Session Item

Hyperthermia (HT) is a cancer treatment that increases the tumor temperature to 39-44°C, and it is a potent radiotherapy sensitizer (RT). HT has various effects on the innate and adaptive immune system and can potentially strengthen systemic antitumor immune responses. However, little is known about how different temperatures and, in particular, the sequence of HT and RT affect the immune phenotype in vitro and in vivo. And how the dendritic cells (DC) which is the main connector of the innate and adaptive immune system get affected by HT and RT needs investigation as well.

To this end, human MDA-MB-231 and MCF7 breast cancer cells were exposed to different temperatures of HT (39, 41, and 44 °C), alone or in combination with RT (2 x 5 Gy) in different sequences. Cell death and expression of immune checkpoint molecules (ICMs) were then analyzed by multicolor flow cytometry. Additionally, human monocyte-derived dendritic cells (hmDCs) were differentiated and co-cultured with the treated cancer cells, then the expression of DC activation markers was analyzed.

Our results showed that, for both cell lines, RT was the main inducer of cell death with apoptosis being the most prominent form of cell death in MCF7 cells, and both apoptosis and necrosis for MDA-MB-231 cells. However, there was no significant difference between the treatment sequences, as determined by measuring cell death. The expression of immune-suppressive ICMs, including PD-L1, PD-L2, and HVEM significantly increased 120h after RT and HT treatment in MCF7 cells. In MDA-MB-231 cells, PD-L2 was upregulated after RT combined with HT (41 and 44°C). Likewise, generally high expression of ICMs is observed after combining RT and HT, however, this was not affected by the treatment sequence. Furthermore, the co-culture of hmDCs with treated cancer cells had no impact on the expression of DC activation markers.

In conclusion, the sequence of HT and RT doesn’t strongly affect the immune phenotype of breast cancer cells. Noteworthy, the combined treatments do result in upregulation of immune suppressive ICMs. This may suggest that, in multimodal treatment settings, immune checkpoint inhibitors could be beneficially combined with HT and RT. Also, as the treatments had no impact on the activation state of dendritic cells, we hypothe that RT and HT combination might affect the immune system in the effector phase rather than in the priming phase of the immune response, although this should be further analyzed in vivo.
In our future experiments, we will investigate the DNA damages caused by RT and HT combination, and how it affects the immune response. The dynamic and different forms of DNA damage will be examined. Specifically, would HT and RT induce micronuclei, which is a result of unrepaired double-strand break, and whether this unresolved DNA damage burden further affects the anti-tumor immunity and the expression of ICMs will be investigated.