Carbon ion irradiation plus CTLA4 blockade elicits therapeutic immune responses in a murine tumor model

Radiotherapy can act as in situ vaccine activating preventive tumor-specific immune responses in treated patients. While carbon ion radiotherapy holds superior biophysical properties over conventional photon irradiation, little is known about the immunological effects induced by this radiation type. Multiple strategies combining radiotherapy with immune checkpoint inhibition (radioimmunotherapy) to enhance anti-tumor immunity have been described, however, results on the immune cell composition in tumors following radioimmunotherapy with carbon ions are lacking. Here, we present a bilateral tumor model based on time-shifted transplantation of murine, Her2+ EO771 tumor cells onto the flanks of immune competent mice followed by selective irradiation of the primal tumor. αCTLA4- but not αPD-L1-based radioimmunotherapy induced complete tumor rejection and mediated eradication of even non-irradiated, distant tumors. Cured mice were protected against EO771 rechallenge indicative of long lasting, tumor-specific immunological memory. Single cell RNA-sequencing and flow cytometric analyses of irradiated tumors revealed activation of NK cells and distinct tumor-associated macrophage clusters with upregulated expression of TNF and IL1 responsive genes. Distant tumors of irradiated mice showed higher frequencies of naïve T cells, which were activated upon combination with CTLA4 blockade. Thus, radioimmunotherapy with carbon ions plus CTLA4 inhibition reshapes the tumor-infiltrating immune cell composition and can induce complete rejection even of non-irradiated tumors. Our data suggest to combine radiotherapy approaches with CTLA4 blockade to achieve durable anti-tumor immunity. Furthermore, evaluation of future radioimmunotherapy approaches should not be restricted to immunological impacts at the irradiation site, but should also consider systemic immunological effects on non-irradiated tumors. Library preparation: Single cells suspensions of eight digested tumors per group were pooled. Afterwards, cells were incubated with LIVE/DEAD® Fixable Dead Cell Stain Kit (Thermo Fisher Scientific) diluted 1:1000 in PBS for 30 min at 4 °C and stained with PE Rat Anti-Mouse CD45 antibody (BD) in FACS buffer for 30 min at 4 °C. Sorting for viable CD45+ cells was performed with a BD FACS Aria III. The cell suspension of CD45 positive cells was adjusted to a concentration of approximately 1000 cells/μl. The cells were then loaded onto the 10x Genomics Chromium controller for droplet-enabled single cell RNA sequencing according to the manufacturer’s instruction manual. Library generation was performed following the Chromium Single Cell 3′ Reagents Kit version 3.1 user guide. The libraries were sequenced on the Illumina NovaSeq 6000 platform.