3D co-culture models demonstrate radiation-mediated activation of tumor-associated macrophages

Document Type


Publication Date


Publication Title

AAI Annual Meeting; May 11-15; Washington DC. 2023


oregon; chiles


Type I IFN signaling mediated by STING pathway is important for the induction of radiation-stimulated adaptive immune responses in responsive tumors. Macrophages recruited to tumors following radiotherapy (RT) can influence the response to RT. Reprogramming immunosuppressive M2-like macrophages in tumors to exhibit a more activated M1-like phenotype can help improve response to therapies. Here, we hypothesize that a 3D co-culture model of cancer cells and macrophages will be more contextually relevant to the events occurring in tumors and can be used to study the crosstalk that occurs between these cells in vivo. We initially optimized seeding densities of murine MC38 colorectal carcinoma cells to develop spheroids of consistent size. Using GFP+ macrophages and Hoechst stained MC38, we imaged the development and localization of these cells during spheroid formation. Next, co-culture spheroids were exposed to 12 Gy radiation on day 1. Viability and phenotype of macrophages in spheroid co-cultures was assessed on day 4 using flow cytometry by staining for CD80, CD86, MHC Class II, CD69 and CD206 expression. STING ligand, cyclic di-AMP (CDA) treated spheroid co-cultures were used as positive controls. CDA treatment significantly increased CD80/86 expression in 3D co-cultures. Treatment with 12 Gy radiation showed similar effect. Interestingly, macrophages derived from MyD88 Lyz2Cre, MyD88fl/fl and STING knockout mice also showed a significant increase in CD80/86 and CD40, suggesting that macrophage activation was independent of MyD88 or STING signaling. Ongoing studies aim to explore the pathways activated on irradiation in these cells and further cross-validate with the macrophage phenotypes observed in vivo in response to RT.

Clinical Institute



Earle A. Chiles Research Institute




Kaur AP, Baird J, Alice AF, Ball MR, Kramer G, Crittenden MR, Gough MJ.

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