Seema C. Parte University of Louisville
Andrei Smolenkov University of Louisville
Surinder K. Batra University of Nebraska Medical Center
Mariusz Z. Ratajczak University of Louisville
Sham S. Kakar University of Louisville
Stem Cells, the Holy Grail for Tissue Regeneration and Repair, Remain an Elusive Phenomenon in Ovarian Biology
Stem cells are considered the holy grail for tissue regeneration and repair, but remain an elusive phenomenon in ovarian biology. Only recently, studies have uncovered the promising potential of “ovarian stem cell differentiation into female gametes”1-3, which could be explored to address female infertility, menopause, and possibly tumor initiation. The possible contribution of stem cells present in adult tissues and organs in cancer scenario is not fully clear especially in ovarian cancer. Stochastic events leading to transformation of normal stem cells and/or progenitors may be implicated4. Epithelial ovarian cancer is the most aggressive and lethal forms of gynecological cancers affecting ∼250,000 women worldwide each year5,6. Approximately 90% of ovarian tumors are epithelial in nature, and the existence of pluripotent stem cells in the ovarian surface epithelium (OSE) layer suggests a strong correlation between origin of epithelial cancer and cancer stem cells (CSCs)7. CSCs represent a novel population (2%–5%) of tumor cells possessing unique characteristics such as self-renewal and transformation into differentiated cells, which constitute the tumor bulk. These properties impart them with an edge to survive chemotherapy, undergo enrichment, and expedite tumor progression resulting in relapse thus contributing to ultimate mortality of the affected individuals8. Existence of CSCs has been reported in various cancers, including ovarian cancer9,10 breast cancer11, Glioblastoma12, and pancreatic cancer13. In this view, their identification and development of novel combination drugs targeting not only cancer cells but also CSCs are immediate requirements9,14.
Cancer initiation and resultant propagation/invasion entail a complex series of cross talk of events at cellular and molecular levels15, while the role played by CSCs requires to be rigorously investigated. There is no consensus regarding true identity of ovarian CSCs and their signaling mechanisms, their location, precise cell(s) of origin, and their individual response to novel drugs. Moreover, the putative CSC populations with ovarian stem cell perspective have never been studied across various stages of ovarian cancer. Tissue specific CSCs have already been identified in ovarian cancer, but it remains to be determined if germline stem cells reported in normal ovaries express these pluripotent stem and CSC specific markers. To reiterate, the goal of present study was to correlate the expression of pluripotent stem cells with germ lineage markers.
Presence of germline stem cells termed “oogonial stem cells” (OGSCs)/“female germline stem cells” (FGSCs) was first put forth by Prof. Jonathan Tilly and his group in adult mice16 and later in adult mice and human ovaries17. Similarly, pluripotent stem cells have been demonstrated in adult human ovaries2,3,18-22 and mice22,23 by certain groups independently. Parallel to these reports, a pluripotent stem cell population with embryonic characteristics named very small embryonic-like stem cells (VSELs) has been identified in various adult tissues and organs. These peculiar, tiny spherical stem cells are remnants of migratory PGCs gone astray during embryogenesis as they share common markers24,25. Similarly mouse hematopoietic stem progenitor cells (HSPCs) show germline connection, and thus, a hierarchical link among murine VSELs, HSPCs, and primordial germ cells (PGCs) was recently speculated26. Likewise a germline connection with the tumors was proposed27 with pluripotent adult stem cells showing embryonic characteristics (VSELs) as the putative connecting link. These VSEL cells have been speculated to differentiate into CSC populations28,29.
VASA/DDX4 is a member of DEAD box protein family involved in processes such as embryogenesis, gametogenesis, cellular growth, division and PGC development, and so on30. Besides its multifaceted cellular functions, recently its expression was linked to tumor formation and maintenance in drosophila brain31, as well as in tumor progression and poor patient prognosis in case of epithelial ovarian cancer cells32,33. DEAD-box helicase 4 (DDX4) is also implicated in cell cycle regulation in multipotent and proliferating cells by influencing 14-3-3 sigma at G2/M checkpoint. Hence it becomes imperative to pinpoint the expression of pluripotent stem cell population(s) with germline characteristics existing within the ovarian tumors in comparison to normal ovaries and investigate their relation with other pluripotent and CSC markers.
Identification of unique gene signatures for detection of early onset, predicting patient response to treatment and their survival, has been the Achilles' heel so far in cancer research. The known pluripotent stem cell population namely ovarian (germline) stem cells and (cancer) stem cells both were studied systematically in various stages of ovarian cancer in comparison to normal ovaries. Single and double immunolabeling experiments interestingly revealed dynamic populations of pluripotent stem cells with germline and CSC specific properties within the OSE and cortex regions of the ovary. Current study provides novel insights into the stem cell profiles in normal versus ovarian tumor tissues and consolidates the germline connection with normal stem cells, as well as CSCs.
Ovarian cancer is most lethal among gynecological cancers with often fatal consequences due to lack of effective biomarkers and relapse, which propels ovarian cancer research into unique directions to establish solid targeted therapeutics. “Ovarian stem cells” expressing germline pluripotent markers serve as novel paradigm with potential to address infertility, menopause, and probably influence tumor initiation. Cancer stem cells (CSCs) pose vital role in tumor recurrence and hence it is extremely important to study them with respect to ovarian stem cells across various cancer stages and normal ovaries. Pluripotent (OCT4, NANOG, SOX2, SSEA1, and SSEA4), germline (IFITM3, VASA/DDX4), and cancer stem (CD44, LGR5) cell specific markers were characterized for protein and mRNA expression in tumor tissues to understand their distribution in the surface epithelium and ovarian cortex in benign, borderline, and high-grade malignant stages. To elucidate whether pluripotent ovarian germline stem cells and CSCs are common subset of stem cells in tumor tissues, VASA was colocalized with known pluripotent stem (OCT4, SSEA1, SSEA4) and CSC (CD44, LGR5) specific markers by confocal microscopy. Single, smaller spherical (≤5 μm), and larger elliptical fibroblast like (≥10 μm) cells (also in clusters or multiples) were detected implying probable functional behavioral significance of cells in tumor initiation and metastasis across various cancer stages. Cells revealed characteristic staining pattern in ovarian surface epithelium (OSE) and cortex regions exclusive for each marker. Co-expression studies revealed specific subpopulations existing simultaneously in OSE and cortex and that a dynamic hierarchy of (cancer) stem cells with germline properties prevails in normal ovaries and cancer stages. Novel insights into CSC biology with respect to ovarian and germline stem cell perspective were obtained. Understanding molecular signatures and distribution within ovarian tissue may enable identification of precise tumor-initiating CSC populations and signaling pathways thus improving their efficient targeting and strategies to prevent their dissemination causing fatal relapse.