Abstract 2187: Functional classification of tumor-associated macrophages and changes in TAM subpopulations in response to various immunotherapies

Despite single-cell omics ushering in many attempts at reclassifying tumor-associated macrophages (TAMs), there remains a lack of a consensus for a widely applicable model that accurately describes their biological functions. Based on single-cell RNA seq data from both mouse and human melanoma tumors, we propose a new model for categorizing TAMs by their function in the tumor microenvironment (TME). We identified eight clusters with various functions including a distinct population of proliferating macrophages with tissue-resident characteristics. Samples from mice with SM1 melanoma tumors that have been treated with ɑ-PD-1 immune checkpoint blockade, the novel HDAC6 inhibitor SS208, combination SS208 + ɑ-PD-1, and adoptive cell therapy (ACT) using HDAC6 inhibition show changes in the proportions of putative subpopulations, most notably a significant decrease in “Pro-Angiogenic/Arginine Metabolism” macrophages within tumors after all treatments, an increase in “Antigen Presenting/Phagocytosis” macrophages in mice treated with ɑ-PD-1 therapy, and an increase in proliferating macrophages in mice treated with ACT. We also identified heterogeneity in the expression of certain markers historically categorized as “M1” or “M2,” indicating that using specific markers interchangeably may be inaccurate in measuring different subpopulations within the tumor. In an attempt to move away from artificial polarization of macrophages in vitro via cytokines, TAMs were cultured in tumor-conditioned media and M-CSF to more accurately retain TME phenotype. The proposed functions of these subpopulations have been further confirmed by in vitro and ex vivo functional assays from isolated TAMs. Additionally, we outline an efficient method for applying the proposed subpopulation gene signature to bulk RNA seq data of macrophages, which allows for more accurate analysis of changes in TAM subpopulation in response to therapy. Citation Format: Bryan T. Weselman, Satish K. Noonepalle, Manasa Suresh, Marie Durr, Nithya Gajendran, Alejandro Villagra, Xintang Li. Functional classification of tumor-associated macrophages and changes in TAM subpopulations in response to various immunotherapies [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 2187.