The Effect of Hyperthermia and Radiotherapy Sequence on Cancer Cell Death and the Immune Phenotype of Breast Cancer Cells

Abstract

Hyperthermia (HT) is an accepted treatment for recurrent breast cancer which locally heats the tumor to 39–44 °C, and it is a very potent sensitizer for radiotherapy (RT) and chemotherapy. However, currently little is known about how HT with a distinct temperature, and particularly, how the sequence of HT and RT changes the immune phenotype of breast cancer cells. Therefore, human MDA-MB-231 and MCF-7 breast cancer cells were treated with HT of different temperatures (39, 41 and 44 °C), alone and in combination with RT (2 × 5 Gy) in different sequences, with either RT or HT first, followed by the other. Tumor cell death forms and the expression of immune checkpoint molecules (ICMs) were analyzed by multicolor flow cytometry. Human monocyte-derived dendritic cells (moDCs) were differentiated and co-cultured with the treated cancer cells. In both cell lines, RT was the main stressor for cell death induction, with apoptosis being the prominent cell death form in MCF-7 cells and both apoptosis and necrosis in MDA-MB-231 cells. Here, the sequence of the combined treatments, either RT or HT, did not have a significant impact on the final outcome. The expression of all of the three examined immune suppressive ICMs, namely PD-L1, PD-L2 and HVEM, was significantly increased on MCF-7 cells 120 h after the treatment of RT with HT of any temperature. Of special interest for MDA-MB-231 cells is that only combinations of RT with HT of both 41 and 44 °C induced a significantly increased expression of PD-L2 at all examined time points (24, 48, 72, and 120 h). Generally, high dynamics of ICM expression can be observed after combined RT and HT treatments. There was no significant difference between the different sequences of treatments (either HT + RT or RT + HT) in case of the upregulation of ICMs. Furthermore, the co-culture of moDCs with tumor cells of any treatment had no impact on the expression of activation markers. We conclude that the sequence of HT and RT does not strongly affect the immune phenotype of breast cancer cells. However, when HT is combined with RT, it results in an increased expression of distinct immune suppressive ICMs that should be considered by including immune checkpoint inhibitors in multimodal tumor treatments with RT and HT. Further, combined RT and HT affects the immune system in the effector phase rather than in the priming phase.

The efficacy of chemotherapy is limited by intratumoural senescent cells that persist through the upregulation of PD-L2

“…However, at present, there is limited information regarding how levels of PD-L2 are regulated in cancer. Two recent reports have found upregulation of PD-L2 in some cancer cell lines in vitro in response to cisplatin 15 or radiation 16 .…”

The potential of gas plasma technology for targeting breast cancer

“…This has also been observed with other physical modalities for breast cancer treatment before, such as radiotherapy or hyperthermia. 200 For those reasons, it is assumed in the field of onco‐immunology that physical modalities may help freeing tumour antigen locally to promote antitumour immunity that a later stage should then be re‐invigorated using immunotherapy, such as checkpoint inhibitors.…”

Biological modeling in thermoradiotherapy: present status and ongoing developments toward routine clinical use

“…In a recent review, Hurwitz discusses the rationale for clinical investigation of hyperthermia and immunotherapy for several tumor sites, including melanoma, bladder, and head and neck cancers [48]. Ongoing research should provide more (pre)clinical data to determine the optimal temperature and timing [45,49].…”