Paul P. Szotek

Normal ovarian surface epithelial label-retaining cells exhibit stem/progenitor cell characteristics

Ovulation induces cyclic rupture and regenerative repair of the ovarian coelomic epithelium. This process of repeated disruption and repair accompanied by complex remodeling typifies a somatic stem/progenitor cell-mediated process. Using BrdU incorporation and doxycycline inducible histone2B-green fluorescent protein pulse–chase techniques, we identify a label-retaining cell population in the coelomic epithelium of the adult mouse ovary as candidate somatic stem/progenitor cells. The identified population exhibits quiescence with asymmetric label retention, functional response to estrous cycling in vivo by proliferation, enhanced growth characteristics by in vitro colony formation, and cytoprotective mechanisms by enrichment for the side population. Together, these characteristics identify the label-retaining cell population as a candidate for the putative somatic stem/progenitor cells of the coelomic epithelium of the mouse ovary.

Adult Mouse Myometrial Label‐Retaining Cells Divide in Response to Gonadotropin Stimulation

Conditional deletion of β‐catenin in the Müllerian duct mesenchyme results in a degenerative uterus characterized by replacement of the myometrial smooth muscle with adipose tissue. We hypothesized that the mouse myometrium houses somatic smooth muscle progenitor cells that are hormonally responsive and necessary for remodeling and regeneration during estrous cycling and pregnancy. We surmise that the phenotype observed in β‐catenin conditionally deleted mice is the result of dysregulation of these progenitor cells. The objective of this study was to identify the mouse myometrial smooth muscle progenitor cell and its niche, define the surface marker phenotype, and show a functional response of these cells to normal myometrial cycling. Uteri were labeled with 5‐bromo‐2′‐deoxyuridine (BrdU) and chased for up to 14 weeks. Myometrial label‐retaining cells (LRCs) were observed in the myometrium and stroma throughout the chase period. After 12 weeks, phenotypic analysis of the LRCs by immunofluorescence demonstrated that the majority of LRCs colocalized with α‐smooth muscle actin, estrogen receptor‐α, and β‐catenin. Flow cytometry of myometrial cells identified a myometrial Hoechst 33342 effluxing “side population” that expresses MISRII‐Cre‐driven YFP. Functional response of LRCs was investigated by human chorionic gonadotropin stimulation of week 12 chase mice and demonstrated sequential proliferation of LRCs in the endometrial stroma, followed by the myometrium. These results suggest that conventional myometrial regeneration and repair is executed by hormonally responsive stem or progenitor cells derived from the Müllerian duct mesenchyme.

Functional analyses of the cancer stem cell-like properties of human endometrial tumor initiating cells

Recent data suggest that rare stem cell populations with the capacity to self renew and drive tumor formation are a feature of solid tumors. Several investigators have identified putative stem cells from solid tumors and cancer cell lines following isolation of a side population (SP) defined by dye exclusion. We investigated this parameter in our efforts to identify an endometrial cancer (EnCa) stem cell population. Multiple EnCa cell lines were assessed and verapamil sensitive SP and non-SP cells were isolated from two human EnCa cell lines. The functional significance of the SP and non-SP derived from AN3CA was evaluated in vitro and in vivo. SP cells proliferated at a significantly slower rate than the non-SP fraction, and a larger proportion of the SP cells were in G1 phase of the cell cycle as compared to the non-SP fraction. The SP fraction was more resistant to the chemotherapeutic agent paclitaxel. The SP comprised ~0.02% of the initial AN3CA cell population and this proportion of SP cells was maintained within the larger heterogeneous population following repeated passages of purified SP cells. These findings suggest that SP cells derived from the AN3CA cell line have the stem cell properties of low proliferative activity, chemoresistance, and self-renewal. We also tested relative tumor formation activity of the SP and non-SP fractions. Only the SP fraction was tumorigenic. Additionally, we identified SP fractions in primary EnCa. Together these results are consistent with the hypothesis that EnCa contain a subpopulation of tumor initiating cells with stem like properties.

Müllerian inhibiting substance regulates its receptor/SMAD signaling and causes mesenchymal transition of the coelomic epithelial cells early in Müllerian duct regression

Examination of Müllerian inhibiting substance (MIS) signaling in the rat in vivo and in vitro revealed novel developmental stage- and tissue-specific events that contributed to a window of MIS responsiveness in Müllerian duct regression. The MIS type II receptor (MISRII)-expressing cells are initially present in the coelomic epithelium of both male and female urogenital ridges, and then migrate into the mesenchyme surrounding the male Müllerian duct under the influence of MIS. Expression of the genes encoding MIS type I receptors, Alk2 and Alk3, is also spatiotemporally controlled; Alk2 expression appears earlier and increases predominantly in the coelomic epithelium, whereas Alk3 expression appears later and is restricted to the mesenchyme, suggesting sequential roles in Müllerian duct regression. MIS induces expression of Alk2, Alk3 and Smad8, but downregulates Smad5 in the urogenital ridge. Alk2-specific small interfering RNA (siRNA) blocks both the transition of MISRII expression from the coelomic epithelium to the mesenchyme and Müllerian duct regression in organ culture. Müllerian duct regression can also be inhibited or accelerated by siRNA targeting Smad8 and Smad5, respectively. Thus, the early action of MIS is to initiate an epithelial-to-mesenchymal transition of MISRII-expressing cells and to specify the components of the receptor/SMAD signaling pathway by differentially regulating their expression.

Müllerian inhibiting substance preferentially inhibits stem/progenitors in human ovarian cancer cell lines compared with chemotherapeutics

Cancer stem cells are proposed to be tumor-initiating cells capable of tumorigenesis, recurrence, metastasis, and drug resistance, and, like somatic stem cells, are thought to be capable of unlimited self-renewal and, when stimulated, proliferation and differentiation. Here we select cells by expression of a panel of markers to enrich for a population with stem cell-like characteristics. A panel of eight was initially selected from 95 human cell surface antigens as each was shared among human ovarian primary cancers, ovarian cancer cell lines, and normal fimbria. A total of 150 combinations of markers were reduced to a panel of three—CD44, CD24, and Epcam—which selected, in three ovarian cancer cell lines, those cells which best formed colonies. Cells expressing CD44, CD24, and Epcam exhibited stem cell characteristics of shorter tumor-free intervals in vivo after limiting dilution, and enhanced migration in invasion assays in vitro. Also, doxorubicin, cisplatin, and paclitaxel increased this enriched population which, conversely, was significantly inhibited by Müllerian inhibiting substance (MIS) or the MIS mimetic SP600125. These findings demonstrate that flow cytometry can be used to detect a population which shows differential drug sensitivity, and imply that treatment of patients can be individualized to target both stem/progenitor cell enriched and nonenriched subpopulations. The findings also suggest that this population, amenable to isolation by flow cytometry, can be used to screen for novel treatment paradigms, including biologic agents such as MIS, which will improve outcomes for patients with ovarian cancer.

Uterine leiomyomas exhibit fewer stem/progenitor cell characteristics when compared with corresponding normal myometrium

Uterine leiomyomas (also known as uterine fibroids) are the most common benign tumors of female reproductive tract and are the single most common indication for hysterectomies. Despite their high prevalence, the exact pathogenesis of these benign tumors is still unknown. One possible mechanism for leiomyoma formation is dysregulation of mesenchymal stem cell activity. Mesenchymal stem cells have been identified in both human and murine uteri and cancer stem cells have been identified in female reproductive malignancies. We compared stem/progenitor cell characteristics in both normal myometrium and the corresponding leiomyoma of patient’s undergoing hysterectomies. We found that leiomyoma cells form fewer mesenchymal stem cell colonies and exhibit less Hoechst dye-excluding side population (SP) activity, which is a function associated with progenitor cells in other tissues, than cells isolated from normal myometrium. Whereas in normal myometrium, we observed heterogeneous expression of CD90, a cell surface marker associated the with differentiation potential of uterine fibroblasts, in leiomyomas, we observed homogenous expression of CD90, suggesting leiomyoma cells are more terminally differentiated. Furthermore, we found that while leiomyoma cells could only produce CD90 expressing cells, both CD90+ and CD90— myometrial cells could reestablish their original heterogeneous CD90 profile when expanded in vitro. These results suggest that normal myometrium contains cells with stem/progenitor cell activities that are absent in leiomyomas.

Recombinant Human Mullerian Inhibiting Substance Inhibits Long-term Growth of MIS Type II Receptor–Directed Transgenic Mouse Ovarian Cancers In vivo

Purpose: Mullerian inhibiting substance (MIS) is a glycoprotein hormone that causes Mullerian duct regression in male embryos. In short-term experiments, recombinant human MIS (rhMIS) inhibits xenotransplanted human ovarian cancer cell lines that are thought to be of Mullerian origin. Because this highly lethal cancer has a high recurrence rate after conventional chemotherapy, new treatments are warranted. We examined whether rhMIS as a novel, nontoxic, naturally occurring growth inhibitor can be an effective anticancer drug in long-term studies in vivo against allograft tumors that recapitulate human ovarian carcinoma. Experimental Design: Mouse ovarian carcinoma (MOVCAR) cell lines expressing the early region of the SV40 virus, including the large and small T-antigen genes under transcriptional control of a portion of the murine MIS receptor type II (MISRII) gene promoter, were derived from TgMISIIR-TAg transgenic mice. rhMIS was tested against MOVCAR cells in growth inhibition assays in vitro, and in vivo in 6-week-old female nude mice. Tumor growth in animals was measured at weekly intervals for up to 20 weeks. Results: MOVCAR cells and tumors express MISRII by Western blot, immunohistochemical, and Northern blot analyses. rhMIS significantly inhibited MOVCAR cell growth in vitro and in vivo in three separate long-term allotransplantation experiments. Conclusions: Because rhMIS is an effective anticancer agent in in vitro and in long-term in vivo preclinical experiments against MISRII-positive tumors, we predict that rhMIS can be used safely and effectively to treat human ovarian malignancies.