| Objective: The simple squamous-to-cuboid single-layered epithelial cell structureof human ovarian surface epithelium (OSE) plays an important physiological roleduring ovulation. The OSE can transform back and forth between epithelial andmesenchymal phenotypes in both follicular rupture and subsequent ovarian remodeling.The fact that the OSE at junction areas, the transitional area between the OSE,mesothelium and oviduct epithelium, contains a stem cell niche that is responsible forOSE regeneration and is prone to malignant transformation was recently identified.About90%of epithelial ovarian carcinomas originated from OSE. Therefore, thehypothesis that “acquisition of mesenchymal and stem cell-like properties involved inthe spontaneous malignant transformation of OSE” was proposed in the study. Takingadvantages of established model on spontaneous malignant transformation of mouseovarian surface epithelial (MOSEC) model, we investigated the alteration ofepithelial-mesenchymal transition (EMT) and stem cells-like properties. The cell andmouse models generated in this work provide new tools for the studies of mechanismsunderlying human ovarian cancer progression.Methods: The mainly work of this study included three sections.(1) Theestablished model of spontaneous malignant transformation of MOSEC: MOSECwere obtained from virgin, mature mice by mild trypsinization and were repeatedlypassaged in vitro. Pan-keratin, an epithelial cell marker, was used to identify MOSEC.A set of experiments including growth curve, plating efficiency andanchorage-independent growth was used to assess the proliferative characteristics ofMOSEC. And cell morphology and the chromosomal number were observed. The oncogenic transformation of MOSEC was further confirmed by tumorigenicity in vivo.(2) The EMT involved in spontaneous oncogenic-transformation of MOSEC:Expression level of mRNA was verified by RT2Profiler PCR Array Mouse EMT kit.The expression of epithelial marker (E-cadherin and Pan-keratin) and mesothelialmarkers (Vimentin, Snail, Slug and β-catenin) was examined by theimmunofluorescence (IF) and Western Blotting assay.(3) The stem cell-likeproperties contributed to spontaneous oncogenic transformation of MOSEC:Stem cell/cancer stem cell markers including CD44, CD117, CD133, Oct-4andNanog were examined using IF and flow cytometry. The capacity of self-renew wasdetected using sphere forming and limiting dilution assay. The properties ofdifferentiation were observed by adhesion of sphere forming cells in10%FBSDMEM/F12medium with non-mEGF or mbFGF. Tumorigenicity of sphere-formingcells was confirmed in vivo.Results:(1) The established model of spontaneous malignant transformation ofMOSEC: According to the alteration in morphology, proliferation and chromosomalnumbers, the progress of MOSEC transition was divided into three distinct stagesmainly: early (P4-P15), intermediated (P20-P85) and late (>P90). The percentage ofMOSEC obtained from mouse ovary was more than90%. Primary MOSEC wasrepeatedly passaged in vitro after the early5-10passages as difficult period. MOSECwas exhibited a typical epithelial cobblestone-like appearance, and formed the closedislands in early passages. Subsequently, MOSEC gradually converted to morespindle-like morphology and formed semi-closed islands and opened-islands in latestage. The malignant foci were appeared at later. Growth curve showed thatproliferation of MOSEC was significantly accelerated in intermediated passages,especially late. Plating efficiency was significantly increased from0%,7.33%to36.00%in late stage (P<0.05); MOSEC in late passages also increasedanchorage-independent growth with1.35%(P<0.05); Chromosome analysis showedthat chromosomal number at different stages was49,58and64, respectively, and theproportion of ultra-tetraploid chromosomal number was increased in the late passages. Tumorigenicity was increased by80%in MOSEC at late stages. These resultssuggested that the spontaneous oncogenic-transformed model was established, and weobserved distinct transitional stages when the MOSEC progressed from a benignphenotype to malignant phenotype. Late-passage MOSEC displays higher tumorigenicpotential in vivo.(2) The EMT involved in spontaneous oncogenic-transformation of MOSEC:MOSEC exhibited a typical epithelial cobblestone-like appearance in early stage, whileit converted to spindle-like morphology in intermediated and late passages. MOSECwas positive for Pan-keratin and Vimentin during long-term culture in vitro, whichindicated that MOSEC had a dual nature, expressing both epithelial markers but alsomesenchymal markers. PCR Array showed that expression of EMT-inducingtranscription factor, specifically Cav2, Cald1, Wnt11and Snail, was markedlyincreased, while the level of mRNA down-regulated during EMT, including Krt19andTspan13, were strongly reduced in progresses of malignant transformed-MOSEC. Theresults of IF and Western Blotting showed a significant up-regulation of Snail1andSlug in oncogenic-transformed MOSEC, compared with their early-stage cells. And weobserved that MOSEC displayed mesenchymal-appearance in intermediate stage,along with up-regulation of E-cadherin and β-catenin. The results indicated that theoncogenic-transformation of MOSEC was accompanied with continuousEMT-inducing signals, specially a notable increase of Snail and Slug.(3) The stem cell-like properties contributed to spontaneous oncogenictransformation of MOSEC: MOSEC in early passages had expressed CD44+,CD117+and CD133+, three cell-surface markers associated with both ovarian cancerstem cells and normal epithelial stem cell. Flow cytometer analysis showed that theproportion of CD44+and CD117+cells was increased with the progresses ofoncogenic-transformed MOSEC. Interestingly, MOSEC in intermediated stageexpressed Nanog, embryonic stem cells/cancer stem cell marker; Frequency of sphereforming by MOSEC in early, intermediated and late stage was0,0.14%and0.69%,respectively (P<0.05). And sphere-forming rate in cells for five consecutivegenerations was markedly higher than that in MOSEC at late stage. Limiting dilution assay suggested that one tumor sphere was generated by96.30MOSEC. The spheroidcells re-attached to the plastic and changed into adherent cells morphologically atserum-containing DMEM/F12medium, suggesting that spheroid cells have thecapacity of multi-differentiation. In addition, CD44expression in subcutaneous tumorsin spheroid cells groups was higher than that in MOSEC at late passage. These resultssupported experimentally the hypothesis that spontaneous malignant transformation ofMOSEC may be explained by the presence of stem cell or cancer stem cell in ovariansurface epithelium. The spheroid cells formed by oncogenic-transformed MOSEC havethe characteristics of self-renew and differentiation, indicated that the stem cell-likeproperties contribute to spontaneous oncogenic transformation of MOSEC.Conclusions:1. We established the spontaneous oncogenic-transformed model and observeddistinct transitional stages when the MOSEC progressed from a benign phenotype tomalignant phenotype. Late-passage MOSEC displayed higher tumorigenic potential invivo.2. The MOSEC exhibited a typical epithelial-like morphology in early andconverted to mesenchymal-appearance in intermediate and late passages. Theyexpressed an increase in the EMT-inducing transcription factors Snail1and Slug,indicated that EMT may account for progresses of spontaneous oncogenic-transformedMOSEC.3. The spontaneous oncogenic transformation of MOSEC may be explained bythe presence of stem cell or cancer stem cell in ovarian surface epithelium. Thespheroid cells formed by oncogenic-transformed MOSEC appeared the characteristicsof self-renew and differentiation, indicated that the stem cell-like properties contributeto spontaneous malignant progresses of MOSEC. |
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