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.

Treatment of locally advanced rectal cancer includes chemoradiation and surgery, but patient response to treatment is variable. Patients who have a complete response have improved outcomes; therefore, there is a critical need to identify mechanisms of resistance to circumvent them. DNA-PK is involved in the repair of DNA double-strand breaks caused by radiation, which we found to be increased in rectal cancer after treatment. We hypothesized that inhibiting this complex with a DNA-PK inhibitor, Peposertib (M3814), would improve treatment response.We assessed pDNA-PK in a rectal cancer cell line and mouse model utilizing western blotting, viability assays, γH2AX staining, and treatment response. The three treatment groups were: standard of care (SOC) (5-fluorouracil (5FU) with radiation), M3814 with radiation, and M3814 with SOC.SOC treatment of rectal cancer cells increased pDNA-PK protein and increased γH2AX foci, but this was abrogated by the addition of M3814. Mice with CT26 tumors treated with M3814 with SOC did not differ in average tumor size but individual tumor response varied. The clinical complete response rate improved significantly with the addition of M3814 but pathological complete response did not. We investigated alterations in DNA repair and found that Kap1 and pATM are increased after M3814 addition suggesting this may mediate resistance.When the DNA-PK inhibitor, M3814, is combined with SOC treatment, response improved in some rectal cancer models but an increase in other repair mechanisms likely diminishes the effect. A clinical trial is ongoing to further explore the role of DNA-PK inhibition in rectal cancer treatment.

Background Histotripsy, a novel image-guided, robotically assisted sonic therapy platform, is a non-invasive and non-thermal tumor ablation modality. We have previously shown that histotripsy potentiates profound innate and adaptive anti-tumor responses in addition to direct tumor destruction.1 In this study, we sought to characterize the biomarkers of tumor cell death pathways immediately after histotripsy and after the induction of adaptive anti-tumor immune responses in preclinical settings. Methods Immunocompetent C57BL/6 mice were inoculated with bilateral subcutaneous flank injections of Hepa1-6 hepatocellular carcinoma to generate 8–10 mm tumors within 8–11 days. Unilateral subtotal histotripsy was then performed. Mice were euthanized at 6h, and 1, 3 and 10–12 days post-treatment (dpt). Tumors were measured, harvested, fixed, sectioned and studied using multicolor immunohistochemistry. Results Histotripsy decreased treated tumor growth by 50% and abscopal tumor growth by 30–40% compared to untreated tumors at 12dpt, evidencing a systemic anti-tumor immune response that inhibited growth of distant untreated tumor. Treated tumors showed immediate tissue liquefaction in the ablation zone with marked extranuclear translocation of the damage associated molecular pattern HMGB1. At 1dpt, 100% of tumor cells within the ablation zone showed HMGB1 translocation, and 70% of tumor cells at the periphery of the ablation zone showed HMGB1 translocation. Caspase 3 cleavage was not observed in the direct ablation zone, but at the junction of the ablated and non-ablated tissue ~40% cells that released HMGB1 showed cleaved Caspase 3. Caspase 9 cleavage was observed in ~50% cells that had cleaved Caspase 3, suggesting early programed cell death with mitochondrial damage and cytochrome C release 1 dpt; the presence of inflammasome integration/activation suggested pyroptosis induction. Areas of tumor well outside the zone of ablation and within untreated tumors contralateral to ablated tumors did not show early DAMP release or apoptotic cell death compared to the control tumors. However, a robust immune cell infiltration was observed in these locations at 10–12dpt, involving CD8 T-cell infiltration and areas of tumoral HMGB1 release in the vicinity of the infiltrating CD8 T cells - indicating the induction of immune rejection of treated and untreated tumors by histotripsy. Conclusions Our results indicate that histotripsy ablation promotes tumor cell destruction through both immediate mechanical disruption, as well as possible adjacent apoptotic and pyroptotic death. Systemic CD8 T-cell mobilization and immunological cell death in the treated and the contralateral tumors is a novel long term therapeutic benefit. Reference Qu S, Worlikar T, Felsted AE, Ganguly A, Beems MV, Hubbard R, Pepple AL, Kevelin AA, Garavaglia H, Dib J, Toma M, Huang H, Tsung A, Xu Z, Cho CS. Non-thermal histotripsy tumor ablation promotes abscopal immune responses that enhance cancer immunotherapy. J Immunother Cancer 2020;8:e000200.

BackgroundDeveloping the ability to use tumor-directed therapies to trigger potentially therapeutic immune responses against cancer antigens remains a high priority for cancer immunotherapy. We hypothesized that histotripsy, a novel non-invasive, non-thermal ablation modality that uses ultrasound-generated acoustic cavitation to disrupt tissues, could engender adaptive immune responses to tumor antigens.MethodsImmunocompetent C57BL/6 mice inoculated with flank melanoma or hepatocellular carcinoma tumors were treated with histotripsy, thermal ablation, radiation therapy, or cytotoxic T lymphocyte-associated protein-4 (CTLA-4) blockade checkpoint inhibition. Lymphocyte responses were measured using flow cytometric and immunohistochemical analyses. The impact of histotripsy on abscopal immune responses was assessed in mice bearing bilateral tumors, or unilateral tumors with pulmonary tumors established via tail vein injection.ResultsHistotripsy ablation of subcutaneous murine melanoma tumors stimulated potent local intratumoral infiltration of innate and adaptive immune cell populations. The magnitude of this immunostimulation was stronger than that seen with tumor irradiation or thermal ablation. Histotripsy also promoted abscopal immune responses at untreated tumor sites and inhibited growth of pulmonary metastases. Histotripsy was capable of releasing tumor antigens with retained immunogenicity, and this immunostimulatory effect was associated with calreticulin translocation to the cellular membrane and local and systemic release of high mobility group box protein 1. Histotripsy ablation potentiated the efficacy of checkpoint inhibition immunotherapy in murine models of melanoma and hepatocellular carcinoma.ConclusionsThese preclinical observations suggest that non-invasive histotripsy ablation can be used to stimulate tumor-specific immune responses capable of magnifying the impact of checkpoint inhibition immunotherapy.