriptional circuits are related to the alteration or reactivation of signaling pathways. 144 Tumor dormancy is a lag phenomenon in tumor growth. Dormancy may occur during primary tumor formation or in the diffusion of some of the constituent tumor cells. However, primary tumor dormancy and metastatic dormancy seem to be different processes. 145 In some cases, cells in the TME produce cytokines, such as Wnt proteins, secreted inhibitors of bone morphogenetic protein (BMP), and Delta, which activate the signaling pathway to maintain the self-renewal ability of CSCs. 146 Activation of Wnt induce the transformation of dormant CSCs into active CSCs to promote cell cycle progression through β-catenin, increasing the expression of downstream cyclin D1 and MYC, and MYC also promotes the expression of the polycomb repressor complex 1 component Bmi-1 and induces the combination E2F with cyclin E. 147 The extracellular matrix (ECM) protein tenascin C often exists in the gap of stem cells, which supports the cell cycle in breast cancer cells by increasing Wnt signals. 148 In addition, aberrant Wnt signaling has also been observed in the self-renewal of CSCs (Fig. 1 ). Many reports have proven that numerous proto-oncogenes stimulate this process through the Wnt signaling pathway. 135 PKM2 catalyzes the last step of glycolysis and plays an essential role in the proliferation of breast CSCs by associating with increased β-catenin levels at regions “−410 to 180 and −2250 to 2000”. 140 , 145 , 149 Enhancer of zeste homolog 2 (EZH2), a key component of the polycomb PRC2 complex, promotes self-renewal of CSCs by activating β-catenin. 150 Moreover, TERT, an RNA-dependent DNA polymerase, acts as a cofactor and forms a complex with β-catenin to activate Wnt downstream targets in prostate CSCs. 141 Capillary morphogenesis gene 2 increases the expression of nuclear β-catenin to regulate the self-renewal and tumorigenicity of gastric CSCs, 151 and SMYD3, which is located downstream of the Wnt pathway, has a similar effect. 152 In addition, long noncoding RNAs and microRNAs also promote self-renewal of CSCs through the Wnt signaling pathway. LncTCF7 recruits the SWI/SNF complex to regulate the expression of the TCF7 promoter in liver CSCs. 153 Lnc-β-Catm associates with the methyltransferase EZH2 to suppress the ubiquitination of β-catenin and promote its stability, 154 and LncTIC1 interacts with β-catenin and maintains its stability, activating Wnt/β-catenin signaling. 155 MicroRNA-1246, miR-19, and miR-92a suppress the expression of AXIN and GSK3β in CSCs. 156 MicroRNA-544a downregulates GSK3β in lung CSCs. 157 MicroRNA-483-5p upregulates the expression of β-catenin in gastric CSCs. 158 In addition, there are still many genes, microRNAs, and noncoding RNAs in CSCs’ self-renewal through the Wnt signaling pathway.

Wnt signaling also plays an important role in the dedifferentiation of CSCs. HOXA5, which is a member of the HOX family, induces the differentiation of colorectal CSCs. However, Wnt indirectly suppresses indirectly via MYC, which is an important direct target of β-catenin/TCF in the intestine. 159 PMP22, an integral membrane glycoprotein in myelin in the peripheral nervous system, induces the differentiation of gastric CSCs, but its mRNA level declines with activation of the Wnt/β-catenin pathway. 160 Moreover, TRAP1, a component of the HSP90 (heat-shock protein 90) chaperone family, inhibits the differentiation of colorectal carcinoma stem cells by modulating β-catenin ubiquitination and phosphorylation. 161 Lgr5, a member of the G protein-coupled receptor (GPCR) family of proteins, is located downstream of the Wnt signaling pathway and restrains the differentiation of esophageal SCC stem cells. 162

Wnt signaling also plays an important role in regulating CSC apoptosis. Dickkopf-related protein 2 induces G0/G1 arrest and cell apoptosis by suppressing β-catenin activity in breast CSCs. 163 DACT1, a homolog of Dapper that is located at chromosomal region 14q23.1, promotes apoptosis in breast CSCs by antagonizing the Wnt/β-catenin signaling pathway. 164 Cadherin-11, a proapoptotic tumor suppressor, reduces the level of active phospho-β-catenin (ser552) to induce apoptosis in colorectal CSCs. 165 Epigallocatechin-3-gallate increases apoptosis by degrading β-catenin in lung CSCs. 166 The small-molecule inhibitor CWP232228 antagonizes the binding of β-catenin to TCF in the nucleus to induce apoptosis in liver CSCs. 167 In addition, temozolomide combined with miR-125b significantly induces apoptosis by targeting the Wnt/β-catenin signaling pathway in glioma stem cells. 168

Wnt/β-catenin signaling has been implicated in CSC-mediated metastasis. 169 In the cytomembrane, Frizzled8 promotes bone metastasis in prostate CSCs. 170 The leucine-rich repeat containing GPCR4 (LGR4, or GPR48), together with its family members LGR5/6, binds to R-spondins 1–4 and leads to Wnt3A potentiation, activating Wnt signaling in breast CSCs. 171 , 172 Increased levels of CD44v6 mRNA in human pancreatic CSCs, lung CSCs, and colon CSCs promote migration and metastasis through the activation of β-catenin. 173 , 174 , 175 In the cytoplasm, TAZ/YAP interacts directly with β-catenin and restricts β-catenin degradation, 176 but TIAM1 antagonizes TAZ/YAP accumulation and translocation from the cytoplasm to the nucleus. 177 Moreover, CDH11 inhibits the migration and invasion of colorectal CSCs by inhibiting Wnt/β-catenin and AKT/RhoA signaling. 165 Wnt signaling decreases the expression of HOXA5 to promote CSC metastasis. 159 These data suggest that amplified Wnt signaling is important for self-renewal, dedifferentiation, apoptosis inhibition, and metastasis of CSCs.

Notch signaling pathway in CSCs

The Notch signaling pathway consists of the Notch receptor, Notch ligand (DSL protein), CSL (CBF-1, suppressor of hairless, Lag), DNA-binding protein, other effectors, and Notch regulatory molecules. In 1917, studies discovered the Notch gene in a mutant Drosophila. Mammals have four Notch receptors (Notch1–4) and five Notch ligands (Delta-like 1, 3, and 4, Jagged 1, and Jagged 2). 178 Notch and DSL ligands are transmembrane proteins that mediate communication between neighboring cells. Under physiological conditions, the ligand binds to a Notch receptor that is expressed on neighboring cells in a juxtacrine manner, thereby triggering proteolytic cleavage of the intracellular domain (ICD) of Notch and its translocation into the nucleus to bind to the transcription factor CSL, forming the NICD/CSL transcriptional activation complex, which activates target genes of the bHLH transcription inhibitor family, such as HES, HEY, and HERP. 179 , 180

The Notch pathway regulates cancer cells in many tumors, such as glioblastoma, leukemia, and those of the breast, pancreas, colon, and lung, among others. 181 Different tumors and tumor subtypes express different Notch ligands and receptors. Therefore, Notch is known to function as both an oncogene and a suppressive gene. As an oncogene, Notch is overexpressed in gastric cancer, 182 breast cancer, 183 colon cancer, 184 and pancreatic cancer. In contrast, Notch expression is downregulated in prostate cancer, 185 skin cancer, 186 non-small-cell lung cancer, 187 liver cancer, 188 and some breast cancers. 189 Whether Notch acts as an oncogene or a tumor suppressor gene is determined by the microenvironment. 190 Moreover, post-translational modifications of Notch receptors change their affinity for ligands and their intracellular half-lives. 191

Many studies on the Notch pathway in CSCs have shown that activation of Notch promotes cell survival, self-renewal, and metastasis and inhibits apoptosis. Aberrant Notch signaling (Notch1 and Notch4) promotes self-renewal and metastasis of breast and HCC stem cells. 192 , 193 However, microRNA-34a downregulates Notch1. 194 Similarly, abundant Delta-like ligand 4 (DLL4) also promotes tumor angiogenesis and metastasis in gastric CSCs. 195 Delta-like 1 activation of Notch1 signaling requires the assistance of the actin-related protein 2/3 complex to maintain the stem cell phenotype of glioma-initiating cells. 196 Additionally, some intracellular genes also regulate the Notch signaling pathway. For example, MAP17 (DD96, PDZKIP1), a nonglycosylated membrane-associated protein, is located on the plasma membrane and the Golgi apparatus. MAP17 interacts with NUMB through the PDZ-binding domain to activate the Notch pathway in cervical CSCs. 197 Inducible nitric oxide synthase promotes the self-renewal capacity of CD24 + CD133 + liver CSCs through TACE/ADAM17 activation to regulate Notch1 signaling. 198 Moreover, tumor necrosis factor-α (TNFα) enhances the CSC-like phenotype by activating Notch1 signaling in oral SCC cells. 199 Overexpression of PER3 decreases the expression of Notch1 and Jagged 1 in colorectal CSCs. 200 In addition, KLF4 and BMP4 also increase Notch1 and Jagged 1 in breast CSCs to regulate cell migration and invasion. 201 , 202 BRCA1 is a key regulator of breast cancer cell differentiation; however, it is localized to a conserved intronic enhancer region within the Notch ligand Jagged 1 gene to maintain the stemness of breast CSCs. 203 Similarly, increased Gli3 also promotes cell proliferation and invasion in oral SCC by increasing Notch2. 204 Hypoxia/hypoxia-inducible factor (HIF)-induced migration and invasion is a well-known phenomenon that has been reported in numerous CSCs. 205 Notch1 can induce the migration and invasion of ovarian CSCs in the absence of hypoxia. 206 Hypoxia-induced Jagged 2 activation enhances cell invasion of breast CSCs 207 and lung CSCs. 208 Moreover, HIF-1α/2α regulates self-renewal and maintenance of glioblastoma stem cells. 209 In addition, increased miR-200b-3p decreases Notch signaling to promote pancreatic CSCs to become asymmetric. 210 MiR-26a directly targets Jagged 1 to inhibit osteosarcoma CSC proliferation. 211 These studies indicate that Notch plays an important role in regulating the self-renewal, growth, and metastasis of CSCs.

Hh signaling pathway in CSCs

The Hh signaling pathway consists of ligands and receptors. The Hh signaling network is very complex, including extracellular Hh ligands, the transmembrane protein receptor PTCH, the transmembrane protein SMO, intermediate transduction molecules, and the downstream molecule GLI. 212 The components of the Hh signaling pathway play different roles. The membrane protein SMO plays a positive regulatory role, while the transmembrane protein PTCH plays a negative regulatory role. PTCH has two subtypes, PTCH1 and PTCH2, 213 and there is 73% homology between the two subtypes. GLI, an effector protein, has three subtypes, Gli1, Gli2, and Gli3, in vertebrates, 214 and these effector proteins have different functions. Gli1 strongly activates transcription, while Gli3 inhibits transcription. 215 Gli2 has dual functions of activating and inhibiting transcription but mainly functions as a transcriptional activator. 216 , 217 Numerous studies have confirmed that Hh signaling is involved in embryonic development and the formation of the nervous system, skeleton, limbs, lung, heart, and gut. 218 As an extracellular signaling pathway, in the presence of ligand signals, Hh ligands bind to PTCH receptors on target cell membranes and initiate a series of intracellular signal transduction processes. 219 When there is no ligand signal, the transmembrane receptor PTCH on the target cell membrane binds to SMO and inhibits SMO activity, which prevents signaling. 220 When the Hh ligand is present, it binds to PTCH, which changes the spatial conformation of PTCH, removing the inhibition of SMO activating the transcription factor GLI and inducing it to enter the cell nucleus, where GLI regulates cell growth, proliferation, and differentiation. 221

Studies have confirmed that abnormal activation of the Hh signaling pathway can be found in human cancers, 222 such as breast cancer, 223 lung cancer, 224 bladder cancer, 225 pancreatic cancer, 226 chondrosarcoma, 227 rhabdomyosarcoma, 228 neuroblastoma, 229 medulloblastoma, 230 and gastric cancer. 231 However, activation of Hh signaling is different in different tumors. Gorlin syndrome (basal cell nevus syndrome), an autosomal dominant condition, is associated with germline loss of the PTCH1 gene. This condition is very common in basal cell carcinoma, rhabdomyosarcoma, and medulloblastoma. 232 , 233 Other Hh pathway components are also mutated in human cancers, such as Gli1 and Gli3 mutations in pancreatic adenocarcinoma, Gli1 gene amplification in glioblastoma, and SUFU (suppressor of fused) mutations in medulloblastoma. 234 , 235 In addition, other genes also regulate the Hh signaling pathway. Speckle-type POZ protein, an E3 ubiquitin ligase adaptor, inhibits Hh signaling by accelerating Gli2 degradation in gastric cancer. 236

Hh signaling plays distinct functions in different types of cancer. 237 During tumor development, Hh signaling has three major roles: driving tumor development, promoting tumor growth, and regulating residual cancer cells after therapy. Based on these functions, the aberrant Hh pathway plays a causal role in CSCs 238 , 239 (Fig. 2 ). The expression level of Hh signaling components is relatively high in CSCs. For example, Hh signaling promotes the maintenance, proliferation, self-renewal, and tumorigenicity of lung adenocarcinoma stem cells. 240 In CD133 + glioma stem cells, SMO, GLI, and PTCH promote cell proliferation, self-renewal, migration, and invasion. The expression of Gli1, PTCH1, and PTCH2 is regulated by histone deacetylase 6. 241 USP48 activates Gli-dependent transcription by stabilizing the Gli1 protein in glioma stem cells. 242 The protein kinase CK2α enhances Gli1 expression and its transcriptional activity in lung CSCs. 243 WIP1 (PPM1D), a nuclear Ser/Thr phosphatase, also enhances the function of Gli1 by increasing its transcriptional activity, protein stability, and nuclear localization in breast CSCs and medulloblastomas. 244 , 245 F-box and leucine-rich repeat protein 17 mediates the release of Gli1 from SUFU for proper Hh signal transduction in medulloblastoma stem cells. 246 Moreover, retinoic acid receptor α2 (RARα2) upregulates the expression of SMO and Gli1 in CD138 + multiple myeloma stem cells. 247 PRKCI, which is regulated by miR-219 in tongue SCC, 248 has a similar function as RARα2 in maintaining a stem-like phenotype in lung SCC cells. 249 Interleukin-27 (IL-27) and IL-6 activate Hh signaling in CD133 + non-small-cell lung CSCs. 250 During self-renewal and maintenance of stemness of BCMab1 + CD44 + bladder CSCs, glycotransferase GALNT1-mediated glycosylation significantly activates Sonic Hh signaling by upregulating Gli1. 251

Hedgehog signaling pathway in cancer stem cells. The Hedgehog pathway plays a key role in stem maintenance, self-renewal, and regeneration of CSCs. The secreted Hh protein acts in a concentration- and time-dependent manner to initiate a series of cell responses, such as cell survival, proliferation, and differentiation. After receiving the Shh signal, the transmembrane protein receptor PTCH relieves the inhibition of the transmembrane protein SMO, which induces Gli1/2 to detach from SUFU and enter the nucleus to regulate downstream gene transcription. During activation of the Hh pathway, some proteins (IL-6, IL-27, Fbxl17 (F-box and leucine-rich repeat protein 17), PPKCI, RARα2, RUXN3, SCUBE2, HDAC6 (histone deacetylase 6), USP48, CK2α, WIP1, GALNT1, VASH2 (Vasohibin 2), BCL6, FOXC1 (forkhead box C1), and p65), microRNAs (miR-324-5p, miR-122, and miR-326), and the long noncoding RNA HDAC2 are involved in the Hedgehog pathway to affect CSC growth

Furthermore, p63, a master regulator of normal epithelial stem cell maintenance, regulates the expression of Shh, Gli2, and PTCH1 by directly binding to their gene regulatory regions, which eventually contributes to the activation of Hh signaling in mammary CSCs. 252 The N-terminal domain of forkhead box C1 binds directly to an internal region (amino acids (aa) 898–1168) of Gli2 to enhance transcriptional activation of Gli2 and determines the stem cell phenotype in breast CSCs. 253 Through recruitment of the deubiquitinating enzyme ATXN3, tetraspanin-8 interacts with PTCH1 and inhibits the degradation of the SHH/PTCH1 complex. In addition, long noncoding microRNAs also activate Hh signaling. For example, lncHDAC2 promotes the self-renewal of liver CSCs by recruiting the NuRD complex onto the promoter of the PTCH1 gene to suppress its expression. 254 In addition, the TME is crucial for the survival of CSCs. Consequently, breast CSCs secrete Shh, which upregulates cancer-associated fibroblasts (CAFs). Subsequently, CAFs secrete factors that promote the expansion and self-renewal of breast CSCs. 255 Hh signaling also promotes self-renewal and metastasis of CSCs by upregulating the expression of related downstream markers of CSCs, such as Bmi-1, Wnt2, ALDH1, CD44, CCND1, Twist1, C-MYC, Nanog, Oct4, PDGFRα (platelet-derived factor receptor-α), Snail, Jagged 1, and C-MET. 231 , 247 , 256 , 257 , 258 , 259 , 260 , 261 , 262 , 263 , 264

Some proto-oncogenes and suppressor genes also directly or indirectly regulate Hh signaling in the proliferation and migration of CSCs. The signal peptide CUB EGF-like domain-containing protein 2 (SCUBE2), a member of the SCUBE family of proteins, inhibits cell proliferation and migration in glioma stem cells by downregulating Hh signaling. 265 BCL6, a transcriptional repressor and lymphoma oncoprotein, directly represses the Sonic Hh effectors Gli1 and Gli2 in medulloblastoma stem cells. 266 The transcription factor RUNX3 suppresses metastasis and the stemness of colorectal CSCs by promoting ubiquitination of Gli1 at the intracellular level. 267 Vasohibin 2 suppresses Smo, Gli1, and Gli2 expression in pancreatic CSCs. 268 β-Catenin stably increases its physical interaction with Gli1, resulting in Gli1 degradation in medulloblastoma stem cells. 269 In addition, microRNAs also target Hh signaling components to regulate CSC proliferation. For example, miR-324-5p significantly decreases SMO and Gli1 in myeloma stem cells. 270 Mir-326 directly downregulates SMO and Gli2 in medulloblastoma stem cells. 271 MiR-326 downregulates SMO in glioma stem cells. 272 Mir-122 targets Shh and Gli1 in lung CSCs. 273 These data demonstrate that amplified Hh signaling is important for the self-renewal, growth, and metastasis of CSCs.

NF-κB signaling pathway in CSCs

Nuclear factor-κB (NF-κB), a rapidly inducible transcription factor, 274 consists of five different proteins (p65, RelB, c-Rel, NF-κB1, and NF-κB2). The main physiological function of NF-κB is the p50-p65 dimer. 275 , 276 , 277 The primary mode of NF-κB regulation occurs at the level of subcellular localization. In the activation stage, transcription factor complexes must translocate from the cytoplasm to the nucleus. 278 The activity of the complexes is regulated by two major pathways (canonical NF-κB signaling and noncanonical NF-κB signaling). In the canonical NF-κB activation pathway, activation occurs through the binding of ligands, such as bacterial cell components, IL-1β, TNF-α, or lipopolysaccharides, to their respective receptors, such as Toll-like receptors, TNF receptor (TNFR), IL-1 receptor (IL-1R), and antigen receptors. 279 Stimulation of these receptors leads to the phosphorylation and activation of IκB kinase (IKK) proteins, subsequently initiating the phosphorylation of IκB proteins. 276 The alternative pathway of NF-κB activation is termed the noncanonical pathway. The noncanonical pathway receptor originates from different classes, such as CD40, receptor activator for NF-κB, B cell activation factor, TNFR2 and Fn14, and lymphotoxin β-receptor. 280 This pathway leads to activation of NF-κB by inducing the kinase (NIK), which then phosphorylates and predominantly activates IKK1. The activity of the latter enzyme induces the phosphorylation of p100 to generate p52. 281

The NF-κB pathway plays an important role in regulating immune and inflammatory responses. In addition, the NF-κB pathway is involved in cellular survival, proliferation, and differentiation. 276 The process of tumor development and progression produces cytokines, growth, and angiogenic factors and proteases to activate NF‐κB signaling. 282 Inflammation has been recognized as a hallmark of cancer. 283 Overactivation of NF-κB signaling has been reported in gastrointestinal, genitourinary, gynecological, and head and neck cancers, breast tumors, multiple myeloma, and blood cancers. 278 , 284 , 285 , 286 However, direct or altered molecular mutations in NF-κB have rarely been reported in human cancers. 287 Based on recent studies, NF-κB regulates many genes and is implicated in cell survival, proliferation, metastasis, and tumorigenesis of cancer. 288 NF-κB activation also directly or indirectly enhances the expression of key angiogenesis factors and adhesion molecules, such as IL-8, vascular endothelial growth factor (VEGF), and growth-regulated oncogene 1. 289

The NF-κB pathway has an essential connection regulating inflammation, self-renewal, or maintenance and metastasis of CSCs (Fig. 3 ). CD44 + cells promote self-renewal, metastasis, and maintenance of ovarian CSCs by increasing the expression of RelA, RelB, and IKKα and mediating nuclear activation of p50/RelA (p50/p65) dimer. 290 High levels of NIK induce activation of the noncanonical NF-κB pathway to regulate the self-renewal and metastasis of breast CSCs. 291 Moreover, stromal cell-derived factor-1 (SDF-1) also has the same effect by regulating the translocation of p65 from the cytoplasm to the nucleus. 292 The inflammatory mediator prostaglandin E2 (PGE2) contributes to tumor formation, maintenance, and metastasis by activating NF-κB via EP4-PI3K (phosphoinositide 3-kinase) and EP4-MAPK pathways in colorectal CSCs. 293 Chemokines, low-molecular-weight proinflammatory cytokines, are important mediators of cell proliferation, metastasis, and apoptosis. 294 C-C chemokine receptor 7 interacts with its ligand chemokine ligand 21 to inhibit apoptosis and induce survival and migration in CD133 + pancreatic cancer stem-like cells by increasing the expression of extracellular signal-regulated kinase 1/2 (Erk1/2) and p65. 295 Furthermore, B cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) also enhances the p65 protein in gastric CSCs. 296 MicroRNAs also play an important role in promoting the proliferation of CSCs. Mir-221/222 promotes self-renewal, migration, and invasion in breast CSCs by inhibiting the expression of PTEN and then inducing the phosphorylation of AKT, resulting in elevated p65, p-p65, and COX2. 297

NF-κB signaling pathway in cancer stem cells NF-κB proteins are involved in the dimerization of transcription factors, regulate gene expression, and affect various CSC biological processes, including inflammation, stress responses, growth, and development of CSCs. The main physiological function of NF-κB is the p50-p65 dimer. The active p50-p65 dimer is further activated by post-translational modification (phosphorylation, acetylation, or glycosylation) and transported into the nucleus, which induces the expression of target genes in combination with other transcription factors. Some proteins (CD44, CD146, TNFRSF19, Bmi-1, FOXP3, and SDF-1) and microRNAs (miR-221 and miR-222) directly regulate the NF-κB pathway. In addition, some proteins (PGE2, GIT-1 (G protein-coupled receptor kinase-interacting protein 1), C-C chemokine receptor 7 (CCR7), and TGF-β) and miR-491 indirectly affect the NF-κB pathway via the ERK and MAPK pathways in CSCs

In addition, other transcription factors also inhibit self-renewal and metastasis in CSCs by the NF-κB pathway. Increased expression of FOXP3 has been identified in different cancers. 298 FOXP3 interacts with NF-κB, inhibits the expression of COX2 located downstream of NF-κB, and affects self-renewal and metastasis in colorectal CSCs. 299 Overexpression of miR-491 blocks the activation of NF-κB in liver CSCs by targeting G protein-coupled receptor kinase-interacting protein 1, which inhibits ERKs. 300 Moreover, some drugs inhibit cell proliferation and metastasis of CSCs by the NF-κB pathway. Disulfiram, an anti-alcoholism drug, inhibits tumor growth factor-β (TGF-β)-induced metastasis via the ERK/NF-κB/Snail pathway in breast CSCs. 301 Sulforaphane preferentially inhibits self-renewal in triple-negative breast CSCs by inhibiting NF-κB p65 subunit translocation and downregulating p52 and its transcriptional activity. 302 Curcumin regulates the proliferation, metastasis, and apoptosis of HCC stem cells by inhibiting the NF-κB pathway. 303 These data demonstrate that amplified NF-κB signaling is important for regulating apoptosis, proliferation, and metastasis of CSCs.

JAK-STAT signaling pathway

The Janus kinase/signal transducers and activators of transcription (JAK-STAT) signaling pathway is a signal transduction pathway that is stimulated by cytokines. This pathway is involved in many important biological processes, such as cell proliferation, differentiation, apoptosis, and immune regulation. Compared with the complexity of other signaling pathways, this signaling pathway is relatively simple. There are three components: the tyrosine kinase-related receptor, the tyrosine kinase JAK, and the transcription factor STAT. 304 Many cytokines and growth factors transmit signals through the JAK-STAT signaling pathway, including interleukin-2-7, granulocyte/macrophage colony-stimulating factor, growth hormone, EGF, PDGF, and interferon. 305 These cytokines and growth factors have corresponding receptors on the cell membrane. The common characteristic of these receptors is that the receptor itself does not have kinase activity, but there is a binding site for the tyrosine kinase JAK in the cells. After binding with ligands, tyrosine residues of various target proteins are phosphorylated through JAK activation to achieve signal transduction from the extracellular to intracellular space. The JAK protein family consists of four members: JAK1, JAK2, JAK3, and Tyk2. 306 JAK proteins have seven JAK homology (JH) domains in their structures. The JH1 domain is the kinase domain, the JH2 domain is the "pseudo" kinase domain, and JH6 and JH7 are the receptor binding domains. 307 STAT is called "signal transducer and activator of transcription". As the name implies, STAT plays a key role in signal transduction and transcriptional activation. At present, seven members of the STAT family (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6) have been identified. The structure of STAT protein can be divided into the following functional regions: N-terminal conserved sequence, DNA-binding region, SH3 domain, SH2 domain, and C-terminal transcriptional activation region. 308 Generally, many cytokines and growth factors integrate with tyrosine kinase-related receptors. After receiving the signal from the upstream receptor molecule, JAK is quickly recruited to and activates the receptor, resulting in JAK activation to catalyze tyrosine phosphorylation of the receptor. The phosphorylated tyrosine on the receptor molecule, which is a signaling molecule, can bind with the SH2 site of STAT. 309 When STAT binds to the receptor, tyrosine phosphorylation of STAT also occurs, which forms a dimer and enters the nucleus. 310 As an active transcription factor, the STAT dimer directly affects the expression of related genes and then changes the proliferation or differentiation of target cells. 311

Constitutive activation of JAKs and STATs was first recognized as being associated with malignancy in the 1990s. 312 Based on current studies, JAK2 mutation and abnormal activation of STAT3 are prone to occur in many tumors. 313 Mutations in JAK2 have been reported in the majority of patients with myeloproliferative neoplasms, 314 such as polycythemia vera, myelofibrosis, and thrombocythemia. 315 , 316 These disorders are caused by the overexpansion of hematopoietic precursors, which are often clonal and can result in leukemia. 314 Several lines of evidence show that constitutive activation of JAK2 and STAT3 in the absence of any stimulating ligand occurs in polycythemia vera. 317 , 318 Moreover, studies have also found aberrant activation of STATs in human cancers, such as head and neck cancer, 319 endometrial cancer, 320 breast cancer, diffuse large B cell lymphoma, 321 HCC, 322 colorectal cancer, glioma, 323 and colon cancer. 324 Furthermore, aberrant STAT5 signaling has been found in the pathogenesis of hematologic and solid organ malignancies. 325 , 326

The JAK/STAT pathway is evolutionarily conserved. This pathway promotes the survival, self-renewal, hematopoiesis, and neurogenesis of ESCs. 327 This pathway is also activated in CSCs. The persistent activation of STAT3 significantly promotes cell survival and the maintenance of stemness in breast CSCs. 328 IL-10 induces cell self-renewal, migration, and invasion in non-small-cell lung CSCs. 329 IL-6 activates the JAK1/STAT3 pathway in ALDH high CD126 + endometrial CSCs. 320 Furthermore, IL-6 also induces the conversion of nonstem cancer cells into cancer stem-like cells in breast cancer by the activating downstream Oct4 gene. 330 Oct4 also activates the JAK1/STAT6 pathway in ovarian CSCs. 331 In CD44 + CD24 − breast and colorectal CSCs, erythropoietin, and IL-6 activate the JAK2/STAT3 pathway. 332 , 333 , 334 Retinol-binding protein 4 activates JAK2/STAT3 signaling by its STRA6 receptor in colon CSCs. 319 HIF-1α enhances the self-renewal of glioma stem-like cells by the JAK1/STAT3 pathway. 335 AJUBA is a scaffold protein that participates in the regulation of cell adhesion, differentiation, proliferation, and migration and promotes the survival and proliferation of colorectal CSCs via the JAK1/STAT1 pathway. 336

Moreover, microRNAs are also involved in activating JAK/STAT signaling by inhibiting negative regulatory factors of JAK2/STAT3. For example, miR-500a-3p targets multiple negative regulators of the JAK2/STAT3 signaling pathway, such as SOCS2, SOCS4, and PTPN, in HCC stem cells, leading to constitutive activation of STAT3 signaling. 322 MiR-30 targets SOCS3 in glioma stem cells. 337 Mir-93 downregulates the expression of JAK1 and STAT3 to induce the differentiation of breast CSCs. Mir-218 negatively regulates the IL-6 receptor and JAK3 gene expression in lung CSCs. 338 In addition, some endogenous or exogenous genes inhibit JAK/STAT signaling in CSCs. Von Hippel–Lindau suppresses the tumorigenicity and self-renewal ability of glioma stem cells by inhibiting JAK2/STAT3. 323 Although there are few studies on JAK in CSCs, there is a role for JAK/STAT signaling in the survival, self-renewal, and metastasis of CSCs.

TGF/SMAD signaling pathway in CSCs

The TGF-β signaling pathway is involved in many cellular processes associated with organism and embryo development, including cell proliferation, differentiation, apoptosis, and homeostasis. Although the TGF-β signaling pathway regulates a wide range of cellular processes, its structure is relatively simple. TGF-β superfamily ligands bind to a type II receptor, which recruits a type I receptor and phosphorylates it. This type I receptor phosphorylates receptor-regulated Smads (R-Smads), which bind to common pathway Smad (co-Smad). The R-Smad/co-Smad complex acts as a transcription factor and accumulates in the nucleus to regulate the expression of target genes. TGF-β superfamily ligands include BMPs, growth and differentiation factors (GDFs), anti-Mullerian hormone (AMH), activin Nodal, and TGF-β. 339 These ligands can be divided into two groups, TGF-β/activin and BMP/GDF. The TGF-β/activin group includes TGF-β, activin, and Nodal, and the BMP/GDF group includes BMP, GDF, and AMH ligands. 340 Based on Smad structure and functions, Smad proteins can be divided into three subfamilies: receptor-activated or pathway-restricted Smad (R-Smads), Co-Smad, and inhibitory Smad (I-Smads), which includes at least nine Smad proteins. 341 , 342 R-Smads are activated by type I receptors and form transient complexes with these receptors. There are two types of Smad complexes: AR-Smads are activated by activin TGF-β, including Smad2 and Smad3, and BR-Smads are activated by BMP, including Smad1, Smad5, Smad8, and Smad9. Co-Smad, including Smad4, is a common medium in various TGF-β signal transduction processes. I-Smads, including Smad6 and Smad7, bind to activated type I receptors and inhibit or regulate signal transduction of the TGF-β family. 343

Many studies have shown that activation of TGF/Smad signaling also occurs in human cancers. Dkk-3, a secreted protein, inhibits TGF-β-induced expression of matrix metallopeptidase 9 (MMP9) and MMP13 to prevent migration and invasion of prostate cancer. 344 Cancer upregulated gene 2 promotes cellular transformation and stemness, which is mediated by nuclear NPM1 protein and TGF-β signaling in lung cancer. 345 TGF/Smad also plays an important role in the cell proliferation of CSCs. Cyclin D1 interacts with and activates Smad2/3 and Smad4, promoting cyclin D1-Smad2/3-Smad4 signaling to regulate self-renewal of liver CSCs. 346 CD51 binds to TGF-β receptors to upregulate TGF-β/Smad signaling in colorectal CSCs. 341 Upregulation of TGF-β1 induces the expression of smad4, p-Smad2/3, and CD133 in liver CSCs. 347 TGF-β1 also upregulates the expression of PFKFB3 through activation of the p38 MAPK and PI3K/Akt signaling pathways to regulate glycolysis in glioma stem cells. 348 Furthermore, silencing ShcA expression also induces activation of STAT4 in breast CSCs. 349 Moreover, miR-148a inhibits the TGF-β/Smad2 signaling pathway in HCC stem cells. 350 Smad7, a newly discovered target gene of miR-106b, is an inhibitor of TGF-β/Smad signaling, which inhibits sphere formation of gastric cancer stem-like cells. 351 Although there are few studies on the TGF/Smad signaling pathway in CSCs, this pathway still plays a very important role.

PI3K/AKT/mTOR signaling pathway in CSCs

Phosphatidylinositol-3-kinase (PI3K) is an intracellular phosphatidylinositol kinase. 352 It consists of the regulatory subunit p85 and catalytic subunit p110, which have serine/threonine (Ser/Thr) kinase and phosphatidylinositol kinase activities. 353 AKT is a serine/threonine kinase that is expressed as three isoforms: AKT1, AKT2, and AKT3. 354 AKT proteins are crucial effectors of PI3K and are directly activated in response to PI3K. One of the key downstream target genes of AKT is the mammalian target of rapamycin (mTOR) complex, which is a conserved serine/threonine kinase. It forms two distinct multiprotein complexes: mTORC1 and mTORC2. 355 mTORC1 consists of mTOR, raptor, mLST8, and two negative regulators, PRAS40 and DEPTOR. 356 , 357 mTORC2 phosphorylates AKT at serine residue 473, which leads to full AKT activation. 358

Studies show that mutations in PTEN lead to the inhibition of PI3K/mTOR signaling in glioblastoma multiforme. However, deletion of PTEN in neural stem cells leads to a neoplastic phenotype that includes cell growth promotion, resistance to cell apoptosis, and increased migratory and invasive properties in vivo. 359 Inactivation of PTEN and activation of protein kinase B have been found in other solid tumors, such as myeloproliferative neoplasia and leukemia. 360 Therefore, the PI3K/mTOR signaling pathway is vital for cell proliferation and survival. Abnormal activation of PI3K/mTOR signaling is found in some cancers, such as non-small-cell lung cancer, 361 breast cancer, 362 prostate cancer, 363 Burkitt lymphoma, 364 esophageal adenocarcinoma, 365 and colorectal cancer. 366

Although PI3K/AKT/mTOR has been extensively studied in cancers, there are few studies in CSCs. 358 PI3K/Akt/mTOR signaling is involved in ovarian cancer cell proliferation and the epithelial–mesenchymal transition. 367 This signaling activation also enhances the migration and invasion of prostate and pancreatic CSCs. 368 , 369 Downregulation of PTEN induces PI3K activation to promote survival, maintenance of stemness, and tumorigenicity of CD133 + /CD44 + prostate cancer stem-like cell populations. 370 PI3K activation promotes cell proliferation, migration, and invasion in ALDH + CD44 high head and neck squamous CSCs. 371 Activation of mTOR promotes the survival and proliferation of breast CSCs and nasopharyngeal carcinoma stem cells. 328 , 372 mTORC1 activation also increases aldehyde dehydrogenase 1 (ALDH1) activity in colorectal CSCs. 373 Activation of mTORC2 upregulates the expression of the hepatic CSC marker EpCAM (epithelial cellular adhesion molecule) and tumorigenicity in hepatocellular CSCs. 374 Nucleotide-binding domain and leucine-rich repeats (NLRs) belong to a large family of cytoplasmic sensors. NLRC3 (also known as CLR16.2 or NOD3) is associated with PI3Ks and blocks activation of PI3K-dependent kinase AKT in colorectal CSCs. 375

In addition, some studies have shown that the mTOR signaling pathway is closely related to the metabolism of CSCs. For example, low folate (LF) stress reprograms metabolic signals through the activated mTOR signaling pathway, promoting the metastasis and tumorigenicity of lung cancer stem-like cells. 376 However, matcha green tea (MGT), an inhibitor of mTOR, inhibits the proliferation of breast CSCs by targeting mitochondrial metabolism, glycolysis, and multiple cell signaling pathways. 377 A link between the PI3K/Akt/mTOR pathway and CSCs is clearly evident.

PPAR signaling pathways in CSCs

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear transcription factors that were first cloned from mouse liver by Isseman and Green. 378 PPARs are also members of the ligand-activated transcription factor superfamily of nuclear hormone receptors that are associated with retinoic acid, steroids and thyroid hormone receptors. PPARs act as fat sensors to regulate the transcription of lipid metabolic enzymes. 379 At present, three subtypes, PPARα, PPARβ, and PPARγ (encoded by the PPARA , PPARD , and PPARG genes, respectively), have been found. 380 PPARα is highly expressed in hepatocytes, cardiac myocytes, intestinal cells, and renal proximal convoluted tubule cells. PPARγ is abundantly expressed in adipose tissue, vascular parietal cells (such as monocytes/macrophages, ECs, and smooth muscle cells), and myocardial cells. 381 PPARβ is expressed in almost all tissues of the body, and its expression level is higher than that of PPARα or PPARγ. 382 In recent years, studies have found that PPARs are closely related to energy (lipid and sugar) metabolism, cell differentiation, proliferation, apoptosis, and inflammatory reactions. 383 PPARs can exert positive or negative effects to regulate target gene expression by binding to a specific peroxisome located at each gene regulatory site and a proliferative response element. 378 Their natural ligands are unsaturated fatty acids, eicosane acids, oxidized low-density lipoprotein, very low-density lipoprotein, and linoleic acid derivatives. 384

To date, there have been many reports about the role of PPARs in cancer cells, including prostate cancer, breast cancer, glioblastoma, neuroblastoma, pancreatic cancer, hepatic cancer, leukemia, and bladder cancer and thyroid tumors. 385 However, the function of PPARs in CSCs is not well understood, except for some reports on PPARγ. As a tumor suppressor, PPARγ binds and activates a canonical response element in the miR-15a gene in breast CSCs to reduce the CD49 high /CD24 + mesenchymal stem cell (MSC) population and inhibit angiogenesis. 386 PPARγ activation also prevents cell spheroid formation and stem cell-like properties in bladder CSCs and induces adipocyte differentiation and β-catenin degradation in adipose tissues. 387 Furthermore, expression of PPARγ restrains YAP transcriptional activity to induce differentiation in osteosarcoma stem cells 388 and melanoma cells. 389 The PPARγ/NF-κB pathway promotes M2 polarization of macrophages to prevent cell death in ovarian CSCs 4. 390 PPARγ activation promotes expression of its target gene PTEN to inhibit PI3K/Akt/mTOR signaling, which stunts self-renewal, tumorigenicity, and metastasis in cervical CSCs, glioblastoma stem cells, and liver CSCs. 391 , 392 However, combined expression of Dnmt3a and Dnmt3b inhibits PPARγ expression by direct methylation of its promoter in squamous carcinomas. 393 PPARs are also closely related to the metabolism of CSCs. PPARα and PPARβ/δ regulate metabolic reprogramming in glioblastoma stem cells, lung CSCs, and mouse mammary gland cancer. 394 The transcription coactivator peroxisome proliferator-activated receptor gamma coactivator 1α (PPARGC1A, also known as PGC-1α) promotes CSC proliferation and invasion by enhancing oxidative phosphorylation, mitochondrial biogenesis, and the oxygen consumption rate of breast CSCs. 395 In addition, the AMPK signaling pathway (adenosine 5′-monophosphate (AMP)-activated protein kinase) is an AMP-dependent protein kinase that is a key molecule in the regulation of bioenergy metabolism and is the core of the study of diabetes and other metabolic-related diseases. AMPK is expressed in various CSCs related to metabolism. Some studies have shown that AMPK is necessary for prostate CSCs to maintain glucose balance. 396 Metformin, an antidiabetic drug that fights cancer, targets AMPK signaling to inhibit cell proliferation and metabolism in colorectal CSCs 397 and HCC stem cells. 398 Therefore, metformin may be a potential therapeutic regent by regulating the energy metabolism of CSCs. These studies suggest that PPARs play an important role in the growth of CSCs.

Interactions between signaling pathways in CSCs

As mentioned previously, these complex signal transduction pathways are not linear. In some cases, crosstalk between and among various pathways occurs to regulate CSCs. 399 Wnt/β-catenin and NF-κB signaling work together to promote cell survival and proliferation of CSCs. TNFRSF19, a member of the TNF receptor superfamily, is regulated in a β-catenin-dependent manner, but its receptor molecules activate NF-κB signaling to regulate the development of colorectal cancer. 400 Knockdown of CD146 results in inhibition of NF-κB/p65-initiated GSK3β expression, which promotes nuclear translocation and activation of β-catenin. 401 In addition, there is negative regulation between Wnt/β-catenin and NF-κB signaling. Studies have revealed a negative effect of β-catenin on NF-κB activity in liver, breast, and colon cancer cells. 402 , 403 Leucine zipper tumor suppressor 2 (LZTS2) is a putative tumor suppressor, and NF-κB activation inhibits β-catenin/TCF activity through upregulation of LZTS2 in liver, colon, and breast cancer cells. 404 , 405 , 406 Wnt/β-catenin and Hh signaling have important functions in embryogenesis, stem cell maintenance, and tumorigenesis. Wnt/β-catenin signaling induces the expression of CRD-BP, an RNA-binding protein, which results in the binding and stabilization of Gli1 mRNA, leading to an increase in Gli1 expression and transcriptional activity, which promotes the survival and proliferation of colorectal CSCs. 407 However, a report showed that noncanonical Hh signaling is a positive regulator of Wnt signaling in colon CSCs. 408

In addition, crosstalk between pathways promotes cell growth and metastasis through maintenance of the CSC population. Downregulation of Notch1 and IKKα enhances NF-κB activation to promote the CD133 + cell population in melanoma CSCs. 409 IL-6/JAK/STAT3 and TGF-β/Smad signaling induce the proliferation and metastasis of lung CSCs. 410 IL-17E binding to IL-17RB activates the NF-κB and JAK/STAT3 pathways to promote proliferation and sustain self-renewal of CSCs in HCC. 411 TGF-β1 silencing decreases the expression of Smad2/3, β-catenin, and cleaved-Notch1 to inhibit the activation of Wnt and Notch signaling in liver CSCs. 346 Activation of TGF-β1 induces lncRNA NKILA expression to block NF-κB signaling, which inhibits metastasis of breast CSCs. 412 TGF-β also directly regulates the expression of Wnt5a in breast CSCs to limit the stem cell population. 413 Furthermore, Notch, IKK/NF-κB, and other pathways together regulate the proliferation and metastasis of CD133 + cutaneous SCC stem cells. 409 PI3K/mTOR signaling upregulates the expression of STAT3 to promote the survival and proliferation of breast CSCs. 328 Inhibition of TORC1/2 increases FGF1 and Notch1 expression. The PI3K/AKT/mTOR and Sonic Hh pathways cooperate to inhibit the growth of pancreatic CSCs. 414 Increasing evidence shows that crosstalk regulates the survival, self-renewal, and metastasis of CSCs.

The microenvironment of CSCs

CSCs interact with the microenvironment through adhesion molecules and paracrine factors. The microenvironment provides a suitable space for the self-renewal and differentiation of CSCs, protects CSCs from genotoxicity, and increases their chemical and radiological tolerance. The TME mainly consists of the tumor stroma, adjacent tissue cells, microvessels, immune cells, and immune molecules. 415 CSCs not only adapt to changes in the TME but also affect the TME. Concurrently, the microenvironment also promotes the self-renewal of CSCs, induces angiogenesis, recruits immune and stromal cells, and promotes tumor invasion and metastasis (Fig. 4 ).

The microenvironment of cancer stem cells. Proliferation, self-renewal, differentiation, metastasis, and tumorigenesis of CSCs in the CSC microenvironment. The CSC microenvironment is mainly composed of vascular niches, hypoxia, tumor-associated macrophages, cancer-associated fibroblasts, cancer-associated mesenchymal stem cells, and extracellular matrix. These cells in response to hypoxic stress and matrix induce growth factors and cytokines (such as IL-6 and VEGF) to regulate the growth of CSCs via Wnt, Notch, and other signaling pathways

Vascular niche microenvironments and CSCs

The normal vasculature is composed of ECs, basement membranes, and parietal cells. ECs are the basis for the formation of the inner surface of blood vessels. 416 Studies reported that glioblastoma stem cells are located around the blood vessels, and the concept of the cancer microvascular environment was first proposed. Calabrese et al. 417 demonstrated that direct contact between ECs and CSCs occurs in brain tumors. CSCs are also found near ECs in other cancers, such as papilloma and colorectal cancer. 418 , 419 A study also showed that CD133 + /CD144 − glioma stem cell-like cells differentiate into cancer cells and endothelial progenitor cells and finally into mature ECs. 420 CSCs differentiate into cancer vascular stem cells/progenitor cells and are directly involved in angiogenesis or form vasculogenic mimicry that is directly involved in the microcirculation of tumors. 421 , 422 ECs also promote CSC-like transformation and cell growth through Shh activation of Hh signaling. 423 Moreover, secreted microvesicles of CSCs promote the proliferation of human umbilical vein ECs and form a tube-like structure in vitro and in vivo in mice. 424 , 425 , 426 This CSC plasticity has also been demonstrated in other tumors, including neuroblastoma, renal, breast, and ovarian cancer. 427 , 428 , 429 , 430

The vascular microenvironment maintains the initial undifferentiated dormancy of stem cells, supports self-renewal, invasion and metastasis of CSCs, and protects CSCs from any injury. 431 The role of the EC signaling system has been proven in maintaining the survival and self-renewal of head and neck SC stem cells. 432 Pasquier and colleagues 433 showed that treatment with EC microparticles in breast and ovarian cancer models increased the number of CSCs and promoted sphere formation of CSCs. The interaction between CSCs and blood vessels promotes the self-renewal of CSCs through the VEGF-Nrp1 loop. 418 CSCs promote cancer angiogenesis by inducing secretion of the cytokines VEGF and hepatocyte growth factor (HGF) from ECs. 434 VEGF receptor 2 plays a key role in vasculogenic mimicry formation, neovascularization, and tumor initiation of glioma stem-like cells. 435 As a result, the secretion of VEGF in stem cell-like glioma cells is higher than that in normal cancer cells 424 and regulates the proliferation of glioma stem cells through the mTOR signaling pathway. 436 Subsequent studies have further shown that multiple signals, such as integrin, Notch, and growth factor receptors, are linked to each other on the cell surface to maintain the stemness of CSCs. 437 , 438

The hypoxia microenvironment and CSCs

Hypoxia is a key component for CSC formation and maintenance. 439 The hypoxic microenvironment maintains the undifferentiated state of cancer cells, enhances their cloning rate, and induces the expression of CD133 as a specific biomarker of CSCs. 440 HIFs are important transcription factors that regulate cellular hypoxia responsiveness 441 and inhibit cell apoptosis. 442 As a heterodimer, HIF is composed of HIFα and HIFβ. 443 HIF-1α regulates the proliferation and fate of CSCs in medulloblastoma and glioblastoma multiforme 444 and activates the NF-κB pathway to promote CSC survival and tumorigenesis. 445 HIF-2α maintains the survival and phenotype of CSCs. 446 HIFα also regulates the expression of the target genes GLUT1, GLUT3, LDHA, and PDK1. Thus, CSCs can adapt to a new method of cell energy metabolism and avoid apoptosis caused by hypoxia. 447

HIFs also regulate the stemness of CSCs. Previous studies have shown that CSCs need to activate HIF-1α and HIF-2α to maintain their self-sustainability under hypoxic conditions 448 and obtain pluripotency by upregulating the Sox2 and Oct4 genes. 440 More importantly, activation of C-MYC by HIF-2α is necessary to ensure undifferentiated CSCs. 449 The Wnt and Notch signaling pathways regulated by hypoxia and can induce the EMT, which promotes the stemness of CSCs and increases the invasiveness and resistance to radiotherapy and chemotherapy. 450 HIF-1α binds the Notch ICD and enhances its transcriptional activity. In the hypoxic microenvironment of glioma, both HIF-1α and HIF-2α require the Notch signaling pathway to ensure the self-renewal and undifferentiated status of CSCs. 451

Tumor-associated macrophages and CSCs

Macrophages are an important component of the innate immune response and are a group of cells with plasticity and heterogeneity. 452 Infiltrating and inflammatory macrophages originate from the precursors of bone marrow mononuclear cells. 453 These precursor cells infiltrate various tissues from blood vessels and differentiate into different subtypes in different microenvironments. There are two subtypes of macrophages: the M1 and M2 phenotypes. The M1 phenotype has anti-inflammatory and anti-tumor effects and secretes proinflammatory factors such as interleukin-1 (IL-1), IL-12, IL-23, TNF-α, chemokine (C-X-C motif) ligand 5 (CXCL5), CXCL9, and CXCL10. M2 macrophages are generally considered to be the phenotype of tumor-associated macrophages (TAMs), 454 , 455 , 456 have immunosuppressive and angiogenesis-promoting effects, and are considered to be a tumor-promoting cell type. 456 , 457 M2 macrophages secrete CCL17 (C-C chemokine ligand 17), CCL22, and CCL24 and have low expression of IL-12 and high expression of IL-10. Cytokines secreted by macrophages affect the proliferation, tumorigenic transformation, or apoptosis of CSCs through various signaling pathways. 458

TAMs are closely related to CSCs or stem cell transformation. Renal epithelial cells cocultured with macrophages induce the EMT to transform renal cancer cells into CSCs expressing CD117, Nanog, and CD133. 459 Another study also showed that mucin-1 secreted by M2 macrophages induces the transdifferentiation of non-small-cell lung cancer cells into CSCs that express CD133 and Sox2. 460 Jinushi and colleagues 461 also reported that TAMs secrete MFG-E8, which maintains the self-renewal ability of colon and breast CSCs, and knockout of MFG-E8 significantly inhibits the tumorigenic ability in SCID mice. 461 TAMs are closely related to glioma stem cell growth. 462 TAMs are mainly distributed near CD133 + glioma stem cells and accumulate in pericapillary and hypoxic areas. 463 Glioma stem cells recruit and maintain macrophages by secreting a potent chemokine membrane protein. 464 The ablation of TAMs inhibits the tumorigenesis of glioma stem cells. 465 Recent studies have shown that the interaction between the TME and CSCs is regulated by a variety of signaling pathways. 466 Macrophages enhance the invasion of glioma stem-like cells through the TGF-β1 signaling pathway. 467 TAMs activate the STAT3/Sox2 signaling pathway in mouse breast CSCs by secreting EGF, which promotes the self-renewal ability of CSCs. 468 IL-8 secreted by TAMs also induces the EMT in hepatocellular cancer cells by activating the JAK2/STAT3/Snail pathway. 469

Cancer-associated fibroblasts and CSCs

CAFs are one of the most important components of the TME and are critical in tumor development and metastasis. 470 The origin of these cells in the stroma is not entirely clear. Current studies hypothesize that there are five possible sources: (1) transference of fibroblasts in the host stroma; 471 (2) EMT; 472 (3) transdifferentiation of perivascular cells; 473 (4) EMT; 474 and (5) differentiation of MSCs derived from bone marrow. 475 In addition, CAFs are also derived from other cell types, such as smooth muscle cells, pericytes, adipocytes, and immune cells. 476 It is not clear whether there are differences in the functions of CAFs from different sources. CAFs affect cancer cell growth through cell–cell interactions and the secretion of various invasive molecules, such as cytokines, chemokines, and inflammatory mediators. 477 , 478 , 479

CAFs in the TME play an indispensable role in the generation and maintenance of CSCs. 480 CAFs transform cancer cells into CSCs. 481 Studies have shown that CAFs promote the EMT and enhance the expression of prostate CSC markers 482 by secreting IL-6 and IL-1β in breast cancer. 483 , 484 CAFs also secrete TGF-β and activate related pathways to increase ZEB1 transcription, which stimulate lung cancer cells to undergo EMT and CSC transformation. 485 CAFs secrete matrix metalloproteinases, which induce the EMT and promote the growth of stem cell-specific components in tumors. 482 Paracrine interaction between CAFs and CSCs is critical for maintaining the CSC niche of lung CSCs. 486 Fibroblast-derived CCL-2 regulates CSCs through gap activation, thus promoting the progression of tumors. 487 CAFs and adipocytes also secrete leptin, which increases the globulation rate of breast CSCs in vitro. 488

CAFs also regulate the proliferation of CSCs by other signaling pathways. For example, CAFs increase the secretion of CCL-2 to activate the Notch1/STAT3 pathway, which increases the expression of stem cell markers and upregulates the globulation rate in breast cancer. 489 CAFs regulate TIC plasticity in HCC through c-Met/FRA1/HEY1 signaling. 490 CAFs secrete high levels of IL-6 to activate Notch signaling through STAT3 Tyr705 phosphorylation, thus promoting the stem cell-like characteristics of HCC cells. 491 Similar studies have shown that CAF-derived exons enhance colon stem cell resistance to 5-fluorouracil by activating the Wnt signaling pathway. 492

Cancer-associated MSCs and CSCs

MSCs have high self-renewal ability and multidirectional differentiation potential. 493 MSCs also specifically migrate to the injured site and tumor tissue and are easy to isolate and expand in vitro. 494 , 495 MSCs are considered to be an ideal vector for gene therapy because of their characteristics of homing to and secreting cytokines in tumors. 496 However, these tumorigenic characteristics of MSCs still need to be studied. MSCs not only promote tumor development 497 , 498 but also inhibit cancer cell growth. 499 Bone marrow MSCs promote tumor growth by promoting angiogenesis, metastasis, and the survival of CSCs. 500 MSCs in the TME are conducive to the proliferation, carcinogenesis, and metastasis of breast CSCs through ionic purinergic signal transduction. 501 MSCs can differentiate into CAFs, and CAFs further regulate CSCs and promote the occurrence and metastasis of cancers. 502 The possible mechanism is related to the spontaneous fusion between cancer cells and MSCs. 503 The fusion of MSCs with breast cancer, ovarian cancer, gastric cancer, and lung cancer cells in vitro and in vivo has been confirmed. 504 , 505 MSCs regulate the TME by secreting IL-6 to maintain the undifferentiated state of osteosarcoma cells. 506 , 507 IL-1 stimulates the secretion of PGE2 via autocrine signaling, which ultimately activates β-catenin signaling in cancer cells in a paracrine manner and transforms cancer cells into CSCs. 508 In the ECM, bone mesenchymal stem cells activate the NF-κB pathway and induce a CSC phenotype by secreting a variety of cytokines and chemokines, such as CXCL12, CXCL7, and IL-6/IL-8. 509 The interaction between MDSCs and CSCs via IL-6/STAT3 and Notch signaling is critical to the progression of breast cancer. 510

Extracellular matrix and CSCs

The ECM is an insoluble structural component of the matrix in mesenchymal and epithelial vessels. The ECM includes collagen, elastin, aminoglycan, proteoglycan, and noncollagen glycoprotein. 511 , 512 At present, increasing evidence shows that the ECM is an integral part of stem cell niches that regulates the balance of stem cells in three different biological states: static, self-renewal, and differentiation. 513 Experiments in vitro and in vivo have shown that ECM receptors can be used to aggregate CSCs 514 and induce drug resistance. 513 , 515 Fibronectin, vimentin, collagen, and proteoglycan in the ECM bind to cytokines such as FGF, HGF, VGF, BMP, and TGF-β in the TME and regulate their activities. 516 In HCC, an increased matrix promotes cell proliferation and chemotherapeutic resistance and increases the expression of CSC-related markers, including CD44, CD133, c-kit, cxcr4, Oct4, and Nanog. Hyaluronic acid in the ECM is a ligand for the CD44 receptor and can regulate the acquisition and maintenance of CSC stemness during mutual contact. 517 The ECM also binds the Wnt ligand Wnt5b via molecular MMP3 and leads to the expansion and proliferation of mammary epithelial stem cells. 518 In addition, tenascin C in the ECM maintains the stability of breast CSCs by increasing the activity of the Wnt and Notch signaling pathways. 519

Exosomes in the TME and CSCs

Exosomes are nanovesicles secreted by various types of living cells (30–100 nm in diameter) 520 and are widely distributed in peripheral blood, saliva, urine, ascites, pleural effusion, breast milk, and other body fluids. 521 Exosomes contain a large number of functional proteins, RNA, microRNAs, DNA fragments, and other bioactive substances. 522 , 523 , 524 , 525 These bioactive substances mediate material transport and information exchange between cells, thus affecting the physiological function of cells. 526 , 527 The exosomes secreted by cancer cells promote angiogenesis, 528 induce differentiation of tumor-related fibroblasts, 529 participate in immune regulation of the TME, 530 and regulate the microenvironment before metastasis. 531 Clinical analysis has revealed that exosomes are released at higher levels in cancer cells. 532

Recent studies have shown that endocytosis of lipid rafts in MSCs is associated with increased secretion of exosomes. 533 Exosome signaling mediates the interaction of CSCs and normal stem cells, thereby regulating oncogenesis and tumor development. 534 Exosomes also regulate CSC growth by targeting specific signaling pathways, such as Wnt, Notch, Hippo, Hh, and NF-κB. 535 , 536 , 537 Extracellular vesicles released by glioblastoma stem cells promote neurosphere formation, endothelial tube formation, and the invasion of glioblastoma. 538 CSCs promote cell proliferation and self-renewal through crosstalk between exosome signal transduction and the surrounding microenvironment. 539 The exosomes released from CSCs affect signal transduction in nearby breast cancer cells 540 and increase the stemness of breast cancer cells. 540 Similarly, fibroblast-derived exosomes contribute to chemoresistance by promoting colorectal CSC growth. 491 Exosomes in the TME promote the transformation of non-CSCs into CSCs. CAF-derived exosomes significantly increase the ability to form mammary globules and promote the stemness of breast cancer cells. 541 Similarly, CAF-derived exosomes also promote sphere formation of colorectal cancer cells by activating Wnt signaling and ultimately increase the percentage of CSCs. 491 Exosomes from glioma-associated MSCs increase the tumorigenicity of glioma stem-like cells by transferring miR-1587. 542 In addition, exosomes regenerate stem cell phenotypes by mediating the EMT or regulating stem cell-related signaling pathways, such as the Wnt pathway, Notch pathway, Hh pathway and other pathways, which convert cells into CSCs. 543 Exosomes have many advantages, such as low immunogenicity, biocompatibility, easy production, cytotoxicity, easy storage, high drug loading capacity, and long life and have become ideal drug carriers for cancer therapy. 544 , 545 , 546 , 547 , 548

Therapeutic targeting of CSCs

Agents targeting csc-associated surface biomarkers in clinical trials.

Monoclonal antibodies (mAbs) that target CSC-specific surface biomarkers have become an emerging technology for cancer therapy. Rituximab, a CD20 mAb, is an active agent for the treatment of follicular lymphoma and mantle-cell lymphoma, but some serious adverse reactions still occur. 549 Subsequently, to improve the availability and affordability of radioimmunotherapy for refractory or recurrent non-Hodgkin’s lymphoma (NHL), a phase II clinical trial for a radioiodine replacement of rituximab was carried out, which showed a response rate of 71% and a complete remission rate of 54% in 35 patients, with only two cases of grade IV hematologic toxicity observed. 550 Encouragingly, alemtuzumab, a humanized CD52 antibody, has been approved for the treatment of chronic lymphocytic leukemia (CLL) in patients who failed to respond to alkylating agents and purine. Furthermore, the combination of the CD20 and CD52 antibodies in the treatment of refractory CLL was safe, nontoxic, feasible, and positive. 551 Another antibody drug, relabeled bivatuzumab, is an anti-CD44v6 mAb, 71 which was found to be safe when it was used for the treatment of head and neck SCC. 552 These results have been obtained in subsequent clinical research 553 and safety/efficacy studies. 554 Unfortunately, in a stage I dose escalation study with the CD44v6 antibody, one patient with head and neck SCC of the esophagus suffered deadly skin toxicity. 555

Several CD123 antibodies have been developed, XmAb14045 and MGD006, and were designed with biospecific effects against CD3 and CD123. Talacotuzumab is also effective against CD16 and CD123. CSL360, another CD123 antibody, was used to treat relapsed, refractory, or high-risk acute myeloid leukemia (AML) and displayed no anti-leukemic activity in most cases. 556 SL-401, another CD123 antibody, was used to treat blastic plasmacytoid dendritic cell neoplasm patients. The results showed major positive responses in seven out of nine patients, including five complete responses and two partial responses. 557 An ongoing phase II study of SL-401 in 29 patients with blastic plasmacytoid dendritic cell neoplasms demonstrated robust single-agent activity with an 86% overall response rate. 558 The latest antibodies against CSC surface markers, such as XmAb14045 (NCT02730312), flotetuzumab (NCT02152956), and talacotuzumab (NCT02472145), are also in clinical study. Furthermore, several other markers that can distinguish LSCs from other cells are under clinical development, such as IL-1 receptor accessory protein, CD27/70, CD33, CD38, CD138, CD93, CD99, C-type lectin-like molecule-1, and T cell immunoglobulin mucin-3.

EpCAM, a common CSC biomarker, has also received attention in clinical trials. 559 Adecatumumab, an EpCAM antibody, was used in patients with hormone-resistant prostate cancer, and the results showed that the EpCAM-specific antibody has great clinical potential. 560 Catumaxomab, a multifunctional mAb against EpCAM, binds and recognizes EpCAM and the T cell antigen CD3 (anti-EpCAM × anti-CD3). 561 Intraperitoneal injection of catumaxomab to treat EpCAM-positive ovarian cancer and malignant ascites has shown high efficacy in killing cancer cells and reducing the formation of ascites. 562 Catumaxomab has been used in non-small-cell lung cancer and also had a good survival rate. 561 However, other types of EpCAM antibodies, such as edrecolomab 563 and adecatumumab, 564 have minimal efficacy in colorectal and breast cancers. Combining EpCAM antibodies with chimeric antigen receptor T cell (CAR-T) technology has also been used in various types of cancers in phase I trials, such as NCT02915445, NCT03563326, NCT02729493, and NCT02725125. With a deeper understanding of CSC surface biomarkers, there has been significant progress in developing antibodies targeting CSCs (Table 2 ). However, CSC surface phenotypes can vary in different patients or different cancers, and different CSC populations with different phenotypes might coexist. CSCs also diverge or evolve into different cancer cells, acquiring distinct phenotypes upon relapse. Therefore, the strategies used in clinical trials should be determined according to the phenotypes of the different cancers. At the same time, combining different surface antibodies with relevant chemotherapy drugs can achieve an ideal therapeutic effect.

Agents targeting CSC-associated signaling pathways in clinical trials

The signaling pathways that regulate the maintenance and survival of CSCs have become targets for cancer treatment. At present, the main signaling pathways are the Wnt, Notch, and Hh signaling pathways, as well as the TGF-β, JAK-STAT, PI3K, and NF-κB signaling pathways. These pathways often interact with each other during tumor development and in CSCs. Considerable progress has been made in early clinical trials for Notch and Hh pathway inhibitors, while targeting the Wnt pathway has proven to be difficult. 10

The Notch signaling pathway plays an important role in the maintenance of CSCs 565 , 566 and can induce CSC differentiation. Abnormal activity of the Notch signaling pathway has been observed in many cancers, such as leukemia, 567 glioblastoma, 568 , 569 breast cancer, 570 lung cancer, 571 ovarian cancer, 572 pancreatic cancer, 573 and colon cancer. 574 At present, there are three major clinical methods used to inhibit Notch signaling, secretase inhibition (γ-secretase inhibitor (GSI)), Notch receptor or ligand antibodies, and combination therapy with other pathways. For example, GSIs have been tested in clinical trials. Among them, MK-0752 (NCT00100152) was the first GSI used to treat T cell acute lymphoblastic leukemia in children in a phase I trial. However, the study was terminated because of poor results. 575 MK-0752 also had no clinical activity in extracranial solid tumors in subsequent phase II trials. Only one complete response with interdegenerative astrocytoma and SD extension out of 10 patients with different types of glioma was observed. 576 MK-0752 is well tolerated and shows targeted inhibition in recurrent pediatric central nervous system tumors. 577 In addition, combining MK-0752 with cisplatin treatment for ovarian cancer, 578 , 579 docetaxel treatment for locally advanced or metastatic breast cancer, 569 and gemcitabine treatment for ductal adenocarcinoma of the pancreas 580 has shown good efficacy. However, the clinical effect was minimal in patients with advanced solid tumors, 576 , 581 including metastatic pancreatic cancer. 582

In addition, RO4929097, another selective GSI, showed good anti-tumor activity in preclinical and early trials, 583 , 584 but was not good for metastatic colorectal cancer, 585 metastatic pancreatic adenocarcinoma, 586 or recurrent platinum-resistant ovarian cancer. 587 Combinations of RO4929097 with gemcitabine, 588 temsirolimus, 587 cediranib, 589 or capecitabine 590 in advanced solid tumors, as well as with bevacizumab in recurrent high-grade glioma, are well tolerated and have modest clinical benefits. However, NCT01154452, the combination of RO4929097 with vismodegib and vismodegib alone for patients with advanced osteosarcoma, showed no significant difference in a phase Ib trial. The third oral GSI, PF-03084014, had good efficacy in desmoid tumors either in phase I or subsequent phase II studies. 591 Preliminary evidence of its clinical efficacy was demonstrated in patients with solid tumors, 592 as well as in patients with recurrent acute T cell lymphoblastic leukemia. 593 Other selective GSIs, such as BMS-906024 (NCT01292655), BMS-986115 (NCT01986218), CB-103 (NCT03422679), LY3039478 (NCT02836600), and LY900009 (NCT01158404), have also entered the clinical trial stage, and the results still need to be verified.

DLL4 plays a vital role in regulating tumor angiogenesis. 594 Therefore, targeting DLL4 is another strategy to block Notch signaling, and this is being tested in the clinic. Demcizumab (OMP-21M18), a humanized IgG2 mAb that targets DLL4 and blocks its interactions with Notch receptors, was tested in a phase I dose escalation study with 55 patients with previously treated solid tumors. 595 The results have shown that demcizumab had good efficacy against solid tumors, but was not good for metastatic pancreatic cancer treatment when combined with gemcitabine and Abraxane (NCT02289898). NCT02259582, a combination of demcizumab with carboplatin and pemetrexed to treat lung cancers (DENALI study), is ongoing in another phase II study. 595 Enoticumab, another fully human IgG1 antibody against DLL4, has promising activity in phase I clinical trials for advanced solid malignancies.

Activation of Hh signaling has been implicated in a variety of cancers. 596 , 597 , 598 Activation of Hh signaling in CSCs contributes to CSC stemness, chemoresistance, and metastatic dissemination. The Hh signaling pathway mainly regulates target gene expression via smoothened (SMO)-mediated nuclear transfer of transcription factors. Three oral SMO antagonists, vismodegib (GDC-0449), sonidegib (LDE225), and glasdegib (PF-04449913), have been approved by the Food and Drug Administration (FDA) and show significant activity in locally advanced and metastatic basal cell carcinoma, as well as in AML. 599 , 600 , 601 Vismodegib was the first proposed Hh pathway inhibitor in cancer research 602 and is approved by the FDA 603 for local or advanced metastatic basal cell carcinoma treatment. 599 Subsequently, phase I and phase II trials targeting recurrent medulloblastoma have shown that the progression-free survival (PFS) of Shh-mb patients treated with vismodegib is longer and more effective than that of non-Shh-mb patients. Vismodegib even has better activity in patients with recurrent Shh-mb but not in patients with recurrent non-Shh-mb. 604 , 605 Vismodegib has also been tested in metastatic colorectal cancer, 606 pancreatic cancer, 607 chondrosarcoma, 608 relapsed/refractory NHL, CLL, 609 and ovarian cancer. 610 Disappointingly, these treatments with vismodegib have not resulted in better survival.

Sonidegib was the second SMO antagonist approved for the treatment of locally advanced basal cell carcinoma that recurred after surgery or radiotherapy and is not suitable for surgery or radiation therapy. 611 In addition, the results of a multicenter, randomized, double-blind phase II trial have shown that 200 mg sonidegib for patients with advanced basal cell carcinoma is the most clinically appropriate dose. 600

In a phase I study of a 3 + 3 dose escalation to treat small-cell lung cancer patients, sonidegib combined with cisplatin and etoposide sustained PFS in patients with Sox2 amplification. 224 These combinations in a phase II trial for patients with recurrent medulloblastoma resulted in a complete or partial response in 50% of patients 612 and have been used for other cancer treatments in phase I/II clinical trials, such as NCT02111187 for prostate cancer, NCT02027376 for breast cancer, and NCT02195973 for recurrent ovarian cancer.

Glasdegib was the first Hh pathway inhibitor approved for the treatment of AML in patients older than 75 years or those unable to use intensive induction chemotherapy 601 and showed good safety and tolerability in a phase I trial for patients with partial hematologic malignancies in Japan. 613 In a phase II trial, glasdegib combined with cytarabine/daunorubicin had a significant efficacy in patients with AML, chronic myeloid leukemia (CML) or high-risk myelodysplastic syndromes. 614 Glasdegib combined with low-dose cytarabine (LDAC) is a potential option for AML patients who are not suitable for intensive chemotherapy. 615 Other selective SMO inhibitors, including taladegib (LY2940680) and saridegib (IPI-926), have also entered clinical trials for other cancers. As single-target agents, these SMO inhibitors have drug resistance problems. To reduce this problem, some novel inhibitors of terminal components of Hh signaling pathway are being developed, such as arsenic trioxide (ATO) 616 and GANT-61. 617

The Wnt signaling pathway is associated with tumor development in breast cancer, 618 ovarian cancer, 619 esophageal squamous cell cancer, 620 colon cancer, 621 prostate cancer, 622 and lung cancer. 623 Until now, several drugs aimed at the Wnt signaling pathway have been in clinical trials, while the majority of Wnt inhibitors are in preclinical testing. Clinical data from initial trials have shown that ipafricept (OMP-54f28/FZD8-Fc) is a first-in-class recombinant fusion protein that antagonizes Wnt signaling. 624 However, its role in patients with desmoid cancers and germ cell cancers is negligible. 625 NCT02050178, ipafricept combined with ab-paclitaxel and gemcitabine in patients with untreated stage IV pancreatic cancer, NCT02092363, ipafricept combined with paclitaxel and carboplatin in patients with recurrent platinum-sensitive ovarian cancer, and NCT02069145, ipafricept combined with sorafenib in patients with HCC, are currently being investigated. PRI-724, a β-catenin inhibitor, inhibits the interaction between β-catenin and its transcriptional coactivators. Safety and efficacy testing of PRI-724 for patients with advanced myeloid malignancies (NCT01606579) and advanced or metastatic pancreatic cancer (NCT01764477) have been completed in phase I studies. CWP232291, another inhibitor of β-catenin activity, has also been shown to be effective for AML (NCT03055286) in a phase I clinical study and for recurrent or refractory myeloma (NCT02426723) in a phase I/II clinical study. 626 Other Wnt signaling inhibitors have also been under clinical trial, including LGK974 (NCT02278133), ETC-159 (NCT02521844), and OMP-18R5 (NCT01973309, NCT01957007, and NCT02005315).

In addition, the mitochondrial glycolysis pathway also plays a key role in regulating the proliferation and apoptosis of CSCs. Venetoclax, a BCL-2 inhibitor, was initially approved by the FDA recently and shows good tolerance and activity for AML patients with adverse reactions. 627 Two arachidonate 5-lipoxygenase inhibitors, VIA-2291 and GSK2190915, might be potent agents for targeting LSCs in CML, 628 as shown in Table 3 .

Other abnormal signaling pathways have also been found in CSCs, such as TGF-β, JAK-STAT, PI3K, and NF-κB. These signaling pathways are not independent of each other but rather form a complex signaling network. Agents targeting CSC-associated signaling pathways in ongoing clinical trials are listed in Table 3 .

Targeting the CSC microenvironment

The CSC microenvironment contributes to the self-renewal and differentiation of CSCs and regulates CSC fate by providing cues in the form of secreted factors and cell contact. CXCR4 has been found in most cancers, especially in CSCs. The most well-characterized drug-targeting CXCR4 is plerixafor (AMD3100), and this drug is an effective hematopoietic stem cell mobilizer for patients with multiple myeloma and NHL. 629 AMD3100 treatment for relapsed or refractory AML resulted in 46% of patients with complete remission with or without white count recovery in a phase I/II study. 630 In addition, plerixafor with high-dose cytarabine and etoposide treatment for children with relapsed or refractory acute leukemia or myelodysplasia syndrome resulted in two patients with complete remission and one patient with incomplete hematologic recovery out of 18 patients in a phase I study. 631 LY2510924, a small cyclic peptide, is a potent and selective antagonist of CXCR4 and is well tolerated with no serious adverse events in a phase I trial. 632 However, the combination of LY2510924 with sunitinib for patients with metastatic renal cell carcinoma has no better effect than sunitinib alone in a phase II trial. 633 The combination of LY2510924 with carboplatin/etoposide for patients with extensive small-cell lung cancer also had no significant effect compared with that of carboplatin/etoposide alone in a phase II study. 634 The combination of LY2510924 with other drugs for gliomas (NCT03746080, NCT01977677, and NCT01288573) and multiple myeloma (NCT00103662, NCT01220375, and NCT00903968) is also under clinical trial.

The microenvironment plays an important role in CSC growth, and it is also a promising target for treatment. Agents targeting the microenvironment in ongoing clinical trials are listed in Table 3 .

CSC-directed immunotherapy

In the early twentieth century, Paul Ehrlich first proposed the idea that an intact immune system suppresses tumor development (advancing cancer therapy with present and Emerging Immuno-Oncology Approaches). Based on the understanding of cellular immune regulation, new methods for cancer treatment have emerged. In addition to the antibodies against the CSC molecules mentioned above, some novel anti-CSC immunotherapeutic approaches, such as immunologic checkpoint blocking or CAR-T cell therapies, have been developed. Some drugs that target the immune checkpoint receptors CTLA-4, 635 PD-1 (nivolumab, 636 pembrolizumab, 637 and cemiplimab, 638 ) and PD-L1 (avelumab, 639 durvalumab, 640 and atezolizumab 641 ) have also been in clinical trials. I ipilimumab, a CTLA-4 antibody, is approved by the FDA, and initial clinical results showed good effectiveness in patients with metastatic melanoma. 642 For CAR-T cell therapy, as shown in Table 4 , CD19, CD20, CD22, CD123, EpCAM, and ALDH have been used for CSC-directed immunotherapy, and most of them are recruited.

Conclusions and perspectives

We can conclude that CSCs are a small population of cancer cells that have self-renewal capacity and differentiation potential, thereby conferring tumor relapse, metastasis, 643 heterogeneity, 644 multidrug resistance, 645 , 646 and radiation resistance. 647 Several pluripotent transcription factors, including Oct4, Sox2, Nanog, KLF4, and MYC and some intracellular signaling pathways, including Wnt, NF-κB, Notch, Hh, JAK-STAT, PI3K/AKT/mTOR, TGF/Smad, and PPAR, as well as extracellular factors, including vascular niches, hypoxia, TAM, CAF, cancer-associated MSCs, the ECM, and exosomes, are essential regulators of CSCs. Drugs, vaccines, antibodies, and CAR-T cells targeting these pathways have also been developed to target CSCs. 648 Importantly, many clinical trials on CSCs have also been performed and show a promising future for cancer therapy.

However, there are also multiple hurdles that need to be solved to effectively eliminate CSCs. First, the characteristics of many CSCs in specific types of tumors are not well identified. 649 Second, since most studies on CSCs are performed in immune-deficient mice in the absence of an adaptive immune system, these models do not recapitulate the biological complexity of tumors in the clinic. 650 Third, CSCs exist in a specific niche that sustains their survival. However, isolated CSCs are used in most current studies that lacks a microenvironment. 651 Fourth, the environmental factors in CSC niches are not well understood, and the relationship between TAMs/CAFs and CSCs has not been well studied. 645 Fifth, since CSCs also share some signaling pathways with normal stem cells, not all the regulatory factors that contribute to CSCs are appropriate for use as therapeutic targets in cancer treatment. Sixth, whether CSCs should be activated or arrested is an open question in cancer therapy. 652 Seventh, novel signaling and more regulatory levels, such as RNA editing, 653 epigenetics, 654 and cellular metabolism, 655 should be considered in cancer therapy because they also contribute to the stemness of CSCs. Eighth, some inhibitors that target CSC signaling are not very specific, and so new inhibitors need to be designed. 656 Ninth, natural products that target CSCs should also be studied in the future. 657 Finally, novel ways of targeting the microenvironment of CSCs are also promising and need to be explored.

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (nos. 2016YFC1302204 and 2017YFC1308600), the National Science Foundation of China (nos. 81672502, 81872071, and 81902664) and the Natural Science Foundation of Chongqing (no. cstc2019jcyj-zdxmX0033).

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These authors contributed equally: Liqun Yang, Pengfei Shi

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State Key Laboratory of Silkworm Genome Biology, Southwest University, 400716, Chongqing, China

Liqun Yang, Pengfei Shi, Gaichao Zhao, Jie Xu, Wen Peng, Jiayi Zhang, Guanghui Zhang, Xiaowen Wang, Zhen Dong & Hongjuan Cui

Cancer Center, Medical Research Institute, Southwest University, 400716, Chongqing, China

Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, Detroit, MI, 48201, USA

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Correspondence to Hongjuan Cui .

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Yang, L., Shi, P., Zhao, G. et al. Targeting cancer stem cell pathways for cancer therapy. Sig Transduct Target Ther 5 , 8 (2020). https://doi.org/10.1038/s41392-020-0110-5

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Published : 07 February 2020

DOI : https://doi.org/10.1038/s41392-020-0110-5

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Cancer stem cells: advances in biology and clinical translation-a Keystone Symposia report

Affiliations.

• 1 PhD Science Writer, New York, New York.
• 2 CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou, China.
• 3 Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
• 4 Guangzhou Regenerative Medicine and Health Guangdong Laboratory (GRMH-GDL), Guangzhou, China.
• 5 Department of Cell Biology and Physiology, University of North Carolina School of Medicine, Chapel Hill, North Carolina.
• 6 Laboratory of Human Carcinogenesis, and Liver Cancer Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.
• 7 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
• 8 Laboratory for Molecular Cancer Biology, Center for Cancer Biology and Laboratory for Molecular Cancer Biology, Department of Oncology, Leuven, Belgium.
• 9 Glioblastoma Translational Center of Excellence, The Abramson Cancer Center and Department of Pathology & Laboratory Medicine, Perelman School of Medicine and Center for Cellular Immunotherapies, University of Pennsylvania, Philadelphia, Pennsylvania.
• 10 Department of Molecular and Medical Genetics, Department of Pediatrics, and Oregon Stem Cell Center, Oregon Health & Science University, Portland, Oregon.
• 11 Department of Cardiovascular and Metabolic Sciences, Lerner Research Institute and Case Comprehensive Cancer Center, Case Western Reserve University School of Medicine, Cleveland Clinic, Cleveland, Ohio.
• 12 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center and Breast Tumor Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China.
• 13 Bioland Laboratory; Program of Molecular Medicine, Zhongshan School of Medicine, Sun Yat-Sen University; and Fountain-Valley Institute for Life Sciences, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China.
• 14 Division of Hematology, Department of Medicine, Stanford University, Stanford, California.
• 15 Translational Cancer Research Center, Peking University First Hospital, Beijing, China.
• 16 Cancer Biology and Genetics, Memorial Sloan Kettering Cancer Center, New York, New York.
• 17 School of Biomedical Sciences and State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong.
• 18 Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.
• 19 Department of Biomedical Sciences, City University of Hong Kong, Hong Kong SAR, China.
• 20 Departments of Medicine, Genetics and Development, Urology, and Systems Biology, Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York.
• 21 Department of Pathology and State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong, China.
• 22 Department of Genetics and Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.
• 23 School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, Hong Kong.
• 24 Institute for the Biology of Stem Cells, University of California, Santa Cruz, Santa Cruz, California.
• 25 Charité - Universitätsmedizin Berlin, Hematology/Oncology, and Max-Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany, and Johannes Kepler University, Kepler Universitätsklinikum, Hematology/Oncology, Linz, Austria.
• 26 Department of Biology, School of Arts & Sciences, University of Pennsylvania, Philadelphia, Pennsylvania.
• 27 MOE Key Laboratory of Biosystems Homeostasis & Protection, and Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China.
• 28 Department of Pharmacology & Therapeutics, Roswell Park Comprehensive Cancer Center, and Experimental Therapeutics (ET) Graduate Program, University at Buffalo, Buffalo, New York.
• 29 OncoRay National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden and Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany.
• 30 Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiooncology-OncoRay, Dresden, Germany.
• 31 German Cancer Consortium (DKTK), Partner Site Dresden, and German Cancer Research Center (DKFZ), Heidelberg, Germany.
• 32 National Center for Tumor Diseases (NCT), Partner Site Dresden, Heidelberg, Germany.
• 33 Neuro-Oncology Branch, National Cancer Institute, Center for Cancer Research, National Institutes of Health, Bethesda, Maryland.
• 34 AbbVie, Inc., Lake Bluff, Illinois.
• 35 Institute for Stem Cell Biology and Regenerative Medicine, Ludwig Center for Cancer Stem Cell Research, Stanford University, Stanford, California.
• PMID: 34850398
• PMCID: PMC9153245
• DOI: 10.1111/nyas.14719

The test for the cancer stem cell (CSC) hypothesis is to find a target expressed on all, and only CSCs in a patient tumor, then eliminate all cells with that target that eliminates the cancer. That test has not yet been achieved, but CSC diagnostics and targets found on CSCs and some other cells have resulted in a few clinically relevant therapies. However, it has become apparent that eliminating the subset of tumor cells characterized by self-renewal properties is essential for long-term tumor control. CSCs are able to regenerate and initiate tumor growth, recapitulating the heterogeneity present in the tumor before treatment. As great progress has been made in identifying and elucidating the biology of CSCs as well as their interactions with the tumor microenvironment, the time seems ripe for novel therapeutic strategies that target CSCs to find clinical applicability. On May 19-21, 2021, researchers in cancer stem cells met virtually for the Keystone eSymposium "Cancer Stem Cells: Advances in Biology and Clinical Translation" to discuss recent advances in the understanding of CSCs as well as clinical efforts to target these populations.

Keywords: cancer stem cell; hepatocellular carcinoma; organoids; pluripotent; progenitors; stemness; tumor heterogeneity; tumorigenesis.

© 2021 New York Academy of Sciences.

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• Research Support, N.I.H., Extramural
• Research Support, Non-U.S. Gov't
• Biomarkers, Tumor / genetics
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• Neoplasms / genetics*
• Neoplasms / metabolism
• Neoplastic Stem Cells / physiology*
• Research Report*
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• Biomarkers, Tumor

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Combining ‘big data’ experimental approaches to understand the changes in signaling networks that drive cancer formation

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Determining how cancer-initiating stem cells continuously renew and seed distant sites to promote metastasis, and understanding the role of these cells in resistance to chemotherapies

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Resources for International Patients

From exceptional medical care to support with housing and other logistics, we offer many services to international patients:

• Remote consults and online second opinions to evaluate a diagnosis or recommended treatment plans/options
• Patient navigators who accompany patients to appointments
• Interpreter services
• Nutrition Services , with attention to special dietary needs
• Dedicated social work resources
• Extensive patient education materials in print, online, and video formats, many of which are translated for non-English speaking patients and caregivers 

Program Accreditations, Registrations and Licenses

• Foundation for the Accreditation of Cellular Therapy (FACT)
• U.S. Food and Drug Administration
• American Association of Blood Banks (AABB)
• Centers for Medicare and Medicaid Services
• The Joint Commission
• Fully accredited with the NMDP

Clinical and Research Affiliations

• Member of the Center for International Blood and Marrow Transplant Research (CIBMTR)
• Charter member of the Blood and Marrow Transplant Clinical Trials Network of the National Institutes of Health (BMT-CTN)
• Member of the Alliance for Clinical Trials in Oncology (formerly CALGB)
• Founding member of Dana-Farber/Harvard Cancer Center, designated a comprehensive cancer center by the National Cancer Institute

The Basics of Stem Cell Transplantation

Joseph Antin, MD, presents "The Basics of Stem Cell Transplantation" at Cancer Survivorship 101 on June 8, 2019, an event produced by Survivor Journeys™ .

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Our Cellular Therapies Program is at the forefront of novel cellular therapies that help the immune system fight cancer such as CAR T-cell therapy, cancer vaccines, NK-cell therapy and other genetically-modified T cells.

Donate Bone Marrow and Stem Cells

Approximately 30 percent of patients in need of a stem cell donor find a match within their families. The remaining 70 percent search a worldwide database of unrelated volunteer donors, who may be their match and are willing to donate life-saving cells. Learn about how you can become a potential stem cell donor.

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Stem Cell Transplantation Topics

Insight Blog 5/18/2023 How Can a Stem Cell Transplant Help a Patient with Cancer? 

Insight Blog 4/19/2023 Is There a Risk of Immune Rejection From Stem Cell Transplants? 

Insight Blog 4/11/2023 Can Stem Cell Transplants Cure Cancer?

BHATIA PROGRAM

McMaster University

Our research program takes a multidisciplinary approach integrating chemistry, robotic automation, and bioinformatics to support our fundamental question - to understand the mechanisms that drive stem cell fate decisions in the human as it relates to the development of disease

Our research consists of several individual projects. Each project is driven by lab members that work with scientific, clinical and private sector collaborators world-wide. Collectively, these projects define our research program

Our program seeks to achieve our long-term goal of impacting human disease, with an emphasis on human leukemias as a translational gateway to apply innovative treatments and interventions for patients with cancer

PROGRAM DIRECTOR

“I’m moved by the notion that in cancer, your own cells are turning on you. I want to understand how that starts and how we can stop it.”

DR. MICK BHATIA

Dr. mick bhatia is a senior scientist of the faculty of health sciences and professor of biochemistry & biomedical sciences at mcmaster university and molecular pathology. he is the principal investigator and director of the experimental therapeutics in human leukemias program, michael g. degroote chair in stem cell and cancer biology, and canada research chair in human stem cell biology.   dr. bhatia began his scientific and clinical journey as a translational clinical research fellow with the national cancer institute of canada with studies at the sick children’s hospital in the department of genetics, toronto, canada. his first appointment was at the university of western ontario and london health sciences in 1998, where he developed a new program and then became the director of stem cell biology and regenerative medicine at the robarts research institute. this role included faculty recruitment, private and industrial collaborations, and represented the first canadian lab to work with human pluripotent stem cells. after engaging in recruitment efforts to centres in the united states, dr. bhatia returned his focus to canada, and became the inaugural director of mcmaster’s first stem cell and cancer research institute in 2006.             as a senior scientist, he developed a broad programmatic approach to address his interests in the molecular processes that govern somatic and pluripotent human stem cell fate decisions from the survival, growth and differentiation of these rare cells. this research was expanded to investigate this topic from numerous angles using a variety of human specific model systems. this includes induced pluripotent stem cells derived from patient skin and blood donated samples, use of cord blood stem cells, and engagement with academics and the private sector developing new single cell imaging and molecular technologies involving high throughput, and automated processing for compound screening in chemical genomics approaches. output from this work continues to translate directly to patients, and he has led and been part of several phase i clinical trials ranging from testing new anti-leukemic compounds to stem cell expansion to improving stem cell transplants via adjuvant drugs identified and discovered by his group. dr. bhatia also continues to serve as an advisory to provincial and federal ministries in both biomedical research and innovation technologies. in addition, he consults with pharmaceutical and small biotechnology companies, and was the founder of two biotechnology companies, regenerative inducing therapeutics inc and actium inc, and is currently developing the new enterprise to serve as a receptor for technologies from his program.   today, dr. bhatia’s program emphasizes disease intervention, biomarkers, and translation to patients. with established methods, tools, collaborative network and expertise in place, the impact of his work is solely measured by improving human health and developing experimental therapeutics for patients whose options are limited by current standard care and therapies..

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Forging a New Future for Human Health

Fostering connections.

We bring together a community of trailblazing researchers who collaborate across disciplines to understand and address complex biomedical challenges. 

SHAPING LEADERS

We invest in early-career scientists to cultivate the next generation of compassionate healthcare providers and leaders in the fields of regenerative medicine and stem cell science. 

ACCELERATING DISCOVERIES

Our scientists leverage stem cells to uncover foundational insights that deepen understanding of human health and disease.

BRIDGING GAPS TO THE CLINIC

Our center provides the infrastructure and resources that empower researchers to make life-saving discoveries and bring them to patients. 

Patient Stories

Ucla clinical trial offers hope for lymphoma patients.

At 39, actor Hirotaka Matsunaga was diagnosed with n on-Hodgkin's lymphoma. Over the next four years, h e endured two courses of chemotherapy in combination with immunotherapy, but the cancer always returned.  After receiving treatment in the first-in-human clinical trial of a cutting-edge CAR-T cell therapy , he's now in remission and doesn't have any symptoms at all. "I feel like myself again," Matsunaga said.

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Stem Cell Breakthrough Offers New Hope to Cancer Patients

Researchers have discovered a new treatment that greatly expands the universe of people who can be successful stem cell donors.

For some people with blood cancers who need a stem cell transplant, finding a donor who is an excellent match can mean the difference between life and death.

Unfortunately, even though there are more than 40 million potential donors in the national registry, finding a perfect match isn’t always possible, especially in underrepresented racial and ethnic groups.

But a new approach using an old chemotherapy drug,  cyclophosphamide , is is opening up new possibilities for people with cancers like leukemia , lymphoma , and multiple myeloma . Researchers have found that by administering the drug several days after transplantation, people receiving blood stem cells from unrelated, partially matched donors can have survival rates comparable with those who received exactly matched cells.

“This innovative approach can greatly expand patient access to safe and effective stem cell transplant, regardless of matching degree with the donor,” says lead coauthor Monzr M. Al Malki, MD , a hematologist and oncologist and director of the Unrelated Donor BMT program at City of Hope, a cancer research and treatment organization with locations across the United States.

That’s exciting because it means more patients will be able to receive this potentially life-extending therapy, says Dr. Al Malki.

Many Black, Hispanic, and Latino People With Cancer Can’t Find a Stem Cell Donor Match

Donor compatibility is determined by a set of protein markers on blood cells called HLAs (human leukocyte antigens), says David Miklos, MD , a professor of medicine and chief of Stanford BMT and Cell Therapy Program at Stanford Medicine in California. Stanford was one of the medical sites of the trial, though Dr. Miklos is not a coauthor of the research.

Why was an exact match needed? Anything less increased the likelihood of a graft failure, as well as graft-versus-host disease — meaning the transplanted cells attack the patient’s own, which can cause serious or even fatal complications, explains Miklos.

The Drug That Transformed Stem Cell Transplants

About a decade ago, researchers started using cyclophosphamide to destroy the parts of a person’s immune system that would reject the transplant. That breakthrough allowed researchers to not only have better outcomes in fully matched donors, it also opened the door for successful transplants between people who were only partial matches.

High Survival Rate in Mismatched Donors

The new study looked at cyclophosphamide treatment in patients receiving peripheral blood stem cell transplantation — meaning healthy stem cells are harvested from a donor’s bloodstream, and then administered via infusion to the person with cancer.

Blood stem cell transplantation has largely replaced bone marrow transplantation, according to researchers. It's an easier way of collecting stem cells from donors, and it’s a little safer, because donors don’t need to be under anesthesia as they would in bone marrow transplantation, says Al Malki.

For this part of the study, the researchers examined data from 70 adults who were 65 years old on average, all with advanced blood cancers. Participants received a “reduced-intensity” conditioning regimen to somewhat suppress their immune system to prepare them for transplantation, followed by an infusion stem cells from unrelated, partially matched donors.

The researchers reported an overall high survival rate of 79 percent at one year — which is comparable to survival rates seen with fully matched donors.

The main side effect or risk of transplantation is graft-versus-host disease, says Al Malki. After one year, 51 percent of participants were free of the disease and had not relapsed, which is also comparable to what would be seen with fully matched donors, he says.

New Findings Mean That ‘All Patients Have Donors’ Now

Historically, barriers in access to transplant have existed due to the low availability of matched, related sibling donors, as well as the substantial variance of matched, unrelated donor availability, especially for patients with diverse ancestry, says study coauthor Steven M. Devine, MD , chief medical officer of NMDP (formerly known as the National Marrow Donor Program and Be The Match).

“These findings advance our ability to offer more options to patients without a fully matched donor, many of whom are ethnically diverse and have been underserved in receiving potentially lifesaving cell therapy,” says Dr. Devine.

These findings are incredibly important and critical in the effort to improve existing inequities, says Miklos.

“In the past, we could not bring some patients forward to receive this lifesaving therapy because they didn’t have a compatible donor, but with the new approach of using post-transplant cyclophosphamide, all patients have donors now,” he says.

Editorial Sources and Fact-Checking

Everyday Health follows strict sourcing guidelines to ensure the accuracy of its content, outlined in our editorial policy . We use only trustworthy sources, including peer-reviewed studies, board-certified medical experts, patients with lived experience, and information from top institutions.

• Post-Transplant Cyclophosphamide-Based Graft-Versus-Host Disease Prophylaxis Following Mismatched Unrelated Donor Peripheral Blood Stem Cell (PBSC) Transplantation. ASCO . May 31, 2024.
• Half-Matches Open Door to Bone Marrow Transplant for More Patients. City of Hope . May 6, 2019.
• NMDP and CIBMTR to Present New, Promising Stem Cell Transplantation Trial Data using Mismatched, Unrelated Donors at 2024 ASCO Annual Meeting. Business Wire . May 23, 2024.
• Shaw B et al. National Marrow Donor Program-Sponsored Multicenter, Phase II Trial of HLA-Mismatched Unrelated Donor Bone Marrow Transplantation Using Post-Transplant Cyclophosphamide. Journal of Clinical Oncology . June 20, 2021.

Tackling the hurdle of tumor formation in stem cell therapies

Pluripotent stem cells (PSCs) are a type of stem cells capable of developing into various cell types. Over the past few decades, scientists have been working towards the development of therapies using PSCs. Thanks to their unique ability to self-renew and differentiate (mature) into virtually any given type of tissue, PSCs could be used to repair organs that have been irreversibly damaged by age, trauma, or disease.

However, despite extensive efforts, regenerative therapies involving PSCs still have many hurdles to overcome. One being the formation of tumors (via the process of tumorigenesis) after the transplantation of PSCs. Once the PSCs differentiate into a specific type for stem cell therapy, there is a high probability of tumor formation after differentiated stem cells are introduced to the target organ. For the success of PSC-based therapies, the need of the hour is to minimize the risk of tumorigenesis by identifying potentially problematic cells in cultures, prior to transplantation.

Against this backdrop, a research team led by Atsushi Intoh and Akira Kurisaki from Nara Institute of Science and Technology, Japan, has recently achieved a breakthrough discovery regarding stem cell therapy and tumorigenesis. "Our findings present advancements that could bridge the gap between stem cell research and clinical application," says Intoh, talking about the potential of their findings. Their study was published in Stem Cells Translational Medicine and focuses on a membrane protein called EPHA2, which was previously found to be elevated in PSCs prior to differentiation by the team.

Through several experiments involving both mouse and human stem cell cultures, the researchers gained insights into the role of EPHA2 in preserving the potency of PSCs to develop into several cell types. They found that EPHA2 in stem cells is co-expressed with OCT4 -- a transcription factor protein which controls the expression of genes which are critically involved in the differentiation of embryonic stem cells. Interestingly, when the EPHA2 gene was knocked down from the cells, cultured stem cells spontaneously differentiated. These results suggest that EPHA2 plays a central role in keeping stem cells in an undifferentiated state.

The researchers thus theorized that EPHA2-expressing stem cells, which would fail to differentiate, might be responsible for tumorigenesis upon transplantation into the target organ.

To test this hypothesis, the researchers prepared PSC cultures and artificially induced their differentiation into liver cells. Using a magnetic antibody targeting EPHA2, they extracted EPHA2-positive cells from a group of cultures prior to transplantation into mice. Interestingly, the formation of tumors in mice receiving transplants from cultures from which EPHA2 had been removed was vastly suppressed.

Taken together, these results point to the importance of EPHA2 in emerging stem cell-based therapies. "EPHA2 conclusively emerges as a potential marker for selecting undifferentiated stem cells, providing a valuable method to decrease tumorigenesis risks after stem cell transplantation in regenerative treatments," remarks Kurisaki.

Further in-depth studies on this protein may lead to the development of protocols that make PSCs safer to use. Luckily, however, these findings pave the way towards a future where we will be able to finally restore damaged organs and even overcome degenerative conditions.

• Prostate Cancer
• Skin Cancer
• Brain Tumor
• Medical Topics
• Immune System
• Embryonic stem cell
• Bone marrow transplant
• Adult stem cell
• Stem cell treatments
• Somatic cell nuclear transfer
• Somatic cell
• Monoclonal antibody therapy

Story Source:

Materials provided by Nara Institute of Science and Technology . Note: Content may be edited for style and length.

Journal Reference :

• Atsushi Intoh, Kanako Watanabe-Susaki, Taku Kato, Hibiki Kiritani, Akira Kurisaki. EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells . Stem Cells Translational Medicine , 2024; DOI: 10.1093/stcltm/szae036

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City of Hope Awarded $5.4-Million Grant to Create New Stem Cell Laboratory

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City of Hope, one of the largest cancer research and treatment organizations in the United States, has been awarded $5.4 million from the California Institute for Regenerative Medicine (CIRM) to build and fund a stem cell research laboratory on its Duarte campus that will further expand its scientific capabilities.

The mission of the unique Stem Cell-Based Disease Modeling Laboratory is two-fold. First, it will advance stem cell-based disease modeling to spur innovation in regenerative medicine. The laboratory leverages City of Hope’s infrastructure and expertise using human cell-derived organoids – a key focus in current efforts to create disease models relevant to understanding biological mechanisms that often lead to new therapies. In particular, the laboratory will supply healthy and cancerous stem cell-based models in brain, heart and breast tissue to the region’s scientists for research.

Second, it will stimulate stem cell research in medically underserved communities by increasing scientists’ access to specialized laboratory equipment services and training and by educating students about regenerative medicine.

“Our new laboratory will expand access to state-of-the-art disease models to researchers at City of Hope and our neighboring institutions, enabling them to pursue impactful scientific questions and to accelerate innovation in stem cell therapies,” said program director Nadia Carlesso, Ph.D., City of Hope’s chair of stem cell biology and regenerative medicine. “This will accelerate research in regenerative medicine, benefiting patients and researchers throughout California. We also aim to plant the seed for future careers in regenerative medicine by educating students about the field.”

Carlesso’s team will focus on Southern California regions, such as the Inland Empire, which is home to some of the fastest-growing, low-income communities that face greater health issues and have access to fewer physicians.

“It is important to emphasize that innovative biomedical research flourishes when people with different perspectives, experience and skills are empowered to explore new ideas and to work collaboratively and inclusively,” Carlesso said. “Thus, recruiting, training, retaining and nurturing a workforce representing all dimensions of diversity is critical for the development and implementation of leading-edge therapies that can reach underserved populations.”

To this end, a 22-person educational team will educate community physicians, researchers and students on how to use stem cell disease models with the goal of cultivating California’s future workforce in regenerative medicine. These programs will partner with higher education institutes in the Inland Empire and with K-12 school districts in Duarte, Monrovia, Charter Oak and Azusa as well as provide hands-on training to undergraduate/graduate students, postdoctoral fellows, researchers and others.

“We are eager to introduce more people to the exciting world of stem cell research and shape their vision for future jobs in science,” said co-program director John Termini, Ph.D., professor of cancer biology and molecular medicine at City of Hope. “Our workshops will explore how to apply the field’s techniques and examine diverse approaches to solving medical problems.”

“Long-term activities supported by the laboratory will accelerate research in regenerative medicine, benefiting patients and researchers throughout California,” said Carlesso, who is also associate director of basic research at the Gehr Family Center for Leukemia Research at Beckman Research Institute of City of Hope.

Led by Carlesso and Termini, the laboratory team includes Michael Barish, Ph.D., professor of stem cell biology and regenerative medicine; Mark LaBarge, Ph.D., professor of population sciences; June-Wha Rhee, M.D., assistant professor of cardiology; and Yanhong Shi, Ph.D., chair of neurodegenerative diseases and Herbert Horvitz Professor in Neuroscience.

The grant to Carlesso’s team adds to the more than $212 million that the Beckman Research Institute at City of Hope has received from CIRM to date, indicating the state-funded agency’s support of City of Hope’s long-standing leadership in stem cell-related therapies.

As a biomedical institution, City of Hope holds deep expertise in developmental and stem cell biology, resulting in strong clinical programs in bone marrow transplantation, cancer immunotherapy and gene therapy research to correct genetic defects as well as cell replacement and tissue regeneration strategies to potentially treat diabetes.

The Stem Cell-Based Disease Modeling Laboratory will operate in a renovated space that will assemble state-of-the-art instruments and technologies and centralize the generation and establishment of organoid models. It joins a wealth of City of Hope core resources, including Biostatistics & Mathematical Oncology and Gene Editing and Viral Vector cores, to both advance science and train others in subspecialized scientific areas.

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Our research program bridges gaps in traditional funding to encourage bold thinking and nurture scientific careers.

We strive to create a highly collaborative environment with multiple channels of interaction, which gives rise to new ideas, new programs, and the resources to support them., disease programs.

We channel world-class resources, both intellectual and technological, toward some of the most prevalent, devastating diseases for which stem cell research holds promise. 

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Early Stage Research

HSCI provides early funding for innovative projects in stem cell research through Seed Grants, Junior Faculty Programs, and the Barry Family HSCI Innovation Award. These focused funds allow scientists to pursue "high risk/high reward" avenues of research that might be difficult to fund from more traditional sources.

HSCI also recognizes the achievements of early-stage researchers at an annual retreat, which is a unique opportunity for our researchers to connect with one another and forge new collaborations.

Seed Grants

We support ideas that are not typically funded by traditional sources, either because the research is too early-stage or the researcher too junior to compete with larger laboratories.

Junior Faculty Programs

We support highly collaborative, “high risk, high reward” projects in which young HSCI faculty with diverse expertise pool their efforts to tackle a major, shared challenge.

Barry Family HSCI Innovation Award

We support innovation by early career investigators that has the potential to transform the field of stem cell research.

Our approach

Stem cells have already changed medicine, saving the lives of leukemia patients for over 50 years. Today, technological breakthroughs have opened up vast potential to use stem cells to understand and treat human disease. HSCI researchers are harnessing the potential of stem cells in a number of ways:

Different types of stem cells can be used to develop cell-replacement therapies. For example, a diabetes patient’s damaged insulin-producing cells could be replaced with healthy ones created from stem cells, which have been corrected using gene editing. We are expanding this approach to address many diseases.

Stem cells can be reprogrammed to become specific tissues, such as neurons or heart tissues. This helps scientists study human diseases directly in a dish, and test a potential treatment safely outside the body before giving it to a patient.

Understanding how different organs repair themselves following injury or disease helps HSCI scientists identify ways to stimulate a patient’s own stem cells to regenerate, for example using drugs to regrow inner ear cells and restore hearing.

Learn more about stem cells and medicine

Comprehensive resources are hosted by the International Society for Stem Cell Research (ISSCR) and the Alliance for Regenerative Medicine.

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Assoc. Prof. Dr. dr. Agung Putra, M.Si., Med.

Director of SCCR Indonesia

Stem Cell and Cancer Research (SCCR) is an independent institution for research and pharmacy industries studies that was established in June 2013 by Assoc Prof. Dr. dr. Agung Putra, M.Si Med. SCCR scope of work covers a wide range of stem cell (regenerative medicine) and cancers stem cell issues, primarily from the research projects. We have 10 years of experience in collaboration research between faculties of clinical departments and basic medical scientists, in other to have an international competitiveness level in Indonesia. SCCR actively work to promote future medicine through research-based evidence. The SCCR is guided by the director and the head of each two main division which are the division of the fundamental and applied research.

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Stem Cell & Cancer Research led by Assoc. Prof. Dr. dr. Agung Putra, Msi.Med had a casual discussion with the

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MoU’s Ceremony between SCCR and Central Java’s Health Office has been held in Conference room, Integrated Biomedical Laboratory’s building at

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Core facilities, original articles.

Suppression of transforming growth factor-β by mesenchymal stem-cells accelerates liver regeneration in liver fibrosis animal model

The Role of Hypoxic Mesenchymal Stem Cells Conditioned Medium in Increasing Vascular Endothelial Growth Factors (VEGF) Levels and Collagen Synthesis to Accelerate Wound Healing

Hypoxia-preconditioned mesenchymal stem cells attenuate peritoneal adhesion through TGF-β inhibition

The Role of Mesenchymal Stem Cells in Regulating PDGF and VEGF during Pancreatic Islet Cells Regeneration in Diabetic Animal Model

Sccr laboratory development process, research in sccr laboratory.

Jalan Kol.R.W Sugiarto – RT 02/RW 05 – Kel.Nongkosawit, Kec.Gunung Pati, Kota Semarang, Jawa Tengah (50223)

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Identifying the initial steps in colorectal cancer formation

By alan dove weill cornell medicine.

Research led by Weill Cornell Medicine provides new evidence that most colorectal cancers begin with the loss of intestinal stem cells, even before cancer-causing genetic alterations appear.

The results, published on May 29 in Developmental Cell, overturn the prevailing theory for colorectal tumor initiation and suggest new ways to diagnose the disease before it has a chance to become established.

This multiplex immunofluorescence image shows a healthy colon lining (left) and a premalignant serrated lesion (right). Colonic stem cells are labeled in green and express aPKC proteins labeled in magenta. Premalignant metaplastic cells are labeled in turquoise and are missing aPKC proteins and stem cells.

“Colorectal cancer is very, very heterogeneous, which has made it difficult for many years to classify these tumors in order to inform therapy,” said senior author Jorge Moscat , the Homer T. Hirst III Professor of Oncology in Pathology and Vice-Chair for Cell and Cancer Pathobiology in the Department of Pathology and Laboratory Medicine at Weill Cornell Medicine. This heterogeneity, the diverse characteristics of colorectal tumor cells in different patients and also within the same tumor, makes treatment particularly challenging.

Colorectal tumors can arise from two types of precancerous polyps: conventional adenomas and serrated adenomas. Conventional adenomas were thought to develop from mutations in the normal stem cells that lie at the bottoms of intestinal crypts, pit-like structures in the lining of the intestine. Serrated adenomas, on the other hand, are associated with a different type of stem-like cell with fetal characteristics that appears mysteriously at the tops of the crypts. Scientists in the field have described these apparently distinct tumor-forming processes as “bottom-up” and “top-down.”

“We wanted to determine how those two routes really start and how they progress, so we can better understand their heterogeneity as the cancer progresses,” said co-senior author Maria Diaz-Meco , the Homer T. Hirst Professor of Oncology in Pathology in the Department of Pathology and Laboratory Medicine at Weill Cornell Medicine and a member of the Meyer Cancer Center at Weill Cornell Medicine. That’s particularly important for serrated tumors, which doctors sometimes miss because of their initial flat shape, and which can become aggressive cancers later.

The co-first authors are Hiroto Kinoshita and Anxo Martinez-Ordoñez, postdoctoral associates in the Department of Pathology and Laboratory Medicine at Weill Cornell Medicine.

The researchers previously found that many human colorectal tumors of both origins have abnormally low levels of proteins called atypical protein kinase C (aPKC). The new study investigated what happens when aPKC genes are inactivated in animal models and cultured intestinal organoids.

“We approached this project with the bottom-up and top-down theories, but we were surprised to find that both tumor types showed loss of intestinal stem cells after aPKC genes were inactivated,” said Moscat, who is also a member of the Sandra and Edward Meyer Cancer Center at Weill Cornell Medicine.

The characteristic top-side stem cells on serrated adenomas only arise after the normal stem cells at the bottom of the crypt die, throwing the structure of the entire crypt into disarray. “So, the conventional cancer is bottom-up, and the serrated cancer is also bottom-up,” Moscat said.

The findings suggest a new unified model for the initiation of colorectal cancer where damage to the intestinal crypts causes a decrease in aPKC protein expression, followed by loss of the normal stem cells at the bottom of the crypt. Without those stem cells, the crypt cells can’t regenerate. To survive, the structure can spawn either a replacement population of regenerative stem cells at the bottom, or more fetal-like stem cells at the top. These replacement cells may then lead to cancer.

“If we can better understand how aPKC protein expression is regulated, we could control and prevent tumor development, and also better understand the progression of tumors,” Diaz-Meco said. The team is now looking at aPKC expression patterns in human tumors at different stages, with hopes of developing molecular tests that could be used to detect tumors earlier, classify tumors in patients and develop better treatments.

This research was supported in part by the National Cancer Institute of the National Institutes of Health.

Many Weill Cornell Medicine physicians and scientists maintain relationships and collaborate with external organizations to foster scientific innovation and provide expert guidance. The institution makes these disclosures public to ensure transparency. For this information, please see profiles for  Dr. Jorge Moscat  and  Dr. Maria Diaz-Meco .

Alan Dove is a freelance writer for Weill Cornell Medicine.

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