Abstract 900: Reprogramming macrophages with HDAC6 inhibitors for anti-cancer macrophage-based cell therapy
Authors: Noonepalle Satish Kumar Reddy , Hernandez Maria Gracia , Delgado Christian Zevallos , Aghdam Nima , Gajendran Nithya , Knox Tessa , Tan Karen , Sotomayor Eduardo , Chiappinelli Katherine B. , Wardrop Duncan , Horvath Anelia , Shook Brett A. , Lee Norman , Dritschilo Anatoly , Fernandes Rohan , Shibata Maho , Musunuri Karthik , Villagra Alejandro 
Published: April 2023

Abstract
Tumor-associated macrophages (TAMs) play a critical role in shaping the tumor microenvironment (TME). Responding to numerous cues from various cell types present in the tumor, TAMs often exhibit a spectrum of phenotypes, often ranging between M1 (inflammatory) and M2 (tumor-supportive) macrophages. Therefore, the M1/M2 macrophage ratio often determines tumor suppression or tumor growth. Higher TAMs infiltration is usually associated with poor prognosis in several cancer types due to their predisposition towards M2 function. Therefore, reprogramming TAMs to the M1 phenotype is a promising avenue to be explored. In this study, we reprogrammed bone marrow-derived macrophages (BMDMs) ex-vivo by treating them with HDAC6 inhibitor (HDAC6i) followed by polarization to M1 phenotype and directly implanted them into immunocompetent tumors. We performed immunohistochemistry of tumors for macrophage markers, immunophenotyping of tumor-infiltrated immune cells by flow cytometry, gene expression analysis by quantitative PCR, and immunoblot analyses. We also performed single-cell analyses, including secretome with Isoplexis platform and CD45+ tumor-infiltrated immune cells by single-cell RNA-seq analysis. To further underscore the translatability of macrophage-based cell therapy, we treated humanized NSG-SGM3 mice bearing melanoma PDX tumors with HDAC6i-treated human M1 macrophages. Adoptive cell therapy (ACT) with HDAC6i-treated M1 macrophages resulted in significant tumor suppression and prolonged survival compared to other cohorts in the study. Two weeks post-transplantation, ACT macrophages were viable, and inhibition of HDAC6 rendered them resistant to M2 polarization. Inhibition of HDAC6 suppressed STAT3 activation and subsequent M2 marker Arg1 expression, further underscoring the role of HDAC6 in macrophage plasticity. M2 re-polarization assay further corroborated that HDAC6i-treated M1 macrophages were resistant to change into M2 phenotype. Single-cell secretome analysis revealed polyfunctionality as HDAC6-treated M1 macrophages were capable of secreting inflammatory cytokines such as Tnfa and t-cell recruiting Cxcl10. Histological analysis of tumor sections for macrophage markers and single-cell transcriptomic analysis of tumor-infiltrated immune cells further corroborated the M1/M2 ratio the increase observed by flow cytometry. Pro-inflammatory gene expression signature from scRNA-seq analysis also correlated with better survival in the SKCM dataset. In both immuno-competent SM1 murine melanoma and humanized NSG-SGM3 melanoma models, ACT enhanced anti-tumor immunity by increasing the M1/M2 ratio and infiltration of CD8 effector T-cells shifting the balance towards anti-tumor immunity. For the first time, we demonstrate that reprogramming macrophages with class-specific HDAC inhibitors is a viable cell therapy option to treat solid tumors.
Citation Format: Satish Kumar Reddy Noonepalle, Maria Gracia Hernandez, Christian Zevallos Delgado, Nima Aghdam, Nithya Gajendran, Tessa Knox, Karen Tan, Eduardo Sotomayor, Katherine B. Chiappinelli, Duncan Wardrop, Anelia Horvath, Brett A. Shook, Norman Lee, Anatoly Dritschilo, Rohan Fernandes, Maho Shibata, Karthik Musunuri, Alejandro Villagra. Reprogramming macrophages with HDAC6 inhibitors for anti-cancer macrophage-based cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 900.