Abstract 1304: Brachytherapy dose heterogeneity primes response to immune checkpoint blockade to generate anti-tumor immunity
Authors: Jagodinsky Justin C. , Jin Wonjong , Vera Jessica M. , Sriramaneni Raghava N. , Clark Paul A. , Lan Keng-Hsueh S. , Chakravarty Ishan , Siegel Noah , Allawi Raad H. , Emma Sarah E. , Arthur Ian S. , Das Rupak K. , Ong Irene M. , Miller Jessica R. , Morris Zachary S. 
Published: June 2022

Abstract
Purpose: The immunologic effects of radiation (RT) are influenced by dose and each may be optimized over a unique dose range. We used brachytherapy (BT) to deliver a heterogeneous RT dose within a single tumor and compared the relative capacities of BT and homogenous dose external beam radiotherapy (EBRT) to enhance the anti-tumor immune response in combination with immune checkpoint inhibition (ICI).
Materials and Methods: We used syngeneic murine models of melanoma (B78) and prostate cancer (Myc-CaP). To evaluate the effect of BT on the microenvironment, mice bearing B78 tumors were randomized to receive BT (192Ir source, 2 Gy to tumor edge), sham insertion, or EBRT (2, 8, or 20 Gy). Tumors were harvested 3 days following RT and in the case of BT, punch excisions were taken from the dissected tumor 1, 3, and 5 mm from the source location for gene expression and immune cell infiltration analysis. To evaluate anti-tumor response, we randomized mice bearing B78 tumors on the right flank (~200 mm3) and left shoulder (~100 mm3) to BT alone, catheter insertion alone, BT + ICI (anti-PD-L1 and anti-CTLA-4, 200 µg IP injection days 3, 6, 9 after RT), EBRT (2 or 8 Gy) + ICI, or ICI alone. Mice bearing MyC-CaP tumors were randomized to sham insertion, BT (2 or 8 Gy to tumor edge), BT + ICI, EBRT (2, 8, or 20 Gy) + ICI, or ICI alone. Immediately following RT, mice were engrafted with a secondary tumor.
Results: Using qPCR analysis, we observed statistically significant differences in gene expression of Mhc-1, Ifnβ, and Vcam between tissue locations in BT treated tumors. Furthermore, in a single tumor BT optimally engaged all three pathways while each EBRT dose only optimally engaged one. Bulk RNAseq analysis revealed unique gene expression signatures between tissue locations in BT treated tumors. We observed significant differences in number of CD8+ and FOXP3+ cells between tissue locations. In B78 tumor bearing mice, we observed a greater anti-tumor response at the secondary untreated tumor with BT + ICI (63% CR) compared to EBRT + ICI (12.5% CR). In mice bearing Myc-CaP tumors treated with BT + ICI, we observed a significant reduction in tumor growth compared to other groups and no engraftment at the secondary tumor. This effect was dependent on both CD4+ and CD8+ cells and was sensitive to BT dose. Splenocytes harvested from disease-free mice co-cultured with tumor cells showed significant increases in expression of T cell activation markers in both CD4+ and CD8+ cell populations.
Conclusions: We report dose-dependent effects of RT on expression of immune susceptibility markers and immune cell infiltration in a murine tumor model. We observe that a heterogeneous dose of BT results in spatial differences in these effects within a single tumor. This spatial heterogeneity in activation of immune mechanisms may underlie a greater capacity of BT to augment anti-tumor immune response when combined with ICI, as compared to homogenous dose EBRT.
Citation Format: Justin C. Jagodinsky, Wonjong Jin, Jessica M. Vera, Raghava N. Sriramaneni, Paul A. Clark, Keng-Hsueh S. Lan, Ishan Chakravarty, Noah Siegel, Raad H. Allawi, Sarah E. Emma, Ian S. Arthur, Rupak K. Das, Irene M. Ong, Jessica R. Miller, Zachary S. Morris. Brachytherapy dose heterogeneity primes response to immune checkpoint blockade to generate anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1304.