Ovarian Cancer Cells Direct Monocyte Differentiation Through a Non-canonical Pathway

Abstract

Background: Alternatively-activated macrophages (AAMs), an anti-inflammatory macrophage subpopulation, have been implicated in the progression of high grade serous ovarian carcinoma (HGSOC). Increased levels of AAMs are correlated with poor HGSOC survival rates, and AAMs increase the attachment and spread of HGSOC cells in vitro. However, the mechanism by which monocytes in the HGSOC tumor microenvironment are differentiated and polarized to AAMs remains unknown. Methods: Using an in vitro co-culture device, we cultured naïve, primary human monocytes with a panel of five HGSOC cell lines over the course of seven days. An empirical Bayesian statistical method, EBSeq, was used to couple RNA-seq with observed monocyte-derived cell phenotype to explore which HGSOC-derived soluble factors supported differentiation to CD68+ macrophages and subsequent polarization towards CD163+ AAMs. Pathways of interest were interrogated using small molecule inhibitors, neutralizing antibodies, and CRISPR knockout cell lines. Results: HGSOC cell lines displayed a wide range of abilities to generate AAMs from naïve monocytes. Much of this variation appeared to result from differential ability to generate CD68+ macrophages, as most CD68+ cells were also CD163+. Differences in tumor cell potential to generate macrophages was not due to a MCSF-dependent mechanism, nor variance in established pro-AAM factors. TGFa was implicated as a potential signaling molecule produced by tumor cells that could induce macrophage differentiation, which was validated using a CRISPR knockout of TGFA in the OVCAR5 cell line. Conclusions: HGSOC production of TGFa drives monocytes to differentiate into macrophages, representing a central arm of the mechanism by which AAMs are generated in the tumor microenvironment.