Chemotherapy (CT) is the standard care for advanced pancreatic ductal adenocarcinoma (PDAC); however, with limited efficacy. Hyperthermia (HT) treatment has been suggested as a sensitizer to improve outcomes. However, the direct effect of the HT and CT combination is not fully understood. Therefore, we aim to assess the direct cytotoxic effect of HT in PDAC cells as monotherapy or in combination with chemotherapeutics. Different temperatures (37-, 40.5-, 41-, and 41.5 °C) and durations (6-, 12-, and 24 h) were tested in PDAC cell lines (BxPC-3, Capan-1, Capan-2, PANC-1, and MIA-PaCa-2). Different concentrations of gemcitabine, 5-fluorouracil, and cisplatin were also tested in these conditions. The impact on cell metabolic activity was determined by an MTS assay. Enhancement of chemosensitivity was assessed by a reduction in half-maximal inhibitory concentration (IC50). HT and chemotherapeutics interactions were classified as antagonistic, additive, or synergistic using the combination index. HT inhibited cell proliferation in a cell type, temperature, and duration-dependent manner. The induction of apoptosis was seen after 6 h of HT treatment, eventually followed by secondary necrosis. The HT and CT combination led to an IC50 reduction of the tested CT. At 12 h of HT, this effect was between 25 to 90% and reached a 95% reduction at 24 h. The additive or synergistic effect was demonstrated in all cell lines and chemotherapeutics, although, again, this depended on cell type, duration, and temperature. HT is cytotoxic and enhances the therapeutic effectiveness of gemcitabine, 5-fluorouracil, and cisplatin on PDAC cells. This result was further confirmed by the decrease in the expression of RRM2, TS, and ERCC1 in BxPC-3 and Capan-2 cells. These observations warrant further study in specific subsets of PDAC patients to improve their clinical outcomes.

In recent years, hyperthermia (HT) is gaining popularity as a therapeutic modality that could potentiate other treatments such as chemotherapy. Although pre-clinical and clinical research do suggest a beneficial effect of moderate HT (41.5°C), more knowledge on the molecular mechanisms is required before it can be routinely implemented in the clinic. In brief, several commercially available PDAC cell lines were subjected exposed to increasing durations of HT at 41.5°C, ranging from for 6, 12, and to 24 hours. Following HT, the effects on apoptotic signaling and chemotherapy sensitivity were evaluated mainly via RT-qPCR quantification of relevant genes. We found that subjecting pancreatic ductal adenocarcinoma (PDAC) cells (BxPC-3, Capan-2) to 41.5 °C for increasing durations, leads to (1) a decrease in cell viability, (2) a decreased mRNA expression of BCL-2 and three genes associated with chemotherapy resistance RRM2, TS, ERCC1, together with (3) an increase in expression of the pro-apoptotic BAX. These effects also appear to be cell-line dependent. More specifically, exposure of BxPC-3 cells to 6, 12 and 24 h of HT led to an increase in BAX/BCL-2 ratio of respectively 1.35, 2.80 and 4.65, whereas for Capan-2 cells a distinct pattern was observed. Here, the ratio of BAX/BCL-2 changed to 0.84, 1.56 and 3.95 for the three durations. Moreover, we confirmed the increased susceptibility towards the chemotherapeutic drugs 5-fluorouracil, gemcitabine, and cisplatin by subjecting the cells to varying doses of the drugs in absence or presence of HT. In general, exposure of 5 different PDAC lines (BxPC-3, PANC-1, Capan-1, Capan-2, MIA PaCa-2) cells to 12 h of HT allowed a dose reduction of 50%, and exposure of 24 h allowed a dose reduction of 75-95% to achieve a similar cell-killing effect as that which was observed at 37 °C. Together, these results show that subjecting the exposure of PDAC cells to long durations of moderate HT induces pro-apoptotic signaling and increases their susceptibility towards commonly used chemotherapeutic drugs, and that this effect is time-dependent. Nevertheless, treatment durations of 12 and 24 h ours would not be feasible very challenging in the clinic. Therefore, future research we will attempt to uncover whether an identical or similar affect can be achieved when the total HT exposure is fractionated into several shorter intervals, as is done with radiotherapy. Lastly, it is unlikely that the effects of HT are limited to those discussed. Future research will also focus on the role of cellular stress by HT-induced reactive oxygen species and non-necrotic, non-apoptotic cell death, such as ferroptosis, in pancreatic cancer cells. Citation Format: Robin Colenbier, Costanza E. Maurici, Ivana Gorbaslieva, Eke van Zwol, Jean-Pierre Timmermans, Elisa Giovannetti, Johannes Bogers. Long-term moderate hyperthermia induces apoptotic signaling and reduces chemoresistance in pancreatic cancer cells in vitro [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B048.