Abstract Tumor-associated macrophages (TAMs) perform a wide range of functions in the tumor microenvironment (TME), several of which contribute to resistance to immune checkpoint therapy. Given their high abundance and diverse functions, altering TAM activity is a promising supplement to immune checkpoint therapy. We demonstrate the potential of a new classification of TAMs, characterized by functional distinctions in gene expression, cell-cell interactions, and spatial localization. We validate the functions suggested by gene expression using ex vivo functional assays with primary TAMs. We compare markers of the eight functional TAM subpopulations in murine melanoma data with those in human data to develop a translatable gene signature for each functional subtype. We demonstrate that treating bone marrow-derived macrophages with different cytokine combinations can replicate TAM subtypes in vitro, advancing the field beyond the conventional M1-M2 model and enabling a more comprehensive approach. To demonstrate the importance of TAM subpopulations in response to immune checkpoint therapy, we show differences in the abundance of each subtype in untreated murine tumor models that are responsive or non-responsive to anti-PD-1 therapy. At the single-cell RNA-seq level, we find changes in the TAM landscape of murine SM1 melanoma tumors after systemic treatment with epigenetic-modifying agents, with or without anti-PD-1 therapy, and compare these changes with those observed in publicly available human melanoma data. Anti-PD-1 therapy in murine tumors results in a clear enrichment of APC-TAMs (an antigen-presenting, phagocytic, and complement-driven subpopulation), which is further enhanced by systemic HDAC inhibitors. The IFN-TAMs, which reflect a more conventional “M1” macrophage, are less strongly associated with response to immune checkpoint therapy. We see a reduction in angiogenic, hypoxia-driven “AFH-TAMs” in response to multiple anti-cancer therapies. In future studies, we will harness the APC-TAM population to overcome resistance to immune checkpoint therapy in multiple murine tumor types. Compared with melanoma tumors, murine breast cancers have a significantly higher proportion of ECM-TAMs, macrophages involved in extracellular matrix remodeling and homeostasis. We will aim to pharmacologically alter the abundance of other tumor-supporting macrophage populations, depending on the characteristics of different tumor types. Citation Format: Bryan T. Weselman, Satish Kumar Reddy Noonepalle, Manasa Suresh, Alexandra Singh, Isabella Duchovny, Xintang Li, Mackenna Ward, Alejandro Villagra. A new classification of tumor-associated macrophages and their roles in resistance to immune checkpoint therapy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3433.
Weselman et al. (Fri,) studied this question.