Abstract 2240: High dose acetaminophen re-polarizes CD11B+ cells in the tumor microenvironment towards an activated macrophage phenotype

Background: High dose acetaminophen (AAP) has demonstrated promising results in early phase clinical trials. When administered concurrently with the CYP2E1 inhibitor fomepizole, AAP can be dose-escalated 100-fold relative to standard dosing without liver toxicity and without compromising anti-tumor activity. The present study aims to characterize the effects of high dose AAP on the tumor immune microenvironment. Methods: The current study used a 23-plex flow cytometry panel to study AAP induced changes in the tumor immune microenvironment of syngeneic mouse breast tumors. AAP-induced changes in macrophage phenotype were studied in vitro using phagocytosis and antigen presentation assays, both alone and in combination with PD-1 antibody therapy. Results: Flow cytometric analysis of the tumor immune micro-environment revealed that the vast majority of CD45+ cells in triple negative breast cancer model are CD11B+ cells of the innate immune system. The CD11B+ cells in the tumor micro-environment of vehicle-treated mice were mostly comprised of GR1+ myeloid derived suppressor cells. On the other hand, the CD11B+ cells in the high dose AAP-treated mice were mostly of the activated macrophage phenotype (F4/80+CD80+MHCII+). Other observed changes within the tumor of AAP-treated mice include a large influx of B-cells, NK-cells and T-cells. Given the increased number of activated macrophages observed in the AAP-treated tumors, in vitro studies were performed to better understand AAP effects on macrophage function. It was demonstrated that AAP enhances phagocytosis as well as antigen presentation by macrophages to antigen-reactive T-cells. These effects are amplified when PD-1 antibodies are combined with AAP in the in vitro antigen presentation assay. Furthermore, AAP has synergistic anti-tumor activity in vivo when combined with PD-1 antibody therapy, an effect that is macrophage dependent. Conclusion: High dose AAP can be administered safely when combined with fomepizole-based rescue in mouse models. The present study demonstrates profound AAP-induced changes in the tumor immune microenvironment, particularly within the phenotype of CD11B+ cells of the innate immune system leading to increased “M1” type macrophages in the tumor immune microenvironment. Citation Format: Allyn Bryan, Pavani Pingali, Bhaumik Patel, Won Sok Lee, Joseph Landry, Alexander J. Neuwelt. High dose acetaminophen re-polarizes CD11B+ cells in the tumor microenvironment towards an activated macrophage phenotype [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 2240.