Spatiotemporal local and abscopal cell death and immune responses to histotripsy focused ultrasound tumor ablation.

Introduction: Histotripsy is a novel focused ultrasound tumor ablation modality with potent immunostimulatory effects. Methods: To measure the spatiotemporal kinetics of local andabscopal responses to histotripsy, C57BL/6 mice bearing bilateral flank B16 melanoma or Hepa1-6 hepatocellular carcinoma tumors were treated with unilateral sham or partial histotripsy. Treated and contralateral untreated (abscopal) tumors were analyzed using multicolor immunofluorescence, digital spatial profiling, RNA sequencing (RNASeq), and flow cytometry. Results: Unilateral histotripsy triggered abscopal tumor growth inhibition. Within the ablation zone, early high mobility group box protein 1 (HMGB1) release and necroptosis were accompanied by immunogenic cell death transcriptional responses in tumor cells and innate immune activation transcriptional responses in infiltrating myeloid and natural killer (NK) cells. Delayed CD8+ T cell intratumoral infiltration was spatiotemporally aligned with cancer cell features of ferroptosis; this effect was enhanced by CTLA-4 blockade and recapitulated in vitro when tumor-draining lymph node CD8+ T cells were co-cultured with tumor cells. Inoculation with cell-free tumor fractions generated by histotripsy but not radiation or freeze/thaw conferred partial protection from tumor challenge. Discussion: We propose that histotripsy may evoke local necroptotic immunogenic cell death, priming systemic adaptive immune responses and abscopal ferroptotic cancer cell death.

RAD51 Mediates Resistance of Cancer Stem Cells to PARP Inhibition in Triple-Negative Breast Cancer.

INTRODUCTION: PARP inhibitors have shown promising results in early studies for treatment of breast cancer susceptibility gene (BRCA)-deficient breast cancers; however, resistance ultimately develops. Furthermore, the benefit of PARP inhibitors (PARPi) in triple-negative breast cancers (TNBC) remains unknown. Recent evidence indicates that in TNBCs, cells that display "cancer stem cell" properties are resistant to conventional treatments, mediate tumor metastasis, and contribute to recurrence. The sensitivity of breast cancer stem cells (CSC) to PARPi is unknown. EXPERIMENTAL DESIGN: We determined the sensitivity of breast CSCs to PARP inhibition in BRCA1-mutant and -wild-type TNBC cell lines and tumor xenografts. We also investigated the role of RAD51 in mediating CSC resistance to PARPi in these in vitro and in vivo models. RESULTS: We demonstrated that the CSCs in BRCA1-mutant TNBCs were resistant to PARP inhibition, and that these cells had both elevated RAD51 protein levels and activity. Downregulation of RAD51 by shRNA sensitized CSCs to PARP inhibition and reduced tumor growth. BRCA1-wild-type cells were relatively resistant to PARP inhibition alone, but reduction of RAD51 sensitized both CSC and bulk cells in these tumors to PARPi treatment. CONCLUSIONS: Our data suggest that in both BRCA1-mutant and BRCA1-wild-type TNBCs, CSCs are relatively resistant to PARP inhibition. This resistance is mediated by RAD51, suggesting that strategies aimed at targeting RAD51 may increase the therapeutic efficacy of PARPi. Clin Cancer Res; 23(2); 514-22. ©2016 AACR.