Characterization of Treatment Effects on the Tumor Microenvironment Using Single Cell RNA Sequencing

Abstract

tumor trafficking, and the formation of a tumor cell/T-cell immune response. This type of immunogenicity is comprised of the release of immune modulating signals, which include the release of high mobility group box 1 (HMGB1), ATP, and the translocation of calreticulin to the cell surface. Previous studies have demonstrated a positive correlation between markers of immunogenic cell death (ICD) and radiation dose with X-rays. Less is known about the effect of higher energies using charged particle irradiation and ICD. In this study, we explore whether varying linear energy transfer (LET) greater than 60 keV/mm with helium ion irradiation would impact immunogenicity in tumor cells as measured by levels of calreticulin translocation, ATP release, and HMGB1 release. Materials/Methods: Reporter cell lines designed to detect ATP luminescence (RLUs), red fluorescence protein tagged HMGB1 (relative fluorescent units [RFUs]), and cell surface calreticulin (percentage of calreticulin positive cells [%CRT+]) were derived from TSA (murine mammary carcinoma; syngeneic to BALB/c mice) cells. Prior to irradiation, the cells were seeded on six micron thick Mylar attached to 1.5 cm metal rings. The cells were then irradiated at the Radiological Research Accelerator Facility (RARAF) through a Track Segment Charged-Particle Irradiator. ATP and HMGB1 levels were measured 72 hours after RT and calreticulin was measured 24 hours after RT. Various doses of 250kV X-rays and LETs of helium ions were delivered and levels of these tumor ICD markers were determined. Results: For ATP measurements, when compared to non-irradiated controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/mm helium ions resulted in RLU fold-changes of 1.00 AE 0.05 (non-irradiated), 2.33 AE 0.08, 3.27 AE 0.19, 3.24 AE 0.10, 3.99 AE 0.33, 4.12 AE 0.32, and 2.91 AE 0.16 respectively. For HMGB1 measurements, when compared to controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/mm helium ions resulted in RFU fold-changes of 1.00 AE 0.02 (non-irradiated), 1.43 AE 0.03, 1.72 AE 0.03, 1.97 AE 0.06, 1.64 AE 0.03, 1.61 AE 0.04, and 1.51 AE 0.03, respectively. For calreticulin measurements, when compared to controls, cells irradiated with 5 and 20 Gy X-rays and with 5 Gy 65, 80, 110, and 160 keV/mm helium ions resulted in %CRT+ fold-changes of 1.00 AE 0.12 (non-irradiated), 2.32 AE 0.15, 3.07 AE 0.32, 3.16 AE 0.17, 3.01 AE 0.12, 2.74 AE 0.39, and 1.61 AE 0.15, respectively. Conclusion: Immunogenicity of tumor cells as measured by immune cell death signals increases with high LET ion irradiation when compared to low LET X-ray irradiation at the same dose. These marker levels appear to decrease at LETs greater than 110 keV/mm. Further investigation is warranted on the effects of high LET charged particle irradiation on the tumor immune response.