Histotripsy-Focused Ultrasound Treatment Abrogates Tumor Hypoxia Responses and Stimulates Antitumor Immune Responses in Melanoma

Histotripsy-focused ultrasound treatment gives rise to systemic antitumor immune responses. We investigated whether histotripsy effects on immunosuppressive tumor hypoxia were a potential mechanism for these immunostimulatory effects. Immunocompetent or CD8-deficient C57BL/6 mice with flank B16F10 or YUMM1.7 melanoma tumors underwent sham or subtotal histotripsy. Tumor growth, immune cell infiltration, and intratumoral hypoxia responses were examined using flow cytometry and fluorescence microscopy. Chemokine receptor CXCR3 and hypoxia-inducible factor-1α (HIF1α) were intercepted with antibodies and inhibitors to assess their roles in immune responses after histotripsy. Histotripsy-treated tumors exhibited rapid loss of intratumoral hypoxia and suppression of HIF1α and downstream prosurvival proteins. Histotripsy was followed by intratumoral upregulation of the CXCR3 ligand CXCL10 and CXCR3⁺/CD8⁺ T-cell infiltration. Tumor growth inhibition by histotripsy was significantly diminished in CD8-deficient mice and mice receiving anti-CXCR3 mAb. Post-histotripsy inhibition of hypoxia and tumor growth eventually receded in parallel with cessation of CD8⁺ T-cell influx, and pharmacologic HIF1α suppression with the MEK inhibitor trametinib substantially augmented the therapeutic effects of histotripsy. Transient abrogation of intratumoral hypoxia and HIF1α-associated hypoxia responses is mechanistically linked with intratumoral infiltration of activated CXCR3⁺/CD8⁺ T cells via CXCL10–CXCR3 engagement. These findings suggest that the immune effects of histotripsy may be regulated by hypoxia abrogation and that pharmacologic hypoxia abrogation could potentiate the immunotherapeutic effects of histotripsy.