Impact of hyperthermia and/or radiotherapy on the immune phenotype of murine mammary adenocarcinoma and melanoma cells

Background: An increased incidence of metastatic melanoma and metastatic breast cancer has led to the search for more effective treatment options. As classical therapy options like surgery, chemotherapy and radiotherapy (RT) alone are not sufficient to lower the high mortality rate, a new focus is multimodal treatment. Especially the combination of immune-modulating therapies like RT and hyperthermia (HT) is promising. That’s why the effects of RT and/or HT on cell death, immune phenotype, cell cycle distribution and the secretion of heat-shock-protein 70 (Hsp70) were investigated in this work. Methods: In-vitro murine mammary adenocarcinoma cells (TSA) and murine melanoma cells (B16-F10) were treated with hypofractionated RT (1x5 Gy or 2x5 Gy) and/or with HT (39°C, 41°C or 44°C) for 60 min. The resulting cell death and induced death forms as well as the change in expression of immune checkpoint molecules on the cell surface and cell cycle distribution were analyzed using multicolor flow cytometry. Immune suppressive molecules (PD-L1, PD-L2, HVEM, Gal-9), immune activating molecules (ICOS-L, Ox40-L, CD70, CD137-L) and epidermal human growth factor (EGFR) were included in the analysis. The secretion of Hsp70 was measured using sandwich enzyme-linked immunosorbent assay (ELISA). Results: RHT had an additive cell killing effect on both cell lines, whereas HT alone did not increase cell death significantly. This was supported by the results of cell cycle distribution with an increased number of apoptotic cells and cell cycle arrest mostly in G2/M-phase after RT and RHT. Overall RT and RHT increased the expression of all measured immune checkpoint molecules. Both cell lines showed the greatest increase with RHT at 41°C or 44°C when analyzed 72 hours after the first treatment. The secretion of Hsp70 was significantly increased by RHT at 41°C or 44°C and 2x5 Gy for both cell lines. Conclusion and discussion: Overall, the results suggest, that HT, even though in this project not relevantly effective on its own, is valuable in supporting RT. RHT might enhance the inconsistent response rates of immunotherapy. However, this is highly dependent on the cell line and its individual pattern of immune checkpoint molecules, as well as on the used temperature and fractionation. The application time of supplementary immunotherapy seems to be important, as the measured results were strongest 72 hours after the first treatment. Further in-vivo studies combining RHT and immunotherapy are therefore necessary.