title: Evaluation of the influence of multimodal cancer therapy with hyperthermia and radiotherapy of breast cancer cells on immune checkpoint molecule expression and on the activation status of dendritic cells.
Creators: name: Walter, Jannik
Publisher: name: Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU)
Year: 2025

description: ABSTRACT Background: Cancer is one of the most common non-communicable diseases in industrial-ized countries, while breast cancer is the most frequent human malignancy concerning women. High incidence rates and a high mortality rate strongly depend on classic prognostic factors and thus fuel the urgent need for new treatment options. Conventional treatment methods e.g., radiotherapy treatment (RT), surgery, and systemic therapies are now often combined with novel options such as hyperthermia treatment (HT) and/or immunotherapy. Methods: The immune-modulating effects of hypo-fractionated RT, HT, or a multimodal cancer therapy (RHT) with breast cancer cells (MDA-MB-231/MCF-7) were determined. Hypo-fractionated RT (2x5 Gy) and HT (39, 41, 44 °C) was performed for 60 min. Number and emerging forms of cell death, cell cycle distribution and immune checkpoints were an-alyzed by multicolor flow cytometric analyses (FACS). Immune-activating (HVEM, Ox40-L, CD137-L, CD70), immune-suppressive (PD-L1, PD-L2), immune-modulating (ICOS-L) and epidermal growth factor receptor (EGFR) expression was considered. The activation status of monocyte derived dendritic cells (moDCs) was evaluated via FACS after co-culture with treated supernatants (TS)/cells (TC). The amount of secreted danger signal heat-shock protein 70 (HSP70) was evaluated using sandwich enzyme-linked immuno-sorbent assay (ELISA). Results: Apoptotic and necrotic cell death was induced by either method. Higher tempera-tures led to increased cell death rates and cell cycle arrests in all examined conditions, espe-cially the MDA-MB-231 cells in the G2-Phase. RT and RHT significantly increased the expression of immune-activating/-modulatory/-suppressive checkpoint molecules and HSP70 danger signal secretion. Co-culture with TS could not increase the moDC activation, while TC significantly increased the expression of co-stimulatory checkpoint molecules. Conclusion: RHT using hypo-fractionated RT and HT led to higher rates of necrotic cell death and results in immune-activating/pro-inflammatory responses. It directly affected the immune-stimulatory/-modulatory-/suppressive immune checkpoints. HSP70 secretion was increased by the addition of HT compared to the stand-alone treatment of HT or RT. There-fore, more moDCs were attracted and activated. Overall, the immune-modulating effects of HT, RT and/or RHT strongly depend on the cell line, heating method, RT scheme (fraction, dose), temperature, treatment time and measured time point. Combining traditional and novel approaches of therapy clearly demonstrated their ability to modulate the immune re-sponse by increasing necrotic cells, increased HSP70 secretion, regulation of immune check-points and cell-cell interaction to activate moDCs. Future studies should build on the herein presented interdisciplinary approach as it seems to play a key role in the treatment of breast cancer.