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

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.

Efficient Radial-Shell Model for 3D Tumor Spheroid Dynamics with Radiotherapy

“…We present a data-driven dynamical model for the growth kinetics of a spheroid that both resolves the radial cell composition of the spheroid and at the same time is efficient enough for manageable parameter optimization; see Figure 1 for an illustration and Appendix C for details. The spheroid is assumed to be rotationally symmetric, as suggested by experimental observations [18,24]. The resulting one-dimensional (1D) dynamics are defined by the average concentrations c T (r, t) of type T cells and the partial oxygen pressure, i.e., the oxygen pressure ρ(r, t) along the radius r of the spheroid discretized into radial shells of width dr.…”

“…This model can be seen as a special one-dimensional Markov chain representing the cells' dynamics in and between radial shells. For this, we assume the spheroids to be rotationally symmetric, as suggested by histological sections [18,24] and exploited by several mathematical models before [1,24,[29][30][31]34]. We demonstrate that this model is (i) spatially sufficiently resolved to describe the effects of radiotherapy, and (ii) has sufficient computational performance to enable multi-parametric optimization.…”

“…State-of-the-art long-term spheroid-based assays assess the curative potential of new treatment strategies by classifying each spheroid within a population as either controlled or relapsed due to its individual growth recovery and kinetics, respectively. The spheroid control probabilities and selected spheroid control doses are computed as analytical endpoints by averaging the therapeutic response over ensembles of spheroids for each treatment dose [17][18][19][20], analogous to the tumor control dose employed for in vivo experiments with mice [21,22]. Growth curves and spheroid control probabilities depend on the spheroid type (cell line), the size of the spheroids at the onset of treatment, and the applied dose.…”

Clinical and Preclinical Outcomes of Combining Targeted Therapy With Radiotherapy

“…Other approaches include generation of radioresistant lines and determining which molecular factors play a role in their resistance. 3D models can aid in recapitulating the cell-cell interactions within tumor and stroma, cytokine signaling, hypoxia response, and combination therapy involving RT and allow us to quantitate the effects on the tumor as well as cells from the TME such as CAFs (136)(137)(138). A study comparing radiosensitive and radioresistant non-small cell lung cancer (NSCLC) demonstrated that pathways previously implicated including DNA repair, apoptosis, and NFkB activation in NSCLC were involved in the cellular response to RT (54).…”

Models for Translational Proton Radiobiology—From Bench to Bedside and Back

“…It allows visualizing culture integrity and subsequently determining volume growth kinetics, e.g., in spheroid growth delay studies [115,137,149] or patient-derived organoids [144]. The state-of-the-art spheroid control probability assay represents a clinically relevant endpoint for experimental radiotherapy [149][150][151][152], analogous to the tumor control probability (TCP) and tumor control dose 50% (TCD50) assessment in vivo [153]. Such analytical endpoints are essential for curative treatments and show great potential for systematic proton irradiation studies [136].…”