Abstract 4751: Identification of mechanism involved in mesothelial clearance by ovarian tumor spheroids
Authors: Rachel Davidowitz, Marcin Iwanicki, Laura Selfors, Gottfried Konecny, Judy Dering, Dennis Slamon, Joan Brugge
Journal: Cancer Research (2011)

Abstract Metastatic dissemination of ovarian tumor cells to organs in the peritoneal cavity involves the intercalation of dissociated tumor cells into the mesothelial monolayer covering those organs. We established a cell culture model that uses time-lapse video microscopy to investigate the mechanisms involved in the initial interactions between tumor cells and the mesothelium. Tumor cells are cultured in suspension, where they associate into multicellular spheroids. When spheroids are incubated with a mesothelial monolayer, the tumor cells attach to the mesothelial cells and then displace the mesothelial cells and gain access to the underlying substratum. We believe that force generation by the ovarian cancer cells is required for mesothelial clearance, as inhibition of myosin IIA, IIB, or talin I in the ovarian cancer spheroids attenuates mesothelial clearance. To further elucidate the mechanisms by which ovarian cancer cell aggregates clear a mesothelial monolayer, we have taken several different approaches. First, 21 ovarian cancer cell lines were classified according to their ability to clear a mesothelial monolayer. 14 of the cell lines were able to induce clearance in the monolayer, whereas 8 could not. Western blot analysis of the expression levels of several integrins in the 21 cell lines revealed a positive correlation between α5 integrin expression and clearance ability. Furthermore, blocking α5 integrin function, using a function-blocking antibody, significantly inhibited mesothelial clearance in 6 out of 8 cell lines tested. In addition, data from RNA microarrays on the 21 cell lines were analyzed to identify genes that are differentially expressed in intercalation-competent and -incompetent tumor cell lines. The microarray analysis revealed several candidate genes, including: SEPT6, GJA1, VIM, and SLIT2 (up) as well as IL23A, INHIBB and LCN2 (down). The functional importance of these genes, and others, in mesothelial clearance is currently being investigated through loss-of-function and gain-of-function genetic manipulations. Finally, we have initiated a targeted RNAi screen using one of the ovarian cancer cell lines, OVCA433, that aggressively clears the mesothelial monolayer. We plan to include siRNAs that were shown to regulate migration in a previous screen our lab had performed, as well as, other siRNAs directed against genes known to be involved in migration. In a pilot screen using 54 siRNAs, we found several genes that decrease mesothelial clearance, including EPHB2, IGFR1, and MAP3K11. We are expanding the screen to include genes that are differentially expressed in clearance competent and incompetent ovarian tumor cell lines. The hits form these screens will be validated and characterized to identify the cellular pathways that regulate mesothelial clearance. Lastly, we will determine, in vivo, if these genes regulate mesothelial invasion in a mouse model of peritoneal metastasis Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4751. doi:10.1158/1538-7445.AM2011-4751