Insights from in vivo preclinical cancer studies with histotripsy.

Histotripsy is the first noninvasive, non-ionizing, and non-thermal ablation technique that mechanically fractionates target tissue into acellular homogenate via controlled acoustic cavitation. Histotripsy has been evaluated for various preclinical applications requiring noninvasive tissue removal including cancer, brain surgery, blood clot and hematoma liquefaction, and correction of neonatal congenital heart defects. Promising preclinical results including local tumor suppression, improved survival outcomes, local and systemic anti-tumor immune responses, and histotripsy-induced abscopal effects have been reported in various animal tumor models. Histotripsy is also being investigated in veterinary patients with spontaneously arising tumors. Research is underway to combine histotripsy with immunotherapy and chemotherapy to improve therapeutic outcomes. In addition to preclinical cancer research, human clinical trials are ongoing for the treatment of liver tumors and renal tumors. Histotripsy has been recently approved by the FDA for noninvasive treatment of liver tumors. This review highlights key learnings from in vivo shock-scattering histotripsy, intrinsic threshold histotripsy, and boiling histotripsy cancer studies treating cancers of different anatomic locations and discusses the major considerations in planning in vivo histotripsy studies regarding instrumentation, tumor model, study design, treatment dose, and post-treatment tumor monitoring.

Impact of immune tolerance mechanisms on the efficacy of immunotherapy in primary and secondary liver cancers.

The liver is a functionally unique organ with an immunosuppressive microenvironment. The liver is the sixth most common site of primary cancer in humans and is a frequent site of metastasis from other solid tumors. The development of effective therapies for primary and metastatic liver cancer has been challenging due to the complex metabolic and immune microenvironment of the liver. The liver tumor microenvironment (TME) in primary and secondary (metastatic) liver cancers is heterogenous and consists of unique immune and stromal cell populations. Crosstalk between these cell populations and tumor cells creates an immunosuppressive microenvironment within the liver which potentiates cancer progression. Immune checkpoint inhibitors (ICIs) are now clinically approved for the management of primary and secondary liver cancer and can partially overcome liver immune tolerance, but their efficacy is limited. In this review, we describe the liver microenvironment and the use of immunotherapy in primary and secondary liver cancer. We discuss emerging combination strategies utilizing locoregional and systemic therapy approaches which may enhance efficacy of immunotherapy in primary and secondary liver cancer. A deeper understanding of the immunosuppressive microenvironment of the liver will inform novel therapies and therapeutic combinations in order to improve outcomes of patients with primary and secondary liver cancer.

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.

Impact of Histotripsy on Development of Intrahepatic Metastases in a Rodent Liver Tumor Model.

Histotripsy has been used for tumor ablation, through controlled, non-invasive acoustic cavitation. This is the first study to evaluate the impact of partial histotripsy ablation on immune infiltration, survival outcomes, and metastasis development, in an in vivo orthotopic, immunocompetent rat HCC model (McA-RH7777). At 7−9 days post-tumor inoculation, the tumor grew to 5−10 mm, and ~50−75% tumor volume was treated by ultrasound-guided histotripsy, by delivering 1−2 cycle histotripsy pulses at 100 Hz PRF (focal peak negative pressure P− >30 MPa), using a custom 1 MHz transducer. Complete local tumor regression was observed on MRI in 9/11 histotripsy-treated rats, with no local recurrence or metastasis up to the 12-week study end point, and only a <1 mm residual scar tissue observed on histology. In comparison, 100% of untreated control animals demonstrated local tumor progression, developed intrahepatic metastases, and were euthanized at 1−3 weeks. Survival outcomes in histotripsy-treated animals were significantly improved compared to controls (p-value < 0.0001). There was evidence of potentially epithelial-to-mesenchymal transition (EMT) in control tumor and tissue healing in histotripsy-treated tumors. At 2- and 7-days post-histotripsy, increased immune infiltration of CD11b+, CD8+ and NK cells was observed, as compared to controls, which may have contributed to the eventual regression of the untargeted tumor region in histotripsy-treated tumors.

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.

Effects of Histotripsy on Local Tumor Progression in an in vivo Orthotopic Rodent Liver Tumor Model.

OBJECTIVE AND IMPACT STATEMENT: This is the first longitudinal study investigating the effects of histotripsy on local tumor progression in an in vivo orthotopic, immunocompetent rat hepatocellular carcinoma (HCC) model. INTRODUCTION: Histotripsy is the first noninvasive, nonionizing, nonthermal, mechanical ablation technique using ultrasound to generate acoustic cavitation to liquefy the target tissue into acellular debris with millimeter accuracy. Previously, histotripsy has demonstrated in vivo ablation of noncancerous liver tissue. METHODS: N1-S1 HCC tumors were generated in the livers of immunocompetent rats (n = 6, control; n = 15, treatment). Real-time ultrasound-guided histotripsy was applied to ablate either 100% tumor volume + up to 2mm margin (n = 9, complete treatment) or 50-75% tumor volume (n = 6, partial treatment) by delivering 1-2 cycle histotripsy pulses at 100 Hz PRF (pulse repetition frequency) with p - ≥30MPa using a custom 1MHz transducer. Rats were monitored weekly using MRI (magnetic resonance imaging) for 3 months or until tumors reached ~25mm. RESULTS: MRI revealed effective post-histotripsy reduction of tumor burden with near-complete resorption of the ablated tumor in 14/15 (93.3%) treated rats. Histopathology showed <5mm shrunken, non-tumoral, fibrous tissue at the treatment site at 3 months. Rats with increased tumor burden (3/6 control and 1 partial treatment) were euthanized early by 2-4 weeks. In 3 other controls, histology revealed fibrous tissue at original tumor site at 3 months. There was no evidence of histotripsy-induced off-target tissue injury. CONCLUSION: Complete and partial histotripsy ablation resulted in effective tumor removal for 14/15 rats, with no evidence of local tumor progression or recurrence.

Histotripsy for Non-Invasive Ablation of Hepatocellular Carcinoma (HCC) Tumor in a Subcutaneous Xenograft Murine Model.

Histotripsy fractionates tissue through a mechanical, non-invasive ultrasonic ablation process that precisely controls acoustic cavitation while utilizing real-time ultrasound (US) imaging guidance. This study investigates the potential, feasibility and tumor volume reduction effects of histotripsy for liver cancer ablation in a subcutaneous in vivo murine Hepatocellular Carcinoma (HCC) model. Hep3B tumors were generated in the right flanks of 14 NSG and 7 NOD-SCID mice. The mice were grouped as follows: A (acute, NSG with n=9 treatment and n=1 control), B (chronic, NSG with n=2 treatment and n=2 control) and C (chronic NODSCID, with n=6 treatment and n=1 control). Treatment was performed when the tumor diameters reached >5 mm. 1-2 cycle histotripsy pulses at 100 Hz PRF (p- >30 MPa) were delivered using a custom built 1 MHz therapy transducer attached to a motorized positioner, which scanned the transducer focus to traverse the targeted tumor volume, guided by real-time US imaging. Tumor ablation effectiveness was assessed by obtaining T1, T2 and T2* weighted MR images. Post euthanasia, treated tumor, brain, and lung tissue samples were harvested for histology. Histology of acute group A showed fractionation of targeted region with a sharp boundary separating it from untreated tissue. Groups B and C demonstrated effective tumor volume reduction post treatment on MRI as the homogenate and edema were resorbed within 23 weeks. However, as the tumor was subcutaneous, it was not possible to set adequate treatment margin and since the mice were immune-compromised, residual viable tumor cells eventually developed into tumor regrowth at 3-9 weeks after histotripsy. Groups B and C showed no signs of metastasis in the lung and brain. Our study successfully demonstrated the potential of histotripsy for non-invasive HCC ablation in a subcutaneous murine model. Additional work is ongoing to study the response of histotripsy in immune-competent orthotopic liver tumor models.

Integrated Histotripsy and Bubble Coalescence Transducer for Rapid Tissue Ablation.

Residual bubbles produced after collapse of a cavitation cloud provide cavitation nuclei for subsequent cavitation events, causing cavitation to occur repeatedly at the same discrete set of sites. This effect, referred to as cavitation memory, limits the efficiency of histotripsy soft tissue fractionation. Besides passively mitigating cavitation memory by using a low pulse repetition frequency (~1 Hz), an active strategy was developed by our group. In this strategy, low-amplitude ultrasound sequences were used to stimulate coalescence of residual bubbles. The goal of this work is to remove cavitation memory and achieve rapid, homogeneous lesion formation using a single phased array transducer. A 1-MHz integrated histotripsy and bubble coalescing (BC) transducer system with a specialized electronic driving system was built in house. High-amplitude ( MPa) histotripsy pulses and subsequent low-amplitude (~1-2 MPa) BC sequences were applied to a red blood cell tissue-mimicking phantom at a single focal site. Significant reduction of the cavitation memory effect and increase in the fractionation rate were observed by introducing BC sequence. Effects of BC pulsing parameters were further studied. The optimal BC parameters were then utilized to homogenize a mm2 region at high rate.