Abstract 1574: Chronotherapy of hyperthermia: Circadian rhythm governs HSP expression and modulates anti-tumor immunity

Abstract Background: Heat shock protein (HSP) expression is a key mechanism underlying hyperthermia-induced anti-tumor immunity. However, whether HSP expression exhibits circadian rhythmicity and how this impacts therapeutic efficacy remains unclear. Methods: Murine tumor models received localized hyperthermia at different times of day (specifically comparing afternoon and nighttime). Core circadian clock proteins (e.g., Bmal1 or Clock) were genetically ablated. Tumor tissues were analyzed for: (1) HSP expression levels post-hyperthermia; (2) release of damage-associated molecular patterns (DAMPs), including HMGB1 and ATP; (3) infiltration of CD8+ T cells. The effect of hyperthermia timing on treatment efficacy, both alone and in combination with anti-CTLA-4 antibody, was evaluated. Results: HSP expression induced by hyperthermia showed significant circadian variation: it was highest when hyperthermia was administered in the afternoon and lowest when administered at nighttime. Genetic ablation of core circadian clock proteins abolished this time-dependent difference in HSP expression, confirming the critical regulatory role of the biological clock. Compared to afternoon hyperthermia, nighttime hyperthermia triggered significantly greater release of immunogenic DAMPs (HMGB1, ATP) from tumor tissue and induced markedly enhanced infiltration of CD8+ T cells.Crucially, nighttime hyperthermia demonstrated a significantly stronger synergistic anti-tumor effect when combined with anti-CTLA-4 immune checkpoint blockade therapy. Conclusion: The circadian rhythm profoundly influences the immunogenic effects of hyperthermia by regulating HSP expression. Although inducing lower HSP levels, nighttime hyperthermia is more effective at releasing immunogenic DAMPs (HMGB1, ATP), promoting CD8+ T cell infiltration, and potently enhancing the efficacy of anti-CTLA-4 therapy. This study identifies timing of hyperthermia administration (specifically nighttime) as a critical factor for optimizing immunogenic cell death and augmenting the effectiveness of combined immunotherapy. Citation Format: Tingting Lin, Chunyan Lin, Zhilian Zhou, Shan Li, Lifeng Zhu, Yingming Sun, . Chronotherapy of hyperthermia: Circadian rhythm governs HSP expression and modulates anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1574.