Immuno-modulation of macrophages by ovarian cancer stem cells in an in vitro 3D spheroid platform
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AbstractThe ascites microenvironment of ovarian cancers is enriched in ovarian cancer stem cells (OvCSCs) and macrophages (MPs). The immune system is complicit in the progression, chemoresistance and metastasis of ovarian cancer, yet little is known about the exact nature of interactions between OvCSCs and MPs, especially in the malignant ascites microenvironment. Here, we describe the use of the 3D in vitro hanging drop spheroid platform to study the reciprocal interactions between OvCSCs and MPs. Hetero-spheroids contained: i) monocyte-derived Macrophages (MP); and ii) OvCSCs (ALDH+ CD133+ OVCAR3). For immuno-modulation, we characterized MP polarization markers by flow cytometry and gene expression analysis. Using qPCR, we quantified changes in IL6, IL10, IL12, and Wnt7A, to understand signaling involved in OvCSC/MP hetero-spheroids. CD68 expression (~50%) was observed in OvCSC/MP hetero-spheroids, indicating the presence of MPs. OvCSCs drove higher CD206 expression in MPs (75%) when compared to unsorted ovarian cancer cells (OvCa; 40%). Even if MPs were pre-polarized to inflammatory M1 (HLA-DR+ CD206-), co-culture with OvCSCs reversed the M1 phenotype, and strongly induced CD206 expression. Pre-polarized M2 MPs resulted in 2-fold increase in ALDH+ cells in hetero-spheroids. Apart from CD206, MPs in hetero-spheroids also expressed iNOS, IFN-, and CD163. Gene expression analysis revealed increased immuno-suppressive IL10 in hetero-spheroids, with more IL10 in OvCSC/MP (3.5 fold) compared to OvCa/MP. OvCSC/MP and OvCa/MP spheroid also had consistently elevated (2-3 fold) increase in IL6 gene expression, consistent with the importance of the IL6/STAT3 axis in both cancer stem cell and macrophage signaling. Lastly, Wnt7A was significantly elevated in both differentiated MPs, and OvCSC/MP spheroids, indicating reciprocal trophic and immuno-suppressive signaling components at play. The 3D spheroid model can simulate biological interactions between OvCSCs and MPs within the malignant ascites. We describe that OvCSCs primarily polarize macrophages into a mixed phenotype that express both inflammatory and pro-tumoral markers. We believe that the putative involvement of Wnt7a in OvCSC/MP interactions drives this phenotype, and could have functional implications for tumorigenicity, response to chemotherapeutics and metastatic potential. This work was supported by the DOD OCRP W81XWH-13-1-0134, MIOCA, Rivkin Center for Ovarian Cancer and NIH T32DE00007057-41 and P30CA046592.Citation Format: Shreya Raghavan, Geeta Mehta. Immuno-modulation of macrophages by ovarian cancer stem cells in an in vitro 3D spheroid platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 118.
