Abstract B075: Antitumor immune responses to different Radiation Therapy doses and fractionation schedules in a preclinical model of hormone receptor positive breast cancer
Authors: Buque Aitziber , Bloy Norma , Formenti Silvia , Galluzzi Lorenzo 
Published: February 2025

Abstract
Hormone receptor (HR)+ breast cancer is poorly infiltrated by immune cells. Cold tumors rarely respond to immunotherapy or to immune checkpoint inhibitors (ICIs). It has been reported that, for certain tumors, radiation therapy (RT) mediates immunostimulatory effects and promotes ICI sensitivity. In the present study, we studied local and systemic antitumor immune responses of different RT doses and fractionation schedules. For this purpose, we harnessed a mouse model of multifocal, metachronous mammary carcinogenesis (MPA, M-accelerated, (DMBA, D)-driven) in immunocompetent female C57BL/6 mice. M/D-driven tumors recapitulate key immunobiological features of human HR+ breast cancer: (1) the oncogenesis depends on the transcriptional functions of estrogen receptor 1 (ER); (2) tumors exhibit a luminal, ER+ vimentin (VIM)- phenotype (3) tumors are poorly sensitive to PD-1 blockers when administered as standalone agents but respond robustly to CDK4/CDK6 inhibitors. Once tumors are detected and tumor area reached 20-60mm2, mice were randomized to receive either: (1) no treatment; (2) 1 fraction of 21 Gy focal RT (on d0); (3) 3 fractions of 10 Gy focal RT (on d0, d1 and d2); (4) 2 fractions of 20 Gy focal RT (on d0 and d1); (5) 6 fractions of 6 Gy focal RT (on d0, d1, d2, d3, d4 and d5); (6) 3 fractions of 14 Gy focal RT (on d0, d1 and d2); (7) 3 fractions of 20 Gy focal RT (on d0, d2 and d4). Focal RT was administered using the Small Animal Radiation Research Platform (SARRP, from Xstrahl). Mice were monitored for tumor growth (TG), detection of secondary lesions, and overall survival (OS). To investigate whether RT may be successfully used as a combinatorial partner for ICIs, the most effective RT schedules were selected from above to be combined with anti-PD1 (on d0, d3 and d6) and/or rFLT3L (on d0 everyday for 10 days). Local immune response was evaluated by immune fluorescence and pathological assessment of irradiated tumors at d7 (early response) and at end point (late response). Systemic response was evaluated by flow cytometry of the spleens. We observed that various focal RT doses and fractionation schedules effectively reduced the TG of primary irradiated lesions, with an optimal effect for a 20 Gy X 2 regimen (which ablated tumors in ∼90% of mice). The degree of primary tumor control, however, did not necessarily correlate with OS extension, due to shifts in the appearance and TG of new lesions that contribute to global disease burden, defining humane endpoint. Adding aPD-1 to the best schedules of focal RT failed to alter OS extension as enabled by RT alone. Similar results were obtained with aPD-1 plus r FLT3LG. Overall, these findings suggest that, in these preclinical settings, while administering focal RT to the primary tumor invariably ameliorates OS (to a an extent that depends on dose and fractionation), increasing local disease control by RT alone or in combination with immunotherapy does not necessarily limit the emergence of subsequent non-irradiated malignant lesions and hence is not associated with an OS benefit.
Citation Format: Aitziber Buque, Norma Bloy, Silvia Formenti, Lorenzo Galluzzi. Antitumor immune responses to different Radiation Therapy doses and fractionation schedules in a preclinical model of hormone receptor positive breast cancer [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B075.