Effects of high linear energy transfer particle irradiation on immunogenic cell death
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OBJECTIVE: Radiotherapy (RT) elicits tumor cell death that leads to activation of dendritic cells (DC) and promotes a T-cell immune response. This type of cell death, termed immunogenic cell death (ICD), results in the release of immune signals (HMGB1 and ATP), and the cell surface translocation of calreticulin (CRT). TSA syngeneic murine model of mammary carcinoma were used in these experiments because of their ability to induce abscopal responses when treated with anti CTLA4 antibody, and therefore, a good “immunogenic” tumor model (Dewan et al. 2009). Experiments were done using wild-type cells, as well as TSA cells transfected with various plasmids that allow for easier recording. It has been shown that RT with X-rays is able to increase these three markers of ICD in a dose-dependent manner (Golden et al. 2014). Less is known about the effects of higher linear energy transfer (LET) radiation and how it may impact tumor ICD. This study investigates the impact of increasing LET particle radiation on the expressed levels of these established ICD markers. High LET particle irradiation offers several potential therapeutic advantages over X-rays: increased cell kill, more focused dose distribution and more densely generated double stranded DNA breaks. This experiment investigates the immunogenic effects of high LET particle radiation compared to X-rays. We have previously demonstrated that ICD markers (ATP, HMGB1 and CRT) are increased with high LET ions when compared to X-ray treatment. In addition to in vitro experiments, a tumor vaccination model has been created to test the differences between X-ray RT and charged particle RT in vivo. Tumor cells were treated with either X-ray RT or charged particle RT. These irradiated tumor cells are then injected subcutaneously into a mouse and serves as a systemic vaccine, protecting a mouse from a subsequent challenge of non-irradiated tumor cells. Cells treated with X-ray RT appear to have a vaccination effect that appears in a dose-dependent matter; with increasing radiation dose, we observed an increase in vaccination strength. The ability for irradiated TSA cells to serve as a vaccine appears to plateau at doses of 24 Gy. RESULTS: Preliminary data suggest that the immunogenicity of tumor cells as measured by immune cell death signals appear to increase with ion irradiation at LET greater than 60 keV/µm when compared to X-ray irradiation at the same dose. These marker levels appear to decrease at LET greater than 110 keV/µm. Preliminary in vivo experiments show that mice injected with irradiated TSA cells are able to provide a systemic vaccination effect that appears to be correlated with the original dose of radiation. As the dose is increased from 2 Gy to 50 Gy X-rays, there is a correlative increase in vaccination effect. For mice that grew tumors with lower doses of radiation, the size of tumor growth was significantly slower than control groups. CONCLUSIONS: Using wild-type TSA cells, irradiated cells showed an increase of ICD markers when compared to non-irradiated cells. There was no significant difference between charged particle RT and X-ray RT in their ability to increase markers of ICD. Using the TSA cell line with plasmid transfections, increasing LET leads to increased release of ICD markers. The degree of release appears to plateau after 100 keV/um. The TSA reporter cell line appears to show an increase in ICD marker expression when irradiated with high LET radiation. Our in vivo model shows that irradiated tumor cells injected subcutaneously into a mouse are able to induce an in situ vaccine effect so that subsequent challenge with non-irradiated tumor cells is able to reject tumor formation. Further studies are warranted to investigate the effects of LET and types of charged particle irradiation on the tumor immune response.