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.

Computed Tomography in Patients With Craniosynostosis: A Survey to Ascertain Practice Patterns Among Craniofacial Surgeons.

BACKGROUND: In patients with craniosynostosis, imaging remains up to the discretion of the plastic surgeon or neurosurgeon. To inform best practice guidelines, we sought to obtain data surrounding the frequency at which craniofacial surgeons order computed tomography (CT), as well as indications. We hypothesized that we would identify considerable variation in both imaging and associated indications. METHODS: We surveyed members of the American Society of Maxillofacial Surgeons and the American Society of Craniofacial Surgeons to measure the frequency of preoperative and postoperative head CTs, as well as indications. Initial items were piloted with 2 craniofacial surgeons and 1 neurosurgeon, using interviews to ensure content validity. χ2 Tests were used to measure associations between operative volume, years in practice, and imaging. RESULTS: Eighty-five craniofacial surgeons responded (13.8% response rate), with the majority (63.5%) having performed a craniosynostosis operation in the last month. Only 9.4% of surgeons never order preoperative CTs. Of those who do, the most common indications included diagnosis confirmation (31.2%) and preoperative planning (27.3%). About 25% of surgeons always obtain postoperative head CTs, usually to evaluate surgical outcomes (46.7%). Only 13.3% of respondents order 2 or more postoperative scans. Higher operative volume was associated with a lower likelihood of ordering preoperative head CTs (P = 0.008). CONCLUSIONS: The majority of surgeons obtain preoperative head CTs, whereas only 25% obtain CTs postoperatively, often to evaluate outcomes. Because outcomes may be evaluated clinically, this is a poor use of resources and exposes children to radiation. Consensus guidelines are needed to create best practices and limit unnecessary studies.

Non-thermal histotripsy tumor ablation promotes abscopal immune responses that enhance cancer immunotherapy.

BACKGROUND: Developing 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. METHODS: Immunocompetent 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. RESULTS: Histotripsy 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. CONCLUSIONS: These 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.