Abstract 2253: HDAC6 inhibitors reprogram macrophages in antitumor-adoptive cell therapy

Abstract Tumor-associated macrophages (TAMs) exhibit hybrid phenotypes ranging between M1-like (anti-tumor) and M2-like (pro-tumor) macrophages. In most tumor types, a higher M1/M2 ratio favors antitumor immunity. Therefore, strategies to enhance the M1/M2 ratio can potentially alter the tumor microenvironment (TME) towards antitumor immunity. These strategies include reprogramming resident TAMs, altering the fate of tumor-infiltrating monocytes towards M1-like macrophages, and finally, adoptive cell therapy (ACT) with M1-like macrophages. A significant challenge with the above strategies is to retain the antitumor properties of M1-like macrophages in an overwhelmingly immunosuppressive TME. In this study, we demonstrate a macrophage-based ACT where treatment with HDAC6 inhibitors (HDA6is) retains the M1-like characteristics while significantly suppressing the tumor growth in syngeneic SM1 murine melanoma and with radiation therapy (RT). We reprogrammed GFP-expressing antitumor M1 macrophages ex-vivo with HDAC6 inhibitors to lock into the M1 phenotype and administered intratumorally as ACT in the syngeneic SM1 murine melanoma model and humanized NSG-SGM3 melanoma model. We performed histological analysis of tumors for macrophage markers, immune phenotyping of infiltrated immune cells, single-cell secretome analysis of tumor macrophages, and single-cell RNA-seq analysis of CD45+ immune cell populations. Initially, tail vein injected (i.v) administration of luciferase-expressing monocytes in combination with HDAC6i reached the tumors and diminished SM1 tumor growth compared to the control group. On the contrary, luciferase-expressing M1 macrophages failed to reach the tumor as visualized by IVIS imaging, suggesting that i.v is not an effective route for macrophage therapy. Whereas, intratumor HDAC6i treated M1 macrophage ACT resulted in diminished tumor growth. Single-cell (Sc) RNA-seq analysis of CD45+ sorted immune cells revealed a significant increase in the M1/M2 macrophage ratio in concurrence with flow cytometry data. NicheNet cell-cell interaction analysis indicated that M1-like TAMs activated infiltrating T-cells and monocytes through ligand-receptor interactions. Cell fate analysis of infiltrated monocytes indicated differentiation toward M1-like macrophages in ACT tumors. Flow cytometry analysis indicated an increase in CD8 effector T-cells. Sc-secretome analysis of F4/80 sorted tumor macrophages by the Isoplexis platform revealed polyfunctionality of HDAC6-treated M1 macrophages. Histological examination of tumor sections for macrophage phenotypic markers suggested that transplanted macrophages retained the M1 phenotype post-ACT in both SM1 murine and NSG-SGM3 melanoma tumor models. Our study comprehensively demonstrated the potential of reprogramming macrophages with HDAC6 inhibitors as a viable macrophage cell therapy for treating solid tumors. Citation Format: Satish Kumar Reddy Noonepalle, Maria Gracia Hernandez, Nima Aghdam, Nithya Gajendran, Manasa Suresh, Xintang Li, Abishek Sehgal, David Quiceno, Michael Berrigen, Tessa Knox, Karen Tan, Eduardo Sotomayor, Katherine Chiappinelli, Duncan Wardrop, Anelia Horvath, Anatoly Dritschilo, Rohan Fernandes, Karthik Musunuri, Alejandro Villagra. HDAC6 inhibitors reprogram macrophages in antitumor-adoptive cell therapy [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 2253.