Figure 6.

The combination of histotripsy with checkpoint inhibition results in additive intratumoral infiltration of CD8+ cells and synergistic induction of cancer cell ferroptosis. Mice bearing bilateral B16F10 tumors were treated with no therapy (control), checkpoint inhibition (CI) with anti-CTLA-4 mAb on days 6, 9 and 12 (“CI”), unilateral partial histotripsy ablation (“HT”) on day 7, or both (“HT+CI”). (A) Non-ablated abscopal tumor growth on day 18 was suppressed in mice treated with contralateral HT and CI, but maximal in mice treated with both. (B) Multicolor immunofluorescence of non-ablated abscopal tumors revealed increases in intratumoral CD8+ cell infiltration, loss of nuclear HMGB1, and 4-HNE accumulation after both CI and HT, with maximal effects seen after combinatorial HT+CI. (C) Quantitation of CD8+ cell density demonstrated an additive effect between CI and the abscopal effect of HT. (D) A similar additive effect was observed between CI and the abscopal effect of HT in extranuclear HMGB1 translocation. (E) The combination of HT+CI appeared to be a greater than additive effect on abscopal 4-HNE expression. The additive effects of CI and HT on abscopal CD8+ cell infiltration, HMGB1 release, and 4-HNE expression are shown in line graph form (F) and dot plot form (G). (n=4-7 mice per group; *=p<0.05 compared with controls; **=p<0.01 compared with controls; ***=p<0.001 compared with controls).