Ovarian cancer (OC) is the most lethal tumor of the female genital tract due to aggressive behavior, late diagnosis and high recurrence potential. Most of the patients worldwide are admitted with advanced disease as the initial steps of cancer growth are usually clinically obscured. This is a reason why the 5-year survival in the whole patient population does not exceed 48% (data of American Cancer Society 2020. https://www.cancer.org/cancer/ovarian-cancer/detection-diagnosis-staging/survival-rates.html, accessed on 20 December 2021). Moreover, ovarian cancer shows chemoresistance to standard platinum-based chemotherapy especially in advanced and recurrent cases, the fact which further influences poor survival. Ovarian cancer disease includes a heterogenous group of neoplasia: among them, about 90% are epithelial (subtypes: mucinous, serous, endometrioid and clear cells), as suggested by several and recent morphological and ultrastructural studies [1]. Ovarian cancer is a heterogeneous disease which comprise malignant tumors of serous, mucinous, endometrial or clear cell histology. According to the differences of biological behavior and malignancy, OC has been divided into two types: type I tumors containing low-grade (LGOC) serous, mucinous and endometroid ovarian cancer with better prognosis and lower rate mortality, and type II highly malignant and rapidly progressing high-grade serous ovarian cancer (HGOC) with poor prognosis and mortality about 90% of all OC cases ^[2][3][2,3]. Genetic expression profiling studies support this clinical classification, as type I tumors are associated with relative genetic stability and mutations of PIK3CA, PTEN, BRAF, KRAS and ARID1A genes, while type II tumors possess high chromosomal instability, defective homologous recombination repair and are characterized mostly by TP53 mutations, but also by BRCA1, BRCA2, RB1 and CTNNB1 gene mutations ^{[4][5][6][7][8][9]}[4,5,6,7,8,9]. Progenitor cells for type I OC are endometrial epithelial cells (for endometroid and clear cell tumors), tubal-peritoneal junction cells (for mucinous tumor) or fallopian epithelial cells and cortical inclusion cyst (CIC) epithelial cells (for LGOC), whereas for type II OC the progenitor cell originate from serous tubal intraepithelial cancer lesions (STIC) localized on tubal fimbriae. Early type I tumors frequently exist as so-called borderline tumors which do not show histologic signs of stromal invasion ^[1][2][1,2]. Recent gene profiling studies allowed for a proposal of a new classification based on both gene expression pattern and histological structure. According to this classification ovarian cancer could be divided into five subtypes: mesenchymal, immunoreactive, proliferative, differentiated and anti-mesenchymal. Mesenchymal and proliferative tumors comprise for 28% and 20% of OC, respectively. Mesenchymal subtype show desmoplasia and mesenchymal gene expression pattern, proliferative subtype show limited inflammatory infiltration and activation of signaling pathways for stemness. Both subtypes have an unfavorable prognosis. Otherwise, immunoreactive and anti-mesenchymal subtypes which comprise 21% and 12% of OC, have a better prognosis. The immunoreactive subtype is characterized by extensive T cell tumor infiltration and T-cell receptor and toll-like receptor signaling, while the anti-mesenchymal subtype shows a genotype which is opposite to the mesenchymal type. Differentiated subtype observed in 17% of OC tumors has gene pattern resembling serous borderline tumors and intermediate prognosis ^[10][11][12][10,11,12]. Extensive surgical debulking followed by platinum and taxane-based chemotherapy is a standard of care for invasive OC patients, however, extensive spread of tumor implants inside the peritoneal cavity, as well as a primary chemo-refractoriness or acquired chemoresistance of the tumor are responsible for unfavorable outcome. Recent studies suggest that a unique population of tumor cells called cancer stem cells (CSCs) are the most probable reason for cancer progression and therapy failure in OC.

2. Ovarian Cancer Stem Cells (OCSCs)—Markers

2.1. Cell Surface Markers

CSCs surface markers are not specific as they are also expressed on normal stem cells. Therefore, CSCs should also be identified by precisely defined behavior, such as spheroid formation or the reconstitution of tumors after transplantation to laboratory animals. Numerous markers have been suggested to identify CSCs, including OCSCs; however, their precise clinical significance is still unknown. Despite this, several surface cell markers identifying OCSCs isolated either from patient samples or experimental animals and cancer cell lines have been described (Table 1).

Table 1.

Markers of OCSCs—function, correlation to clinicopathological features and their cell/tissue origin.

Marker	Function	Origin of Studied Cells	Reference	Association to Clinicopathological Features	Cell/Tissue Origin	Reference

CD44+	Increased tumorigenicity, sphere-formation, cells self-renewal	Primary EOC tumors, cell cultures	[13][14][15][16]	[49,50,51,52]	Number of CD44+ cells higher in early stage EOC and correlated with shorter PFS Expression correlated with high-grade, advanced (III/IV FIGO) EOC in younger (<60) patients Higher number of CD44+ cells correlated with chemoresistance and shorter DFI CD44+ correlated with Ki67 index, p53 positivity and tumor grade in HGSOC, mucinous and endometroid EOC	EOC-isolated cells Recurrent EOC (88% HGSOC) Primary and recurrent EOC (78% HGSOC) EOC (HGSOC 62%) and BOT	[17][18][19][20]	[105,139,140,141]
CD44 v6+	Increased tumorigenicity, recapitulation of tumors	Xenotransplantation model	[21]	[57]	Distant metastases more frequent and metastasis free survival shorter in CD44v6+—high group of patients Increased number of CD44v6+ cells in primary tumors correlated with shorter OS	EOC FIGO I–III tumors EOC FIGO III–IV tumors (71% HGSOC)	[22][21]	[56,57]
CD44+/MyD88+	Increased tumorigenicity, sphere-formation, resistance to apoptosis, chemoresistance	Cell lines, ascites	[23]	[142]	Expression of MyD88 protein was an unfavorable prognostic factor for EOC patients	Benign ovarian tumors, BOT and EOC (54% HGSOC)	[24]	[97]
CD44+/CD117+	Increased tumorigenicity, sphere-formation, recapitulation of tumors, chemoresistance	EOC tumors, xenograft models	[13]	[49]	CD44+CD117+ cell lines were less prone to paclitaxel-induced apoptosis	EOC cell lines	[23]	[142]
CD44+/CD24-	Increased tumorigenicity, sphere-formation	Cell lines	[25]	[143]	>25% CD44+/CD24- cells in ascites correlated with higher risk of recurrence and shorter PFS	Ascites-isolated cells from advanced EOC	[26]	[104]
CD44+/CD24+/ EpCAM+	Increased tumorigenicity, chemoresistance	Cell lines, EOC-isolated cell lines, ascites	[27][28]	[100,110]	Ovarian cancer stem cells expressing EpCAM+ are less prone to chemotherapy and are a source of recurrent tumor after the treatment	EOC I-IV FIGO stage (45% HGSOC, 14% clear cell, 17% endometroid, 12% mucinous)	[27]	[100]
CD44+/CD166+	Increased tumorigenicity, sphere-formation	Cell lines	[29]	[108]	Population of platinum-resistant cells is enriched in CD44+/CD166+ population	EOC-isolated and standard cell lines	[30]	[144]
CD44+ALDH1+	Increased tumorigenicity, chemoresistance	Cell lines	[31]	[145]	>50% ALDH1+ cells correlated with shorter OS	Advanced EOC (73% HGSOC)	[31]	[145]
CD44+/CD133+/ALDH1A1+	Chemoresistance	Cell lines, EOC-isolated cell lines	[32]	[116]	Expression of markers increased in recurrent compared to primary tumors	Advanced primary and recurrent EOC	[32]	[116]
CD133+	Increased tumorigenicity, enhanced vasculogenesis	Cell lines, EOC tumors, xenograft models, ascites	[33][34][35][36]	[72,78,81,146]	Expression of CD133+ correlated with presence of HGSOC, higher FIGO stage, ascites, chemoresistance, shorter PFS and OS No correlation with prognosis Expression of CD133+ correlated with shorter PFS and OS Expression of CD133+ correlated with shorter OS and platinum chemo-resistance	EOC (67% HGSOC) EOC FIGO III–IV (72% HGSOC) Advanced metastatic HGSOC Advanced primary HGSOC	[37][38][39][40]	[73,147,148,149]
CD133+/ALDH1A+	Increased tumorigenicity, cells self-renewal, chemoresistance	EOC tumors, cell lines, xenograft models	[41][35]	[80,81]	Expression of CD133+ correlated with III/IV FIGO stage, expression of CD133+/ALDH1A+ correlated with shorter PFS and OS	HGSOC	[42]	[150]
CD117+	Increased tumorigenicity, sphere-formation, recapitulation of tumors, chemoresistance	EOC-isolated cell lines, xenograft model, ascites	[43][44][45][46][47]	[62,63,64,151,152]	Expression of CD117+ correlated with shorter PFS 40% of HGSOC were CD117+ and expression correlated with chemoresistance	Advanced metastatic HGSOC HGSOC	[44][39]	[63,148]
CD24+	Increased tumorigenicity, stimulation of EMT	Cell lines	[48]	[92]	Expression of CD24+ correlated with FIG stage and the presence of peritoneal and lymph node metastases	27% HGSOC 12% mucinous 18% clear-cell 18% endometaroid 23% others	[48]	[92]

BOT—borderline ovarian tumor; DFI—disease-free interval; EMT—epithelial-mesenchymal transition; EOC—epithelial ovarian cancer; FIGO—International Federation of Obstetrics and Gynecology; HGSOC—high-grade serous ovarian cancer; OS—overall survival; PFS—progression-free survival.

2.1.1. Glycoprotein CD44

Molecule CD44 is a cell-surface glycoprotein that is a receptor for the hyaluronic acid receptor. Its stimulation activates signaling pathways including epidermal growth factor receptor (EGFR)/Ras small GTPase protein (Ras)/extracellular signal-regulated kinase (ERK) and transcription factor homeobox protein NANOG-dependent signaling pathways, followed by cell proliferation, differentiation, and increased motility and stemness. Interaction between NANOG and STAT3 results in the up-regulation of multidrug resistance protein-1 (MDR1) and the effective efflux of chemotherapeutic drugs from OCSCs ^[49][13][48,49]. The population of CD44+ OC cells possess self-renewal, tumor initiating and sphere-forming capacities. Recurrent OC shows a higher expression of CD44-positive cells compared to primary tumors which is correlated with poor prognosis, although not all studies are consistent in this matter. CD44 exists in alternatively spliced variants which could be better correlated to the clinical history of OC. Between them CD44v6 was found in excess on OCSCs from distant metastases indicating metastasis-initiating activity via a hematogenous spread. In patients with FIGO stage I-III OC, distant metastasis-free survival was better in patients with CD44v6-low tumors. Silencing of the CD44-regulatory gene with siRNA resulted in tumor shrinkage in a mouse model of OC ^{[13][14][15][16][50][51][52][22][21][53]}[49,50,51,52,53,54,55,56,57,58].

2.1.2. Receptor Tyrosine Kinase CD117

CD117 is receptor tyrosine kinase coded by c-kit proto-oncogene. It has an external ligand-binding domain specific for a stem cell factor (SCF) and a cytoplasmic domain with intrinsic tyrosine kinase activity. Binding CD117 to SCF causes the receptor proteins to dimerize, phosphorylation and activation. This activates several signaling pathways, mainly Ras/ERK, phosphoinositide-3-kinase (PI3K)/protein kinase-B (AKT), non-receptor tyrosine kinase Src and Janus kinase (JAK)/signal transducer and activator of transcription (STAT), responsible for regulating cell proliferation, differentiation, apoptosis and adhesion. CD117 identifies a population of sphere-forming non-adherent OC cells and a so-called “side population” of OC cells having the capacity to harbor a specific position on flow-cytometric panels due to the selective expression of ATP-binding cassette drug transporters (ABC membrane transporters) using Hoechst 33,342 dye-staining. The basis of the “side-population” assay is the ability of ABC transporters to provide a rapid efflux of lipophilic fluorescent dye, and they are identified using special gating strategies during the analysis of flow-cytometric plots. Since its first use for hematopoietic stem cell identification, the “side-population” assay has been successfully used for the identification of progenitor and stem cells in different tissues, including stem-like cancer cells displaying increased capacity of self-renewal, tumorigenicity and chemo-resistance ^[54][59]. The “side population” shows an increased expression of stemness genes. OC cells bearing both CD44 and CD117 markers are abundant in chemo-resistant OCSCs ^{[52][53][55][56][43][44][45][57]}[55,58,60,61,62,63,64,65]. The presence of CD117+ OC cells are correlated with resistance to standard platinum-taxane-based chemotherapy and shorter recurrence intervals in treated OC patients, while the inhibition of CD117 results in the restitution of chemo-sensitivity. The level of CD117 expression correlates with both disease-free and overall survival. The expression of CD117 could be augmented by hypoxia and HIF-1α activation, and is followed by Wnt/β-catenin signaling ^[44][58][59][63,66,67]. SCF growth factor is expressed in high concentrations in the ascitic effusions collected from EOC patients. The soluble SCF form is produced by TAMs and fibroblasts (TAFs), whereas a minority of tumor cells only express the membrane-associated SCF form. C-kit is expressed by OCSCs and binds to both soluble and cell-associated SCF, as well as functionally responding to the ligand ^[60][68].

2.1.3. Prominin-1 CD133

Surface protein CD133 is also called prominin-1 and is a transmembrane glycoprotein activating the PI3K/Akt pathway. It is responsible for tumor maintenance, vascularization, and chemoresistance, and hence is considered to be an OCSC marker. It was found that CD133 mediates metastatic homing to peritoneal tissue in OC ^[61][69]. Endothelin receptor-A (ETRA) is expressed on CD133+ cells and the chemotherapy-induced inhibition of ETRA decreases the ability of CD133+ cells to form spheres ^[62][70]. The activity of nuclear factor-κ-light chain enhancer of activated B cells (NF-κB) and p38 MAPK pathways in response to IL-17 enhances the self-renewal capabilities of CD133+ cells. The expression of intracellular stemness markers octamer-binding transcription factor-4 (OCT4) and sex-determining region Y—box 2 (SOX2) is higher in CD133+ than CD133- cells ^[63][71]. Ovarian C-X-C chemokine receptor type-4 (CXCR4)+/CD133+ OC cells showed more effective platinum efflux and lower platinum sensitivity compared to CD133-negative cells ^[33][72]. The correlation between CD133 expression and advancement of the tumor (presence of HGOC, advanced clinical stage, presence of ascites, tumor non-responsive to chemotherapy), as well as patients’ survival has been pointed out ^[37][73]. Moreover, the population of CD133+ cells was more abundant in recurrent platinum-resistant tumors compared to primary OC tumors ^[64][74]. However, according to some studies, OCSCs indicate an inconsistent expression of the CD133 marker and CD133+ cells do not always have particular pro-tumorigenic properties. It is possible that OCSCs plasticity and some heterogeneity could account for this controversy. Another possibility is an alternating expression of the CD133 molecule in cytosolic and membrane compartments of OCSCs populations ^{[65][66][67][34][68][41][35][69][70][71]}[75,76,77,78,79,80,81,82,83,84].

2.1.4. Heat-Stable Antigen CD24

CD24 is a transmembrane adhesion molecule also called a heat-stable antigen CD24. Through the activation of the STAT3 signaling pathway, it is able to stimulate cell adhesion and augment cell malignancy. The inhibition of the JAK2/STAT3 pathway in OC results in a loss of cancer stemness and reduced tumor growth ^[72][85]. The inhibition of the over-expressed JAK2/STAT3 pathway in patient-derived CD24+ OCSCs correlated with a better survival of OC patients ^[73][86]. The CD24 molecule may also regulate NANOG and OCT4 expression, thus stimulating tumor growth and resistance to chemotherapy. CD24-positive OC cells were isolated both from tissue specimens and from cellular cultures, and were shown to overcome anoikis and to form spheroid structures. CD24+ OC cells also displayed the up-regulation of genes regulating cellular stemness. The abundance of CD24+ cells was increased in metastatic peritoneal implants compared to the primary tumor, where it contributed to the attachment of OC cells to fibronectin and collagen of the peritoneal stroma. STAT3 signaling following CD24 stimulation is a well-established stimulator of stemness of OCSCs cells, while PI3K/AKT and mitogen-activated protein kinase (MAPK) signaling following CD24 stimulation could activate EMT. However, there are also scanty opinions that CD24 expression has no correlation with OCSCs’ self-renewal and chemoresistance ^{[74][75][76][77][78][48][79][80][81]}[87,88,89,90,91,92,93,94,95]. Clinically, high CD24+ expression is a predictor of poor survival in OC patients ^[82][96].

2.1.5. MyD88 Protein

Myeloid differentiation primary response gene 88 (MyD88) is an adaptor protein for signals generated by toll-like receptor-type 4 (TLR-4). TLR-dependent signaling activates NF-κB which moderates the inflammatory pathway in tumor carcinogenesis. The TLR-4/MyD88 pathway has been modified in OC and is responsible for OC chemo-resistance. MyD88-positive ovarian cancer cells equate to OCSCs cells due to the resistance to pro-apoptotic signals and the ability to create a pro-inflammatory tumor microenvironment through leukocyte recruitment. MyD88-negative cells are more differentiated and less aggressive. The expression of MyD88 protein was found to be an unfavorable prognostic factor for OC patients ^[24][83][97,98].

2.1.6. Epithelial Cell Adhesion Molecule EpCAM

Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein regulating intercellular adhesion, present on a subset of normal epithelia, as suggested by several recent studies ^[84][99]. EpCAM-positive OC cells have greater tumor-initiating potential compared to EpCAM-negative cells. The EpCAM/B-cell lymphoma-2 (Bcl-2) signaling pathway prevents platinum-dependent apoptosis of cancer cells resulting in chemo-resistance; therefore, EpCAM expression is increased in tumors of chemo-resistant patients and correlates with unfavorable outcome ^[27][100]. Similarly, the B-cell lymphoma extra-large (Bcl-xL) anti-apoptotic protein present in mitochondria was found to be over-expressed in recurrent chemo-resistant OC. The inhibition of Bcl-xL restored chemo-sensitivity of OC cells ^[85][101].

2.1.7. Multipositivity of Cell Surface Markers

Effective isolation of OCSCs usually demands the identification of two or more cell markers. Double positive CD44+/CD117+ cells are highly capable to recapitulate the original tumor after being transplanted into experimental animals, and are the main component of sphere-forming cells in ascites. This OCSC population also showed high mitochondrial concentrations of reactive oxygen species (ROS) suggesting that mitochondrial respiration is used to sustain OCSC’s viability in stress conditions and during starvation ^[86][102]. The level of CD44+CD24-OCSCs in OC patients has been suggested to have a prognostic value. In patients having more than 25% of CD44+CD24-OCSCs, a greater probability of recurrence and a shorter progression-free survival were observed. Similarly, primary tumors showed either a low or high expression of CD44+ALDH1+ OCSCs. Those exhibiting low expression had a better response to chemotherapy and longer progression-free survival ^{[87][26][17][88]}[103,104,105,106]. Recurrent platinum-resistant ovarian tumors compared to primary tumors are enriched in the population of CD44+CD133+ALDH1A1+ OCSCs. The population of CD44+/E-cadherin-/CD34- inside ovarian tumors identify OCSCs cells with the ability to recapitulate the tumor and support neovascularization ^[89][107]. The population of CD44+CD166+ has stem cell characteristics through the increased capacity of forming spheres and the high activity of histone deacetylases regulating the OCSC’s phenotype ^[29][108]. CD133+/ALDH1+ cells have tumor initiating properties and induce neoangiogenesis. CD44+/MyD88+ cells are highly resistant against apoptosis and chemotherapeutic drugs. They can grow as adherent cells or are able to undergo EMT and form spheroid cell structures ^[90][109]. Similarly, CD44+/CD24+/EpCAM+ cells show OCSCs’ properties having increased migratory and invasive potential and chemo-resistance to platinum, taxane and doxorubicin ^[28][110]. Cells of CXCR4+CD133+ phenotype isolated from OC cell lines also have OCSC properties ^[67][77]. Neoadjuvant chemotherapy is a therapeutic option for patients in whom primary optimal cytoreductive surgery is unavailable due to extensive peritoneal carcinomatosis. However, studies have revealed that this form of management is associated with the enrichment of metastatic tumors in OCSCs defined as ALDH1+ cells showing chemo-resistance and correlated with bad prognosis ^[91][92][111,112]. Recent studies have shown that not only standard platinum-taxane-based chemotherapy contributes to the proliferation of OCSC population; targeted therapy with poly-(ADP) ribose polymerase (PARP) inhibitors which disturb tumor DNA repair systems also result in an enrichment of tumors with OCSCs followed by a restored ability to repair DNA ^[53][58].

The variegated opinions about OCSC markers could have several origins. The simplest explanation is that different OCSC populations are characterized by different markers. Another possibility is that due to ovarian cancer histological and genetic heterogeneity, the observed populations of OCSCs follow this heterogenic pattern. Moreover, OCSCs could differ between distinct localizations and in different stages of the disease. Finally, the marker diversity could result from OCSCs’ molecular and functional plasticity, where cells with different properties share stemness and tumor propagating abilities ^[93][94][113,114]. In this context, patients could have multiple populations of OCSCs which may vary between distinct tumors and patients. This possibility creates an unfavorable perspective for the success of therapy directed against OCSCs.

2.2. Intracellular and Functional Markers

2.2.1. Aldehyde Dehydrogenases ALDH

Aldehyde dehydrogenases (ALDH) are a group of enzymes converting aldehyde substrates to carboxylic acids via the oxidation process. The protective and detoxifying function of the ALDH1 subgroup is involved in the maintenance of cancer cells, especially OCSCs, against chemotherapeutics and radiation. In this subgroup, the most supporting role for the creation of the OCSC phenotype is assigned to ALDH1A1 and ALDH1A2 isotypes ^[95][115]. ALDH1-positive cell phenotype identifies the OCSC populations possessing self-renewal and stemness properties that are capable of sphere formation and restoring the tumor. ALDH1 exerts its function via the Wnt/β-catenin signaling pathway and ABC-transporters. ALDH1 activity is also regulated by NF-κB/transcription factor RelB non-canonical pathway ^[95][115]. Moreover, ALDH1 is engaged in the activation of OCSCs quiescence program by slowing down the proliferation of the cells and in the protection of DNA-repair programs that both contribute to ovarian cancer resistance. It was found that primary OC tumors contain less ALDH1+ cells compared to tumors pre-treated with neoadjuvant chemotherapy ^{[91][32][96][97][98][99][100][101][102]}[111,116,117,118,119,120,121,122,123]. The observation that, in cultures of OC cells, the percentage of ALDH1+ cells was growing along with the dose escalation of platinum was a very alarming phenomenon, indicating great viability and endurance of this cell population ^[35][81]. Epidermal growth factor-like domain-6 (EGFL6), which functions as a stem cell regulatory factor activates an asymmetric division of ALDH1+ OC cells stimulating their proliferation ^{[88][103][104]}[106,124,125]. In HGOC patients, tumors characterized by the higher percentage of double-positive ALDH+/epidermal growth factor receptor (EGFR) + cells, are associated with poor outcomes as compared with tumors that are either ALDH or EGFR negative ^[105][126]. In clear-cell carcinoma population of ALDH1^high OCSCs, cells show markedly higher tumorigenic potential than ALDH1^low cells. The expression of ALDH1^high OCSCs is increased in advanced tumors and correlates with unfavorable prognosis and chemo-resistance ^{[88][99][106]}[106,120,127].

2.2.2. Transcription Factors

A group of transcription factors have also been considered to be markers of OCSCs. These include NANOG, SOX2, forkhead-box protein M1 (FOXM1), and OCT4. NANOG is physiologically responsible for the maintenance of self-renewal and the pluripotency of embryonic stem cells (ESCs), the same role it plays in OC, where it additionally regulates EMT and chemo-resistance via the STAT3 signaling pathway. The expression of NANOG in OCSCs cells correlates with clinical stage and high grade, as well as resistance to standard chemotherapy ^[14][50]. Similar to NANOG, SOX2 is also responsible for the maintenance of self-renewal and the pluripotency of ESCs. The over-expression of SOX2 is related to the stemness of cells via the inhibition of the PI3K/AKT pathway, resulting in resistance to apoptosis. In OC, pathological SOX2-positive cells were identified in the epithelium of the tubal fimbriae of patients with HGOC tumors and patients with germline BRCA1/2 mutations ^[107][108][128,129]. OCT4 is involved in embryonic development and cellular pluripotency. Its function is to stabilize the higher-order structure of chromatin in the NANOG locus ^[109][130]. Up-regulation of OCT4 in OCSCs was correlated to tumor initiation and again, chemo-resistance. The cytoplasmic expression of OCT4 regulates EMT transformation and is a recognized predictor of adverse clinical outcomes in cancer. The co-expression of OCT4 with RNA-binding protein Lin28 was connected to advanced tumor growth and grade ^[110][131]. Increased levels of OCT4 were observed in OCSCs’ CD24-positive cells ^[75][88]. NANOG, OCT4 and SOX2 were over-expressed not only in tumor tissues but also in both ascites and spheres built from OCSCs cells ^{[108][111][112]}[129,132,133]. FOXM1 is a member of the FOX family of transcription factors. It plays an important role in cell cycle control and progression, and in the maintenance of genomic stability. The over-expression of FOXM1 protein was observed in OCSCs exposed to elevated concentrations of lysophosphatidic acid (LPA) present in ascites fluid in OC patients. Increased FOXM1 levels were followed by the activation of wingless and Int-1 (Wnt)/β-catenin signaling and chemo-resistance. Alternatively, FOXM1 suppression resulted in the restitution of chemo-sensitivity and the loss of ability to spheroid formation in the peritoneal environment ^{[113][114][115]}[134,135,136].

The cells characterized by OCSC phenotypes were identified both in ovarian surface epithelium (OSE) and fallopian tube epithelium (FTE) in mice, including ALDH1+, ALDH1A1+, ALDH1A2+, CD133+ and NANOG+ cells. These OCSCs markers were detected especially in the distal portion of the tube (fimbria) supporting the observation of HGOC origin ^[116][117][137,138].

3. Anti-OCSC Therapy

Cancer stem cells constitute a very attractive target for therapy. The effective elimination of this cell population would probably improve the treatment of advanced cancer and protect against recurrent lethal disease. Therefore, many different approaches to CSC-targeted management have been proposed and tested in vitro, in experimental settings and in clinical trials. CSCs are under extensive investigation in practically all known types of cancer. The most popular and tested targets for anti-CSC therapy are signaling pathways regulating the origin and function of CSCs, the surface and intracellular markers of CSCs, drugs changing the epigenetic regulation of CSCs’ function, and CSCs’ metabolism. Among them, there are inhibitors of Wnt (Ipafricept), Hedgehog (Vismodegib, Sonidegib), NOTCH (enoticumab, demcizumab, navicixizumab), MAPK (Salinomycin), and other signaling pathways (Metformin). Another group are drugs targeting CSC markers (Imatinib mesylate), epigenetic regulation by DNA-(cytosine-5)-methyltransferase-1 (DNMT1) (Decitabine, Guadecitabine, Azacitidine) or by histone deacetylase (HDAC) (Vorinostat, Belinostat, Etinostat). There are also some natural compounds being tested, such as curcumin, which has indicated anti-cancer activity in in vitro and animal studies. Another approach is to combine drugs or toxic agents with nanoparticles capable to transport them precisely into the tumor (examples are glucose-coated gold particles, paclitaxel albumin-bound nanoparticles, doxorubicin or mangostin encapsulated poly D/L lactide-co-glicolide acid—PLGA) ^[118][412]. Immunotherapy directed against tumor antigens with the use of chimeric antigen receptor T cells (CAR-T lymphocytes) has also been tested in many malignancies ^[119][413]. However, the immunosuppressive environment of solid tumors represents a barrier to this therapy’s success, due to low antigen expression on tumor cells ^[120][414]. A combination of several methods, for example, CAR-T cells and oncolytic viruses (Ovs), can allow the targeting of CSCs and the surrounding cancer niche. An OV-based strategy to overcome the mechanism of CAR-T cell evasion is to encode CAR-targeted TAAs in OVs to increase TAA expression more homogeneously across the tumor ^[121][415].