The Immunomodulatory Effects of Carbon-Ion Therapy on Solid Tumors

Abstract

Radiation-induced immunostimulation is of great interest as a focal strategy to reinvigorate or augment an immunotherapy-induced tumor-specific response. While evidence supports photon therapy (PhRT) as a tumor immunomodulator, pre-clinical data suggest that carbon-ion therapy (CiRT) may be more potent. Here we evaluate the immunomodulatory effects of CiRT, compared to biologically equivalent doses (BEDs) of PhRT, in a breast cancer mouse model. Using the orthotopic 4T1 mouse breast cancer model, we evaluated the immunomodulatory effects of very low (0.4 Gy), low (1 Gy) and high (4 Gy) doses of CiRT (290 MeV/u, SOBP) delivered to mammary tumors at the Heavy Ion Medical Accelerator in Chiba (HIMAC), Japan compared to BEDs (RBE 2) of PhRT (SARRP, Xstrahl; 220 kV). Primary tumors were treated 7-days post implantation and harvested 72-hours later for flow cytometric characterization of tumor-infiltrating lymphocytes. Low (1 Gy) and high-dose (4 Gy) CiRT resulted in a 32% and 51% (p<0.01) decrease in CD4+ T-cells, with no significant change following very low-dose (0.4 Gy) CiRT. Whereas, very low, low, and high-dose PhRT reduced CD4+ T-cells by 50%, 32% and 44% (p<0.05) and very low and high-dose PhRT reduced CD8+ T-cells by 55% and 47% (p<0.05), compared to control. Very high-dose (4 Gy) CiRT induced a 184% and 117% (p<0.05) increase in proliferation (Ki67+) by CD4+ and CD8+ T-cells, respectively, compared to control. While, very low, low and high-dose PhRT increased proliferation of CD4+ T-cells by 170%, 129% and 216% (p<0.05) and CD8+ T-cells by 84%, 86% and 99% (p<0.05), compared to control. High-dose (4 Gy) CiRT induced pro-inflammatory cytokines production including granzyme B (88%, 205% increase; P<0.005), IL-2 (112%, 165% increase, p<0.01) and TNF-a (230%, 117%, p<0.005) by CD4+ and CD8+ T-cells, respectively, with no change in IFN-g expression. Whereas, high-dose PhRT increased IFN-g expression by 102% (p<0.05) by CD8+ T-cells, with no change in granzyme B, IL-2, or TNF-a. Only high-dose (4 Gy) CiRT induced a 127% increase (p<0.005) in abundance and a 70% (p<0.05) increase proliferation of immunosuppressive regulatory T-cells (Tregs), whereas very low and high-dose PhRT increased proliferation of Tregs by 82% and 99% (p<0.05), respectively, with no increase in abundance. These data suggest that at low doses, lymphocytes differ in their sensitivity to CiRT compared to PhRT, whereby very low dose CiRT is lymphocyte-sparing. Very high-dose CiRT and PhRT induced immunostimulatory effects by differing degrees and through different mechanisms, CiRT being more potent and inducing changes in a diverse set of pro-inflammatory cytokines including granzyme B, IL-2 and TNF-a, whereas PhRT induced only IFN-g. Combining less lymphotoxicity at low doses with a more potent and mechanistically diverse pro-inflammatory effects at high doses, CiRT may be a more optimal synergistic tool to augment an immunotherapy-induced, tumor-specific response.