Spontaneous malignant transformation of ovarian surface epithelial cells correlates with EMT alteration and stemness acquisition [post]

2019 unpublished
KEYWORDS 2 epithelial-mesenchymal transition, cancer stem-like cells, ovarian surface epithelial, malignant transformation, epithelial ovarian carcinomas 3 Abstract Background In order to complete the physiological function of postovulatory repair during repeated ovulation, the ovarian surface epithelium (OSE) not only has to undergo epithelialmesenchymal transition (EMT), but also possess the properties of somatic stem-like cells. However, there is no evidence to indicate that both EMT
more » ... at both EMT alteration and stemness acquisition are linked to epithelial ovarian carcinomas.Methods In this study, we established a cell model of spontaneous oncogenic transformation of mouse OSE (MOSE). The cell proliferation was assessed using clonogenic survival and soft agar. The self-renewal of cancer stem-like cells (CSCs) was determined by spheroid culture. CD44 + /CD117 + cells were analyzed using flow cytometer. The PCR array was used to determine the EMT-related mRNA level. Expression of pan-keratin, vimentin, Ecadherin, Snail1 and Slug were detected using western-blotting and immunofluorescences, respectively. The tumorigenesis were monitored by limiting dilution assay in vitro and in vivo . Results Based on morphological change, chromosomal number and proliferating ability, we defined three sequential stages of transformed cells as early, intermediate and late MOSE cells, respectively. We found that MOSE cells had dual characteristics of not only epithelial but mesenchymal nature as well. Over time, MOSE cells spontaneously developed characteristics of malignant cells and generated tumor nodules expressing both Pankeratin and Vimentin. Furthermore, we found that the neoplastic transformation of MOSE was accompanied by continuous EMT-inducing signals including Snail1 and Slug. Concurrently, the increase of CD44 + /CD117 + cells and their self-renewal ability were associated with the progression of spontaneous neoplastic transformation of MOSE cells in vitro. Conclusion et al [1-3], and modified by us. Briefly, ovaries from female C57BL/6 mice (Experimental Animal Center for Soochow University, ) were resected and, were incubated for 20 min in Dulbecco's Modified Eagle's Medium/Nutrient Mixture F-12 (DMEM/F12, Invitrogen, Carlsbad, CA) supplemented with 0.25% trypsin (Invitrogen, Carlsbad, CA). Single cells and clumps of MOSE were collected, resuspended in MOSE cell growth medium, and seeded onto 35 mm dishes in a humidified atmosphere of 5% CO 2 at 37 ℃. The MOSE cell growth medium consisted of DMEM/F12 medium supplemented with 5% fetal bovine serum (FBS), 10 ng/ml mouse epidermal growth factor (mEGF), 100 U/mL penicillin and 100 µg/mL streptomycin (Beyotime Biotechnology, shanghai, China), 1% insulin-transferrinselenium (Invitrogen, Carlsbad, CA). During early passages of cells (pass 1 through 20), medium was changed with the mixed medium containing a 1:1 of the fresh growth medium and culture supernatant. Intermediated (pass 21-90) and late passage (great than 90) cells were routinely passaged. This animal experiment was performed according to the protocols of the Institutional Animal Care and Use Committee of the Soochow University (IACUCSU). Cell proliferation assay Cell proliferation was detected by Cell Count Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). In brief, cells were plated in 96-well plates at a density of 1000 cells per well and cultured in growth medium. At specific points in time after plating (0, 7, 24, 48, 72, 96, and 120h), the cell counts were determined according to the manufacturer's instructions. For clonogenic survival assay, five hundreds cells were seeded into dishes 35 mm in diameter. After two weeks, the colonies formed were fixed with formaldehyde, stained with Giemsa, and counted using a colony counter soft at GBOX XR-5 (Gene Company Limited, HongKong, China). The plating efficiencies (PE) were determined using the 7 following formula: PE (%) = number of colonies formed / 500 plated cells ×100%. All data points in figures represent three independent experiments. Soft agar colony formation assay Two thousand cells were suspended in 1 mL of 0.35% agarose in the growth medium, plated into 35 mm dishes with a bottom layer of 0.7% agarose. Cells were fed every 3 days with 1 ml growth medium. Colonies (>15mm) were counted two or three weeks after seeding. Data are presented as CFU (percent colony forming units in soft agar assay) = mean of the number of colonies per dish/number of cells seeded per dish ´ 100%. Data points in figures represent three independent experiments. In vivo tumorigenicity assays Ten female BALB/c nude mice (SPF, 4-6weeks, 18-20g) were bought from Experimental Animal Center for Soochow University, and they were housed under temperaturecontrolled conditions, underwent a reverse dark-light cycle, and provided with standard mouse pellets and tap-water ad libitum. The mice were randomly divided into three groups (n=3) including M-E group, M-I group and M-L group for injection MOSE cells. After animals were lightly anesthetized with isoflurane, 5×10 6 of cells were injected subcutaneously into the left and right flanks of each animal. Animals were palpated weekly for tumor formation. Tumor size was measured once per week and tumor volume was calculated using the formula, tumor volume = 0.5 × length × width 2 . Animals were killed as soon as tumor nodules reached a size of 1.2-1.5 cm. The euthanasia/sacrifice of the mice was used by excessive 3% pentobarbital sodium (0.1 ml/10 g), following cervical dislocation to ensure the death of mice. This animal experiment was performed according to the protocols of the IACUCSU. PCR Array Conclusions In conclusion, we established a model representing early, intermediate, and late stages of neoplastic transformation of ovarian surface epithelium. Alteration of EMT and acquisition of stemness are closely correlated to spontaneously oncogenic transformation of OSE cells. The model is a powerful tool for in vitro mechanistic studies, as well as for efficacy studies in vivo of both chemotherapeutic treatment regimens and chemopreventive strategies. Abbreviations CSCs: cancer stem-like cells; EMT: epithelial-mesenchymal transition; MOSE cells: mouse ovary surface epithelial cells; M-E: early stage of MOSE cells; M-I: intermediated stage of MOSE cells; M-L: late stage of MOSE cells. Declarations Ethics approval and consent to participate The performance of the animal experiments followed the 3R principle, in order to minimize the number of animals recruited for the experiments and to reduce their burden. All animal experiment was performed according to the protocols of IACUCSU. The approval number is ECSU-201800049. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. clinicians 2013, 63(1):11-30. 2. Bast RC, Jr., Hennessy B, Mills GB: The biology of ovarian cancer: new opportunities for translation. Nature reviews Cancer 2009, 9(6):415-428. 3. 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doi:10.21203/rs.2.15828/v2 fatcat:3zwqvwvwjzc6vegr36i7wi6zm4