Abstract Background: Androgens stimulate androgen receptor (AR) signaling, driving prostate cancer (PC) cell proliferation and progression. Androgen deprivation therapy (ADT) effectively treats primary disease, but 20-30% of patients relapse after ~5 years, resulting in castration-resistant prostate cancer (CRPC), which has limited treatment options. We have previously shown in a murine model of CRPC that macrophages promote AR signaling in CRPC and that depleting tumor macrophages extends survival in response to ADT with Lupron. This correlates with reduced androgen levels in macrophage-depleted tumors and the ability of macrophages to transfer cholesterol, the anabolic precursor of androgens, to prostate cancer cells. However, the mechanism by which this transfer occurs, and whether cholesterol exchange mediates resistance to ADT, is currently unknown. Methods: To assess cholesterol transfer from macrophages to cancer cells, we established a culture system involving prostate cancer cell lines and either the RAW264.7 macrophage cell line or bone marrow-derived macrophages (BMDMs). Macrophages were pre-loaded with cholesterol using fluorescently labeled low-density lipoprotein (LDL) and were co-cultured or placed in a transwell system with tumor cells for 24 hours. Cholesterol transfer was then measured by flow cytometry. Results: Fluorescent signal from LDL was detected in 50-70% of tumor cells after 24 hours of co-culture. Cell contact was required for cholesterol transfer, as no fluorescence was detected in prostate cancer cells during transwell experiments. Macrophages were also able to internalize and transfer acetylated LDL, which is poorly taken up by tumor cells compared to unmodified LDL. Macrophage polarization with interleukin-4 did not impact cholesterol transfer and uptake of acetylated LDL occurred independently of the scavenger receptor CD36. The scavenger receptor SR-B1, which is often upregulated in prostate cancer cells, was dispensable for transfer, as knocking out SR-B1 via CRISPR/Cas9 in cancer cell lines did not reduce LDL uptake. Conclusion: Cholesterol transfer between macrophages and prostate cancer cells occurs in a contact-dependent manner, independently of the scavenger receptors most commonly associated with cholesterol accumulation. Citation Format: Olabisi Osunmakinde, Brian Ruffell. Investigating cholesterol transfer between macrophages and prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2881.