Abstract 6647: Established 3D tumor-spheroid co-culture models demonstrate macrophage plasticity and unveil potential supportive roles of macrophages in the T cell-tumor-macrophage crosstalk post radiation therapy
Authors: Kaur Aanchal Preet , Alice Alejandro F. , Potuznik Megan R. , Kramer Gwen , Baird Jason , Friedman David J. , Crittenden Marka R. , Gough Michael J. 
Published: March 2024

Abstract
Ionizing radiation therapy (RT) has been characterized as an immunogenic event that in some tumors results in cell-death mediated activation of innate inflammatory and immunostimulatory mediators. These inflammatory mediators in turn modulate lymphocyte effector function in the tumor environment that can contribute to radiation-mediated control of tumors. We aim to model these interactions ex vivo, to identify the critical signals regulating immune function. We demonstrate that it is necessary to establish a three-dimensional model for crosstalk between tumor cells and immune cells to recapitulate myeloid and T cell responses to radiation. We have established 3D tumor spheroid co-culture models using murine MC38 colon carcinoma and Panc02-SIY pancreatic carcinoma cell lines with bone-marrow derived macrophages (BMDM). The spheroid cultures were exposed to 12 Gy RT and maintained in culture over a period of 5 days. Analyses were done using live-cell imaging and flow cytometry. Our findings demonstrate a decrease in hypoxia post RT, and exposure to RT increased the phagocytosis of dying cancer cells by macrophages. This was followed by a significant increase in CD80 and CD86 expression on the surface of macrophages in co-cultures on day 3 and day 4 post RT. These data matched our analyses of tumors treated in vivo with radiation and analyzed on day 4 post RT. In addition to RT-mediated upregulation of costimulatory molecules on macrophages, we also observed an increase in PD-L1 expression that may also impact T cell responses. To test T cell function in the 3D model, antigen-specific T cells were added to the Panc-02-SIY and BMDM spheroid co-cultures, which resulted in enhanced T cell proliferation in RT-treated groups. To assess the mechanism of macrophage activation in co-cultures, we evaluated HMGB1 as an innate adjuvant that may cause TLR4-mediated macrophage activation; however, the pattern of activation did not match that of RT. The involvement of the nucleic acid sensing cGAS-STING pathway was assessed by using BMDM from STING knockout (KO) mice in the 3D co-cultures. Similar upregulation of CD80 and CD86 were observed on STING KO macrophages in 3D co-cultures on day 4 post RT, indicating the observed phenomena to be independent of STING signaling. Ongoing studies aim to decrypt the innate downstream signaling pathways involved in RT-mediated polarization of macrophages so as to identify potential targeted therapies for tumor-associated macrophages, and also translate this to patient tumors.
Citation Format: Aanchal Preet Kaur, Alejandro F. Alice, Megan R. Potuznik, Gwen Kramer, Jason Baird, David J. Friedman, Marka R. Crittenden, Michael J. Gough. Established 3D tumor-spheroid co-culture models demonstrate macrophage plasticity and unveil potential supportive roles of macrophages in the T cell-tumor-macrophage crosstalk post radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6647.