Ascites Microenvironment Facilitates Ovarian Cancer Peritoneal Dissemination

Objective:The purpose of this study was to identify the particular gene phenotype of ascitic tumor cells compared with that of the paired cancer cells from either primary or metastatic tumor site in advanced serous ovarian cancer patients. And further into the mechanism by which the ascites microenvironment influence of the phenotype and malignant behavior of ascitic tumor cells.Methods:After magnetic sorting of epithelial tumor cells from paired primary tumor, omental tumor as well as ascities, adhesion assay was performed to compare the adhesive capacity of these pure tumor cells, invasion assay was done to show the relative invasive capability, intraperitoneal implantation was carried out to check their peritoneal dissemination ability. High-throughout Gene-profiling was performed in5pairs of purified tumor cells to differentiate the gene patterns and conclude the pathway enrichment. Suspension culture in ascites was done to compare the influence of cancer cell phenotype exerted by malignant ascites. In in-vivo peritoneal metastasis model of ovarian cancer, the strategies targeting the malignant cancer cells or other cellular components were performed to demonstrate their effects in inhibiting the diseses exacerbation.Results:The ascitic tumor cells presented the most aggressive adhesive, invasive and intra-peritoneal tumor-formation capacity in immunodeficiency mice. The mRNA microarray data revealed that the ascitic tumor cell phenotype was notably different with that of the paried primary and metastatic tumor cells, and the highest differentially expressed genes were enriched in celluar adhesive pathway. Subsequent analysis showed that Integrin family member integrin a5(ITGA5) was paticulary higher in ascites tumor cells. Interestingly, the ITGA5high phenotype tumor cells reprented the mesenchymal characteristic sub-population and contributed to the peritoneal dissemination process. The cytokines in malignant ascites especially EGF and TGF-β1elevated the expression of ITGA5in ascitic tumor cells. In addition, malignant ascites faciliated tumor cells and cancer-associated fibroblast (CAF) cells in ascites to form compact spheroid architecture, which resulted in the promotion and maintainance of ITGA5high phenotype. The intervention of Fn mimics CH50could specifically bind those ITGA5high phenotype cells presented in the ascites and further retarded ovarian cancer peritoneal metastasis. Morover, the strategy against CAFs in the ascites with imatinb, could abrogate the influence of CAFs on ascitic tumor cells and finally attenuated the peritoneal propagation process of ovarian cancer.Conclusions:The malignant behavior assay and high-throughput microarray data unanimously figured out that the ascitic tumor cells reprented the most aggressive phase during ovarian cancer progression from primary tumor to newly formed metastasis. And the celluar component as CAFs and acellular ingredient as massive cytokines in the ascites microenvironment mediated and maintained the particular malignant phenotype of tumor cells presenting in the ascites. In consideration of the pivotal role of ascites tumor cells in ovarian cancer disease exacerbation, it’s feasible that strategy targeting the malignant tumor cells suspending in the ascites and measures againt the microenvironment that feed those cells could cast some shine in controlling peritoneal metastasis of ovarian cancer. Objective:Advanced Ovarian cancer is characterized by massive intraperitoneal dissemination and malignant ascites. The carcinoma cells exfoliated from the ECM and multi-cellular spheroids are formed fundamental to metastatic dissemination. Activation of EGFR signaling is involved in increased cell metastasis and decreased apoptosis of ovarian cancer. This study investigated the effect of EGFR inhibition in targeting the tumor cells spheroids in ovarian cancer.Materials and Methods:Cell Counting kit-8assay was used to detect cell proliferation and examine cell viability after various treatments. In vivo and in vitro adhesion assay was performed to check the adhesive capacity of cells to ECM. Immunoblotting, immunohistochemstry or Immunofluorescence was performed to detect the protein expression. Autophagosome assay was detected to compare the status of autophagy. KMplot online analysis was performed to predict the clinical impact of EGFR and autophagy markers in ovarian cancer. Pearson correlation analysis of EGFR and autophagy markers in RNA-sequencing data from The Cancer Genome Atlas (TCGA) was performed. The cells in different cell cycle phases were detected by flow cytometry. Tumor cell proliferation was measured by colony formation assay in vitro. Tumor growth was monitored by bioluminescence imaging in vivo, using an i.p. xenograft model of ovarian cancer.Results:The obtained sphere cells acquired elevated proliferation, adhesion, invasion capacity and showed tolerance to typical chemotherapeutical agents of ovarian cancer. Similarly, OC sphere cells were resistant to EGFR inhibition and autophagy was particularly induced in sphere cells after AG1478treatment. Tumor cells suspended in the ascites presented the highest basal level of autophagy and were relatively chemoresistant to EGFR inhibition, which was accompanied with the escalated autophagy response. EGFR and autophagy related genes acted as poor outcome prediction genes, they presented a negative correlation relationship.3MA amplified the cytotoxicity of AG1478in ovarian cell spheroids in vitro. Correspondingly, in the i.p. xenograft model of ovarian cancer,3MA facilitated the effect of AG1478in controlling the tumor peritoneal dissemination.Conclusions:The tumor cells detached from ECM were in relative high status of autophagy, and EGFR inhibitio accelerate the autophagy response. Targeting autophagy held the potential to improve EGFR inhibition benefit in the treatment of ovarian cancer cells during detachment from ECM, and that this combination strategy might provide a new treatment option in controlling peritoneal metastasis of ovarian cancer.