Efficient Heat Shock Response Affects Hyperthermia-Induced Radiosensitization in a Tumor Spheroid Control Probability Assay

SIMPLE SUMMARY: Resistance to therapy and subsequent relapse of the disease are common in patients with cancers in the head and neck region (HNSCC). Recent technological advancements have revitalized the concept of combining hyperthermia (HT) with radio(chemo)therapy for treating these patients. Heat inherently affects multiple cellular components and destroys protein structures, thereby influencing the DNA damage response. However, the plethora of adverse mechanisms in HT-induced radiosensitization is still not fully elucidated. We uniquely evaluated the radiosensitizing potential of HT in HNSCC cells using a sophisticated spheroid assay platform, which turned out as a powerful tool to compare different treatment modalities and gain new mechanistic insight. We show that HT disrupts vital cellular proteostasis and affects global stress response signaling. This triggers massive heat shock and proteotoxic stress responses contributing to the cancer cells’ protection against HT-induced radiosensitization. Selected molecules in this scenario may serve as new targets for combination with hyperthermia and radiotherapy. ABSTRACT: Hyperthermia (HT) combined with irradiation is a well-known concept to improve the curative potential of radiotherapy. Technological progress has opened new avenues for thermoradiotherapy, even for recurrent head and neck squamous cell carcinomas (HNSCC). Preclinical evaluation of the curative radiosensitizing potential of various HT regimens remains ethically, economically, and technically challenging. One key objective of our study was to refine an advanced 3-D assay setup for HT + RT research and treatment testing. For the first time, HT-induced radiosensitization was systematically examined in two differently radioresponsive HNSCC spheroid models using the unique in vitro “curative” analytical endpoint of spheroid control probability. We further investigated the cellular stress response mechanisms underlying the HT-related radiosensitization process with the aim to unravel the impact of HT-induced proteotoxic stress on the overall radioresponse. HT disrupted the proteome’s thermal stability, causing severe proteotoxic stress. It strongly enhanced radiation efficacy and affected paramount survival and stress response signaling networks. Transcriptomics, q-PCR, and western blotting data revealed that HT + RT co-treatment critically triggers the heat shock response (HSR). Pre-treatment with chemical chaperones intensified the radiosensitizing effect, thereby suppressing HT-induced Hsp27 expression. Our data suggest that HT-induced radiosensitization is adversely affected by the proteotoxic stress response. Hence, we propose the inhibition of particular heat shock proteins as a targeting strategy to improve the outcome of combinatorial HT + RT.