To date, many surface markers have been identified on CSCs, secreting specific molecular, interacting specific signal pathway. Many signal pathways mediate various CSC traits including the Janus-activated kinase/signal transducer and activator of transcription, Hedgehog, Wnt, Notch, phosphatidylinositol, 3-kinase/phosphatase and tensin homolog, and nuclear factor-kB signaling pathways.16

Ovarian cancer is the most lethal gynecologic malignancy. Globally, it is a significant reason of morbidity and mortality in females, with rising rates in economically underdeveloped countries and increasing case numbers in high-income countries as a result of population aging, with sharp recurrence and improving malignancy and 5-year survival rates below 45%.17 OCSCs have been found in human epithelial cancer more than a decade ago.2 Both biological and clinical characterizations of ovarian cancer prove it as a prototypical example of CSC-driven disease.18 OCSCs can explain primary tumor growth, metastasis, relapse, and conventional therapy resistance of ovarian cancer.19 There is also a relationship between OCSCs and epithelial–mesenchymal transformation (EMT), which leads to the genetic alteration in cancer and is a mechanism under angiogenesis and chemotherapy resistance.20 Some surface markers have been found in OCSCs, including CD133, CD44, CD47. Some functional markers such as ALDH1 are under intensive research, too. Indeed, there is no stringent distinction between surface markers and functional markers because they all participate in tumorigenesis and progression and are called “marker.” Various signal pathways that play a significant role in ovarian cancer have been researched, including Wnt/β-catenin, NOTCH, IL6/JAK/STAT3, Hedgehog, NFκB, PI3K/AKT, TLR2-MyD88-NFκB, HMGA1, PKCι/Ect2/ERK, YAP/TEAD, and hypoxia/NOTCH1/SOX2.18 Traditional therapy strategies based on cytoreductive surgery, chemotherapy, and radiotherapy are effective to some extent; however, recurrence and resistance to them are inevitable consequences in advanced ovarian cancer. Nowadays, immunotherapy has been used in ovarian cancer showing prospect. OCSCs are subpopulation in a tumor, and are regarded as the initiation cells with the ability to induce resistance to conventional therapy, relapse, and dissemination and should be major target for therapy.


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THE EPIGENETIC MODIFICATIONS INVOLVED IN THE CSCs AND OCSCs

Epigenetics refers to heritable traits that can be inherited from parent cells by daughter cells and are not caused by changes in the DNA sequence through meiotic or mitotic division. Epigenetic regulation mechanisms include DNA methylation, histone modification, chromatin remodeling, and modulation by noncoding RNAs.21 During cancer growth, the epigenome of cancer cells is consistently determined or regulated by cell-intrinsic/extrinsic mechanisms. Meanwhile, a subpopulation of cells (CSCs) in the tumor are conferred stemness, which refers to self-renewal and differentiation. First, epigenetic dysregulation plays an important role in the formation of CSCs, and then epigenetic mechanisms maintain the hierarchical organization of cancer controlled by CSCs.22 Epigenetic modifications play two distinct roles, namely inhibiting the majority of cancer cells to self-renew to constitute hierarchies and sustaining the subsection of CSC stemness. Dynamic epigenetic states have been implicated in the reversible transition from CSCs to non-CSCs and vice versa which is called plasticity and may be a confusion of the deterministic identity of CSCs. Among epigenetic mechanism, DNA methylation is integral to the formation of CSCs in cancer biology. For example, promoter methylation and histone modifications controlled the transcription of CD133, which is a cell surface marker protein of CSCs in many cancer types and is widely detected to identify OCSCs. The extent of DNA methylation of P2 promoter of CD133 is inversely relevant for CD133 transcription level, and CD133 cells sorted from epithelial ovarian cancer are treated with DNA methyltransferase and histone deacetylase inhibitors expressed increasing CD133 surface markers.23 Methylation abnormalities are essential in the process of CSC formation in the first step of cancer.

THE SURFACE MARKERS OR FUNCTIONAL MARKERS ON OCSCs AS THE IMMUNOTHERAPY TARGETS

Many markers on OCSCs have been reported so far, and some of them have been targets for immunotherapy. Considering the physiological and pathological traits of these markers and their current implication is stable evidence for further innovated therapy.

CD133

CD133 (prominin-1) is a transmembrane glycoprotein and was first described as a specific marker of human hematopoietic progenitor cells.24 It is a surface marker to identify various cancer types, such as brain cancer,25 colorectal cancer,26 breast cancer,27 neck and head cancer,28 and liver cancer.29 It can define a TIC population in human ovarian cancer as CD133+ and CD133 cells are sorted, respectively and primary uncultured ovarian cancer cells are injected into non-obese diabetes/SCID mice and then the CD133+ cells increasingly generated a heterogeneous tumor as the primary cancer.30 In addition, CD133 mRNA levels were inversely correlated to the DNA methylation status of the CpG sites for ovarian cancer which is regulated by epigenetic mechanism.31 CD133 expression is an independent predictor of poor prognosis which can reduce disease-free survival time for patients with ovarian cancer.32 Other investigations also described an inconsistent phenomenon that CD133 cells can induce the same characteristics as CD133+ cells, which questions whether the specificity of CD133 to identify CSCs and the glycosylation status of CD133 may be more relevant to the function of CD133 in CSCs.24

CD44

CD44 is a cell surface glycoprotein and the receptor of hyaluronan, a component of the extracellular matrix and the interaction of them has been proved to be associated with a variety of signal transduction pathways such as Nanog and EGFR-Ras-ERK.18 CD44 can be expressed on both somatic cells and CSCs, which is associated to the sphere forming, self-renewing, chemo-resistance, tumor-initiating, proliferation, and invasiveness of CSCs in preclinical experiments.33,34 The expression of CD44 is promoted during EMT, which is involved in the acquisition of stemness of epithelial tumor cells.35 CD44/CD44v isoforms have been identified to isolate and enrich CSCs in various types of malignancies, such as head and neck cancer,36 lung cancer,37 gastric cancer,38 pancreatic cancer,39colon cancer,40 and ovarian cancer.41–43 As for ovarian cancer, a study had suggested that CD44+CD24identifies a group of CSC-like cells from ovarian adenocarcinoma cell line 3AO, and these cells can differentiate into CD44+CD24+ cells and are obviously resistant to carboplatin and paclitaxel therapy.42Especially, CD44 splice variants have exclusive roles in tumorigenesis, such as CD44v6 expression is associated with poor prognosis and metastasis.44 CD44v7/8 has been employed as engineered cytotoxic T-cell immunotherapy target for cervical cancer and has shown a promising prospect.45However, some studies have suggested that CD44+ tumor cells induce a significantly shorter disease-free survival than the CD44 group. On the contrary, other studies have suggested high CD44 expression resulting in improved ovarian cancer outcome, although these inconsistences could be attributed to technical factors, and there is still an ambiguous inherence of CD44 in the CSC model.33

CD47

CD47 is a transmembrane protein which is the ligand for signal regulatory protein-alpha (SIRPα) and secretes matricellular protein thrombospondin-1.46 Substantially, CD47 is an overexpressed surface marker on all human cancers, because its combination with SIRPα sends a “don’t attack me” signal for phagocytic cells, which inhibits macrophage-mediated destruction to evade immune surveillance that is the foremost step in CSC tumorigenesis.47 Higher CD47 expression is significantly correlated with poor prognosis of high-grade serous ovarian carcinoma patients.48

ALDH1

Aldehyde dehydrogenases (ALDHs) are enzymes that promote the oxidation of aldehyde substrates to their corresponding carboxylic acids. ALDH1 is a functional marker on OCSCs, while there is no obvious barrier when considering the specificity to identify OCSCs provided this marker can be detected by modern assays, but the functional marker which is not expressed on the cell surface may not be adapted to direct target therapies. Within the ALDH family, which is composed of 19 isoforms with similar catalytic functions, the ALDH1 subgroup is particularly active in normal cells and CSCs. Particularly, ALDH1A1 is exploited more widely in many different cancer types of CSCs than ALDH1A2 and ALDH1A3 in ALDH1 isozymes.49 ALDH1 is expressed on the cancer cell surface as a molecular pump to efflux chemotherapeutic agents which is the mechanism underlying chemo-resistance in CSCs.18 A recent study has shown that the evolution of surface markers (CD133 and ALDH1) expressed on high-grade serous OCSCs has changed from primary ovarian cancers to recurrent ones and CD133 and ALDH1 coexpression is an independent factor for poor prognosis.50 However, a study suggested that ALDH is superior to CD133, serving as a marker to identify OCSCs, as ALDH is correlated with spheroid formation and tumor forming capability.51

Others

There are some other surface markers to distinguish OCSCs such as epithelial cell adhesion molecule (EpCAM), CD117, and CD24 or functional factors such as transcription factor SOX2 and VAV3.52–55EpCAM (also named CD326) is a Ca2+-independent cell adhesion molecule expressed on the basolateral surfaces of most epithelial cells.56 It can distinguish CSCs of colon cancer,57 prostate cancer,4 ovarian cancer,58 pancreatic cancer,59 lung cancer, breast cancer, and gastric cancer.60

A marker found to distinguish OCSCs is never limited in ovarian cancer but overlaps many cancer types. In another context, to identify OCSCs always needs combined coexpression “marker pairs,” such as Lgr5/ALDH1,61 CD44/CD117,41 and ALDH/CD133.62 The dual diversity reveals the commonness of various malignancies and the heterogeneity within one tumor. It is a drawback that none of these markers are exclusively within CSCs/OCSCs, but nowadays taking advantage of great disparity of expression levels on CSCs/OCSCs when compared with normal tissues gives greater knowledge into immunotherapeutic strategies targeting these markers which is discussed in “Immune system and immunotherapeutic strategies relevant to CSCs/OCSCs”.