Formation Of Vasculogenic Mimicry Determined By Plasticity Of Ovarian Cancer Cells In Vivo And Effects Inhibited By Rapamycin And Sulindac

Epithelial ovarian cancer has the worst prognosis of all gynecological cancers andis a leading cause of death from cancer in women. Recent study has found that someaggressive tumor cells generate vasculogenic-like channels in the absence ofendothelial cells or fibroblasts. The formation of the patterned microeirculation istermed vasculogenic mimicry. Ovarian cancer cells have been recently reported toexpress endothelium-associated genes to form de novo vasculogenic-like networks inthree-dimensional culture. Rapamycin, HIF-1αinhibitor, prevented formation ofvasculogenic mimicry in vitro in our preliminary study. However, little is knownabout vasculogenic mimicry of ovarian cancer in vivo.In the present study, female nude mice were implanted intraperitoneally withGFP-labled ovarian cancer cells. Tumor tissues were studied by H&E staining,electron microscopy and fluorescence microscopy. Expression of vascular epitheliummarkers were investigated by immunohistochemical staining, immunofluorescenceassay, RT-PCR, western blot and flow cytometry. These experiments were dividedinto 3 parts, as following: 1. Formation of vasculogenic mimicry determined byplasticity of ovarian cancer cells in vivo; 2. Correlation between vasculogenicmimicry in vivo and expression of HIF-1α/VEGF; 3. Effects of rapamycin andsulindac on plasticity of ovarian cancer cells and formation of vasculogenic mimicryin vivo.SectionⅠFormation of vasculogenic mimicry determined by plasticity ofovarian cancer cells in vivoObjective: To determine the plasticity of human epithelial ovarian cancer cells and formation of vasculogenic mimicry in vivo.Methods: Lentiviral vector carrying green fluorescence protein(GFP) transducedSKOV3ip and ES-2 cells. GFP-labled tumor cells were implanted into peritonealcavity of nude mice. When the transplanted tumor reached a volume of approximately1 cm~3, tumor tissues were studied by H&E staining, electron microscopy andfluorescence microscopy. Expression of vascular cell markers CD31,factorⅧ,VEGF were detected by flow cytometric analysis after primary cell culture. CD31was also investigated by immunochemical staining, immunofluorescent method,RT-PCR, western blot. DiL-acLDL was incubated with the primarily cultured tumorcells.Results: Ovarian cancer cells in vivo formed patterned networks with erythrocytes inthem. CD31 and factorⅧ, but negative VEGF were detected in SKOV3ip by flowcytometric analysis. CD31, factorⅧand VEGF were detected in ES-2 cells by flowcytometric analysis. CD31mRNA and protein were present in both SKOV3ip andES-2 cells. A little ES-2 cells were capable of uptaking DiL-acLDL.Conclusions: The present study has shown that human ovarian cancer cell lines maybe able to express some specific markers of vascular epithelial cells, and haveplasticity to form vasculogenic mimicry in vivo.SectionⅡcorrelation between vasculogenic mimicry in vivo and expression ofHIF-1α/VEGFObjective: To detect HIF-1α/VEGF expression in the center area and the marginalpart of the transplantable ovarian cancer of nude mice and to investigate the possiblecorrelation between hypoxia and vasculogenic mimicry in vivo.Methods: The transplantable ovarian cancer model of nude mice was established.The tumor tissue without necrosis in the center area and the marginal part wereobtained respectively. Serial sections were cut and stained with H&E. HIF-1α/VEGFmRNA was detected by realtime PCR. HIF-1α/VEGF protein was detected byimmunochemical staining.Results: Tumor cells-lined channels were observed in the center area of the ovariancancer tissue, in the absence of endothelial cells. In the marginal part of the tumortissue, only endothelial cells-lined vascular networks were observed. The level of HIF-1α/VEGF mRNA and protein was significantly higher in the center of the tumortissue than those in the marginal area (P＜0.05). A significant positive correlationbetween HIF-1αprotein and formation of vasculogenic mimicry was found bySpearman's coefficient of correlation (P＜0.05).Conclusions: HIF-1αexpression in the center part of the tumor tissue might berelated to vasculogenic mimicry through upregulating VEGF. Vasculogenic mimicrymight be a pathway for ovarian cancer perfusion to adapt hypoxic microenvironment.SectionⅢEffects of rapamycin and sulindac on plasticity of ovarian cancercells and formation of vasculogenic mimicry in vivoObjective: To investigate the effects of rapamycin and sulindac on phenotypetransformation of ovarian cancer cells and vasculogenic mimicry in vivo.Methods: The transplantable ovarian serous adenocarcinoma model of nude micewas established and divided into 6 groups. Rapamycin and sulindac were injected intothe tumor model 24h after implantation of tumor cells and after the transplantedtumor reached a volume of approximately 1 cm~3, respectively. The negative controlgroup were injected with saline. The DDP control group were injected with DDP.Tumor tissue were subjected to histological examination. Expression of CD31, factorⅧwere detected by flow cytometric analysis. HIF-1α, VEGF and MMP2 mRNAwere detected by RT-PCR. HIF-1α, VEGF and MMP2 protein were detected bywestern blot.Results: Rapamycin injected at early time could significantly inhibited the growth oftransplanted tumor. The level of HIF-1α/VEGF mRNA and protein was reducedsignificantly (P＜0.05). The tumor cells-lined channels could not been observed andexpression level of CD31 and factorⅧwere down-regulated significantly(P＜0.01).But rapamycin injected at later time could not significantly reduce the level ofHIF-1α/VEGF mRNA and protein. Sunlindac used early could significantly inhibitedthe growth of transplanted tumor and the tumor cells-lined channels could not beenobserved. The level of VEGF and MMP2 expression were down-regulatedsignificantly ( P＜0.05). Level of HIF-1αprotein expresson showed correlation withCD31 (P=0.025, r=0.733) and factorⅧexpression(P=0.047, r=0.997).Conclusions: Rapamycin, HIF-1αinhibitor, down-regulated phenotype transformation of ovarian cancer cells and vasculogenic mimicry in vivo. Sulindac,MMP2 inhibitor, might inhibited vasculogenic mimicry. MMP2/VEGF regulationcapacity of sulindac declined when used at later time.In summary, we demonstrated evidence for some epithelial-related phenotypes ofthe aggressive human epithelial ovarian cancer cells in the hypoxic center area of theovarian cancer tissue, suggesting that tumor cell plasticity might allow vasculogenicmimicry to occur. Rapamycin could down-regulate phenotype transformation ofovarian cancer cells and vasculogenic mimicry in vivo. Sulindac, MMP2 inhibitor,might inhibit vasculogenic mimicry. The plastic cell phenotype poses challenge foridentifying and targeting ovarian cancer cells that could masquerade as other celltypes.