Intratumoral radiation dose heterogeneity augments antitumor immunity in mice and primes responses to checkpoint blockade

The immunologic effects of radiation (RT) are influenced by dose and each may be optimized over a unique dose range. We hypothesized that delivering a heterogenous dose of RT would enhance anti-tumor immunity compared to homogenous RT in combination with immune checkpoint blockade (ICI). Using a point source, we delivered a spectrum of dose (~2-32 Gy) to the primary tumor and observed superior systemic immunity in models of metastatic cancer compared to low (2 Gy), moderate (8 Gy), or high (20 Gy) dose homogenous RT. Heterogeneity in RT dose resulted in spatial heterogeneity in gene expression and immune cell infiltration. Moreover, heterogenous RT optimally engaged multiple immune signaling pathways with diverse dose response relationships whereas responses to homogenous RT treatment groups were limited. Single cell RNAseq demonstrated unique enrichment of immune cell populations between each homogenous RT dose group, with several of these dose-dependent changes also represented in the heterogeneously treated tumors, with engagement of both lymphoid and myeloid compartments. This optimized activation of multiple immune mechanisms with distinct dose-dependent profiles within a single tumor may underlie a greater capacity of RT heterogeneity to augment anti-tumor immune response when combined with ICI, as compared to homogenous dose RT. Overall design: B78 melanoma cells were engrafted by subcutaneous flank injection of C57BL/6 and FVBn mice. BT+ICI samples received subcutaneous brachytherapy with 192Ir high dose rate source prescribed as 2 Gy to the proximal edge of the tumor and anti-CTLA-4 and anti-PD-L1 immune checkpoint inhibitors. BT+ICI treated tumors were harvested three days post-radiation treatment.