Hyperthermic Enhancement of Immunotherapy: Findings of In Vitro Modeling

Abstract

Despite advancement in understanding and manipulation of immune checkpoint molecules in immunotherapeutic design, limitations in treatment efficacy persist. Strategies to enhance effectiveness include use of multiple immunotherapeutic agents or combination with radiation therapy. Prior studies have also shown potential for hyperthermia to augment response to both of these therapeutic modalities. We hypothesized that in vitro assessment of moderate hyperthermia effects on the anti-tumor immune response will aid in development of targeted strategies that best combine hyperthermia with other immune manipulating therapies. To understand the consequences of temperature on carcinogenic phenotypes in vitro, B16-F10 melanoma cells were grown at 37°C or 41°C and biochemical profiles including protein expression were evaluated. Impact of hyperthermia on cell migration and proliferation were also assessed as were changes in the immune milieu including cytokine expression in response to heat. Data obtained was used to define ongoing in vivo experiments in which B16-F10 cells are implanted into C57BL/6 mice, grown to palpable tumors than treated with infrared radiation in combination with either anti-PDL1, anti-PD-1, or IL-15. Future studies based on these initial in vivo studies will explore integration of radiotherapy with hyperthermia and immunotherapy. B16-F10 cells grown at 41°C decreased cell migration by 70% in 24 hours, and decreased proliferation by 62% at 48 hours and 94% at 72 hours. To assess biochemical orchestrations exemplified by these data, protein expression profiles were evaluated. Expression of pERK and ERK decreased by 86% and 50% and caspase-3 increased by 31% at 41°C. Activation of sphingomyelinase and caspase-3 both rely on caspase-8. Sphingomyelinase activation results in CD95 receptor translocation, leading to cell death initiation in melanoma cells. Cell stress can induce death pathways and the heat shock protein response simultaneously. Of note, Hsp70 has an established role in fostering a tumor specific immune response. Thus, we investigated inducible hsp70 expression. Hsp70 expression increased by 188% at 41°C vs. 37°C. To evaluate the immune milieu, cytokine array data from conditioned media showed that at 41°C, TNFa expression was increased and IL-4 expression was decreased, suggesting a proinflammatory shift in cytokine profiles at hyperthermic temperatures. In support of our data, hyperthermia-induced TNFa apoptotic responses have been reported. In direct relation to clinical practice, we observed that hyperthermic potentiation decreased PDL1 expression in B16-F10 by 35%. Our work to date supports the hypothesis that hyperthermia can enhance immunotherapy via several mechanisms. In vivo study of the ability of hyperthermia to augment immune modulating therapies such as checkpoint blockade and radiation therapy is warranted.