Abstract 6643: Hyperthermic induction of immunotherapeutic response in <i>in vitro</i> melanoma model
Authors: Tara Jarboe, Danielle Quaranto, Nicole R. DeSouza, Kaci Kopec, Jan Geliebter, Raj K. Tiwari, Mark D. Hurwitz
Journal: Cancer Research (2024)

Abstract Immunotherapy has revolutionized the treatment of advanced melanoma, yet challenges persist with non-responders, development of resistance, and dosage concerns. Our strategy to enhance checkpoint inhibitor efficacy is to combine it with hyperthermia. Moderate hyperthermia, 39-45°C, is an interesting anti-neoplastic therapy which induces production of tumor associated antigens rendering tumor sites more amenable to immunotherapy. To investigate temperature-induced alterations on carcinogenic phenotypes in vitro, B16-F10 melanoma cells were grown at 37°C under normal culture conditions, or incubated at 41°C for 2 hours for moderate, febrile hyperthermic induction. Hyperthermia-induced changes were analyzed by trypan blue exclusion assay, scratch wound assay, collection of conditioned media for cytokine array, and protein isolation for Western Blot. Conditioned media from the hyperthermia-treated group showed an inflammatory shift in cytokine and chemokine expression, with increased TNFa and decreased IL-4 expression. Prior studies found the hyperthermia-induced apoptotic effect is directly correlated to endogenous TNFa levels, elucidating a potential molecular mechanism. Treatment for two hours at 41°C decreased proliferation of B16-F10 cells by 62% after 48 hours and 94% after 72 hours. Additionally, hyperthermia decreased cellular migration by 70% after 24 hours. Further evaluation of cellular interactions in important pathways responsible for these hyperthermia-induced changes were investigated via Western blot. Expression of constitutively active MAPK signaling cascade effectors pERK and ERK was decreased by 86% and 50%, respectively, helping to regulate pro-tumorigenic proliferative signaling. Some cancer cells have been demonstrated to resist hyperthermia via mechanisms to inhibit induction of caspase-3, however, in our model caspase-3 expression increased by 31% following 41°C treatment, thus allowing for induction of apoptosis via caspase-3 in this model. Cellular stress may induce these cell death pathways, as well as heat shock responses. Following febrile hyperthermic induction, hsp70 expression increased by 188% compared to cells cultured at 37°C. Hsp70 has a well-established role in supporting tumor-specific immune responses. Induction of hyperthermia in combination with anti-PDL1, anti-PD-1, or IL-15 immunotherapy is currently being evaluated in a C57BL/6 mouse model by our group. These studies aim to assess if hyperthermia augments response to immunotherapies in vivo. Our current works suggests that moderate, non-ablative hyperthermia helps initiate anti-tumor immune responses, assisting in the induction of anti-tumorigenic cell programs and, potentially, the production of additional tumor associated antigens, thereby making it an interesting candidate for combination with immune checkpoint inhibition and other immunotherapeutic approaches. Citation Format: Tara Jarboe, Danielle Quaranto, Nicole R. DeSouza, Kaci Kopec, Jan Geliebter, Raj K. Tiwari, Mark D. Hurwitz. Hyperthermic induction of immunotherapeutic response in in vitro melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6643.