Abstract Whole-body hyperthermia (WBHT) is under investigation as a strategy to enhance chemotherapeutic efficacy in PDAC. Although localized HT (often 42 °C) synergizes with radiotherapy, these temperatures are not compatible with systemic application. Here, we evaluate the effects of moderate HT (41. 5 °C, 4h) on the S-phase and its implications on the efficacy of gemcitabine (GEM) in pancreatic cancer cell lines. Due to the pervasive character of HT, we probed several cellular pathways in vitro via immunofluorescence microscopy (IFM), nucleoside labeling and metabolic viability assays. BxPC-3 and AsPC-1 cell lines cell lines consistently showed an antagonistic effect of simultaneous HT on the efficacy of GEM as measured by resazurin assays. Nor did HT as monotherapy induce significant cytotoxicity. In all evaluated PDAC cell lines, nuclear 5-Ethynyl-2′-deoxyuridine (EdU) incorporation decreased during, and following HT. This could signify that moderate HT also attenuates the incorporation of analogs such as GEM, leading to decreased drug efficacy. Although the evaluated cell lines exhibited similar S-phase attenuation, PANC-1 cells responded to HT with a distinctly larger decrease in EdU incorporation. PANC-1 has the highest ENT-1 (SLC29A1) expression of all cell lines evaluated; which may suggest that cellular uptake of EdU via ENT-1 is affected by HT. Indeed, the ENT-1 inhibitor nitrobenzylthioinosine (NBTI), was able to further reduce EdU incorporation in a dose-dependent manner for PANC-1, whereas this was not observed for BxPC-3 and AsPC-1 cells. Therefore, ENT-1 could be involved in both EdU uptake and responses to HT. Intriguingly, IFM revealed that the increase in Hsp70 levels post-HT in EdU+ nuclei is less pronounced in PANC-1 compared to BxPC-3 and AsPC-1 cells. This underlines a potential role for Hsp70 as a requirement for proficient EdU incorporation under increased temperatures. The involvement of DNA damage responses (DDR) was evaluated via IFM for 53BP1 and phospho-γH2AX (pSer139). In AsPC-1, PANC-1 and BxPC-3 nuclei, levels of 53BP1 were unaltered, but increased levels of phospho-γH2AX (pSer139) were observed. ATR inhibition with VE-821 or ATM inhibition (InSolution®) could not restore the decreased EdU incorporation in all cell lines, suggesting that the S-phase attenuation may not be entirely caused through excessive ATR/ATM-signaling. Summarized, we observe a consistent decrease in EdU incorporation in PDAC cells subjected to HT, without complete S-phase arrest. Although likely involved, it is unclear how the heat-shock response and DDR may drive or mitigate this attenuation. Additionally, cellular uptake of nucleosides may be affected by HT in a negative manner, especially in cell lines with high ENT-1 expression. Our findings underscore the complexity and potential cell line–specific impact of HT on nucleoside uptake and DNA replication, with important implications for chemotherapeutic efficacy. Careful drug selection and optimization of treatment timing will be essential to harness potential synergy in combinatorial regimens. Citation Format: Robin Colenbier, Denisa L. Jurescu, Tine Logghe, Gaëlle Boulet, Jean-Pierre Timmermans, Johannes Bogers. Moderate hyperthermia as adjuvant therapy for Pancreatic Ductal Adenocarcinoma (PDAC): Pleiotropic effects may modulate drug cytotoxicity abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85 (18Suppl₃): Abstract nr B017.
Colenbier et al. (Sun,) studied this question.