Abstract 7277: Development of a macrophage-based screening pipeline to evaluate the immunomodulatory effects of small molecule inhibitors

Abstract Introduction: Immunotherapies targeting adaptive immunity have shown promising results in solid tumors; however, many patients are either unresponsive or develop resistance. Emerging interest in innate immunity has driven novel strategies to target myeloid cells, including tumor-associated macrophages (TAMs). TAMs primarily have an anti-inflammatory, pro-tumoral M2-like phenotype secreting growth and angiogenic factors to promote an immunosuppressive tumor microenvironment (TME). Modulating the TAMs towards a more proinflammatory M1-like elicits anti-tumor responses with enhanced antigen presentation and phagocytic activity. Therefore, understanding the molecular mechanisms driving macrophage phenotype plasticity is essential to maintaining the phenotypic switch from M2-like to M1-like in an immunosuppressive TME. Current high-throughput pipelines for screening and optimizing anticancer drugs focus on the cytotoxic and antiproliferative effects of these compounds, underestimating or ignoring their immunomodulatory effects. In this study, we developed drug screening pipelines to identify small molecule inhibitors impacting the macrophage functional phenotype to identify leading candidates with immunoregulatory properties. Methods and Analysis: Mouse bone marrow-derived macrophages were polarized to M1-like or M2-like phenotypes and applied in bulk RNA sequencing to identify specific genes associated with each phenotype. We created stable RAW264.7 macrophages expressing luciferase under the control of phenotype-specific promoters to identify targets modulating the macrophage phenotype. Additionally, THP-1 reporter cell lines driven by response elements of inflammatory signaling pathways were used to further evaluate the mechanistic role of small molecule inhibitors on the phenotypic switch in macrophages[MS1] following stimulation of the pathway with appropriate cytokines. Results and Conclusions: RNA sequencing data identified exclusive genes upregulated with M1 and M2 polarization of macrophages. Based on the gene expression changes and successful validation of the plasmids in RAW264.7 cells, we selected TNF and CXCL5 as genes for M1 and Arg1 and CCL24 for M2 markers. Using the stable reporter RAW264.7 cell lines and the THP-1 reporters for specific pathways, we screened several epigenetic modifiers, including novel HDAC inhibitors. Using this approach, we found selective HDAC6 inhibitors diminished the anti-inflammatory phenotype of macrophages and, upon stimulation with appropriate cytokines, increased pro-inflammatory pathways, such as NFkB. These efforts could enable high-throughput screenings of novel small molecule inhibitors for their immunomodulatory effect on macrophages. Citation Format: Marie Durr, Manasa Suresh, Bryan Weselman, David Quiceno-Torres, Megan Winakur, Satish Noonepalle, Alejandro Villagra. Development of a macrophage-based screening pipeline to evaluate the immunomodulatory effects of small molecule inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7277.