Abstract Ovarian cancer is the most lethal gynecological malignancy in the United States, with a known predilection for metastasizing to the omentum, a sheet of fatty tissue that encloses the abdomen. While it has been shown that ovarian cancer cells invade towards mature adipocytes in vitro, the contribution of stromal cells that reside in the omentum is not well understood. We hypothesize that preadipocytes, the precursor cells that give rise to omental adipocytes, are important mediators of cancer progression in the omental tumor microenvironment as they have a pro-inflammatory secretome that is distinct from that of mature adipocytes. To evaluate this hypothesis, we tested whether preadipocyte secreted factors altered tumorgenicity of ovarian cancer cells using colony formation and cell viability in vitro and a limiting dilution in vivo in a subcutaneous mouse model. The mouse studies showed that low dilutions of cancer cells require preadipocytes for engraftment and tumor formation. A transplantation assay showed that cancer cells require the presence of preadipocytes for sustained tumor formation capacity. In vitro co-culture assays revealed that preadipocytes secrete factors that increase clonogenicity and extend cell viability in serum-free conditions. To identify signaling pathways induced by preadipocytes we co-cultured cancer cells with either primary human omental preadipocytes or differentiated mature adipocytes derived from the same female donor and performed RNA sequencing and gene set enrichment analysis (GSEA). We identified differentially expressed genes in cancer cells that were unique to preadipocyte co-culture, including DCN, MMP2, COL6A2 and COL12A1, genes involved in extracellular matrix organization. The most significantly upregulated gene was insulin-like growth factor binding protein 5 (IGFBP5), which has functions in insulin-like growth factor signaling and ECM interactions. Ongoing studies using CRISPR edited cancer cells suggest IGFBP5 mediates the enhanced tumorigenesis of cancer cells in the presence of preadipocytes. Our findings highlight the role of preadipocytes in the ovarian tumor microenvironment and implicate IGFBP5 as a preadipocyte-mediated gene. Future studies will determine if there is a link between the upregulation of IGFBP5 and modulation of extracellular matrix proteins that promote cancer progression. Understanding these processes will enable the design of more effective therapies for treating ovarian cancer. Citation Format: Jennifer A. Waters, Mikella Robinson, Samuel F. Gilbert, Ixchel Urbano, Carrie D. House. Omental preadipocytes support ovarian cancer tumorigenesis by mediating genes important for extracellular matrix reorganization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2535.