Synergistic Effect of Thermoneutral Housing and Chronotherapeutic PD-1 Blockade Overcomes Melanoma Resistance

BackgroundImmune-checkpoint blockade transformed melanoma therapy, yet the B16-F10 model resists PD-1 inhibition. Sub-thermoneutral housing (22{degrees}C) causes cold stress, and dosing outside circadian immune peaks dampens anti-tumour immunity. We hypothesised eliminating both would render B16-F10 curable. MethodsC57BL/6 mice (n=40) with B16-F10 tumours were randomised to conventional (22{degrees}C; ZT22 PD-1 timing), chronotherapy only (22{degrees}C; ZT4), thermoneutrality only (30{degrees}C; ZT22), or "double-hit" (30{degrees}C; ZT4). Anti-PD-1 was given on days 3, 6, 9, 12. Tumour volume, weight, survival, and day-14 serum IFN-{gamma} were recorded. Synergy threshold {Delta} [≥] 20% was prespecified. ResultsDay 14 control tumours were 1207{+/-}48 mm3. Chronotherapy and thermoneutrality slowed growth (737{+/-}55 mm3 and 565{+/-}28 mm3 respectively; p<0.0001). The double-hit nearly halted progression (43{+/-}12 mm3, -96%; p<10{square}1{square}), yielding seven complete responses and 100% survival. IFN-{gamma} increased 8.8-fold versus control (430{+/-}24 vs 48{+/-}7 pg mL{square}1; p<10{square}1{square}). Bliss {Delta} was 25%, confirming synergy. ConclusionsRemoving cold stress and circadian mistiming unmasks curative PD-1 response in refractory melanoma. Available warming blankets and morning infusions suggest a practical tactic to rescue PD-1 efficacy in "cold" tumours. A factorial multicentre trial is warranted.