Abstract 71: Caloric restriction promotes antitumor immunity through polarization of intratumoral macrophages to M1 phenotype via downregulation of miR-21
Authors: Simone Brittany A. , DeAngelis Tiziana , Palagani Ajay , Ko Kevin , Harshyne Larry , Hooper D. Craig , Dicker Adam P. , Simone Nicole L. 
Published: August 2018

Abstract
Tumor-associated macrophages (TAMs) are the most abundant inflammatory cells in the breast cancer (BC) tumor microenvironment (TME) and suppress antitumor immune function, contributing to treatment resistance. These M2-polarized macrophages are characterized by fatty acid oxidation as opposed to the glycolytic pathways associated with M1-polarization. MiR-21 is known to affect several metabolic pathways relevant to macrophage polarization. We have previously shown that caloric restriction (CR) can decrease miR-21 expression and increase response to radiation therapy (RT). We propose CR as a means of metabolically reprogramming TAMs toward M1 phenotype to increase efficacy of radiation therapy (RT).
To assess the effects of CR in vivo on the composition of the TME in breast cancer, 40 female Balb/c mice were orthotopically injected at 12 weeks old with 4T1 cells and assigned to one of four cohorts: ad lib (AL) fed, 8 Gy RT to the primary tumor (RT), 30% reduction in caloric intake (CR) or CR+RT. MiR-21 levels were significantly decreased in both the CR and CR+RT arms; however, miR-21 was increased in the RT arm. Increased expression of M2 markers was noted in tumors in the RT group as compared to the CR and CR+RT groups, which favored M1 markers. On microarray analysis, the top pathways affected by CR discovered by gene ontology pathway analysis were: inflammation mediated by chemokine and cytokine signaling (31/509 hits), Wnt signaling (21/509 hits) and apoptotic signaling (18/509 hits). Multiple genes related to macrophage polarization were altered by CR including Ccl2 and IL12A. IL-12 is a known target of miR-21, and CR decreases miR-21 expression, therefore we sought to determine if miR-21 may be the mechanism behind the shift in chemokine and cytokine expression in the TME. Cohorts of MMTV-PyMT mice with spontaneously developed breast tumors were bred with a miR-21-/- mouse to generate breast tumors that lack expression of miR-21. These tumors exhibited high levels of IL-12 as well as IL-23 and Cxcl10, all of which are associated with M1 phenotype. MiR-21+/+ tumors exhibited high levels of IL-4 and TGF-β, both associated with M2 phenotype. In addition, peripheral monocytes (PBMCs) were co-cultured with sera from each of the following mice: miR-21-/-, miR-21+/+, AL, RT, CR and CR+RT. M1-polarization was induced by sera from miR-21-/- mice as well as mice receiving CR alone and CR+RT. Ionizing radiation seemed to induce M2-polarization in this manner, as did the miR-21+/+ serum.
Taken together, these data suggest that CR can counteract the M2-polarization caused by RT in the TME. This is likely due, in part, to miR-21 downregulation leading to enhanced M1 response. By increasing M1-polarized macrophages and enhancing antitumor immunity, we believe CR is a viable adjunct to standard cytotoxic therapies that could help overcome resistance to chemotherapy and immunotherapy.
Citation Format: Brittany A. Simone, Tiziana DeAngelis, Ajay Palagani, Kevin Ko, Larry Harshyne, D. Craig Hooper, Adam P. Dicker, Nicole L. Simone. Caloric restriction promotes antitumor immunity through polarization of intratumoral macrophages to M1 phenotype via downregulation of miR-21 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 71.