Insights from in vivo preclinical cancer studies with histotripsy

Figures & data

Figure 1. Preclinical investigation of histotripsy for cancer treatment. Histotripsy is a noninvasive, non-ionizing, and non-thermal ablation technique that mechanically fractionates target tissue into acellular homogenate via controlled acoustic cavitation. Intrinsic threshold histotripsy, shock-scattering histotripsy, and boiling histotripsy approaches are being investigated preclinically for treating cancers of various anatomic locations in large and small animal tumor models. This review discusses the important findings from in vivo histotripsy cancer studies reporting on the safety, feasibility, survival outcomes, characterization of post-treatment tumor response, and immune effects.

Table 1. Histotripsy treatment parameters and study highlights.

Study	Histotripsy approach and parameters	Tumor model	Study highlights
Worlikar et al. [40]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency 1–2 cycle pulse duration 100 Hz PRF 50 pulses/point 0.5–0.75 mm spacing Estimated p‒ ∼30 MPa	Liver SQ Hepatocellular carcinoma Hep3B SQ in NSG/NOD-scid mice	Histotripsy was used successfully to treat subcutaneous tumors. Tumor burden reduction and improved survival were observed in histotripsy-treated animals compared to controls.
Hendricks-Wenger et al. [41]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency <2 cycle pulse duration 250, 500, and 1000 pulses/point, 1.5 mm axial, 0.5 mm lateral spacing Estimated p‒ 19–33 Mpa	Liver SQ Cholangiocarcinoma patient derived xenograft SQ in NSG mice	The dosimetry study demonstrated that all doses generated complete ablation; however, increasing the histotripsy dose resulted in more well-defined treatment margins.
Hendricks-Wenger et al. [42]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency <2 cycle pulse duration 500 Hz PRF 500 pulses/point, 0.5 mm axial, 0.25 mm lateral/elevational spacing	Liver SQ Cholangiocarcinoma patient derived xenograft in NSG mice	Improved tumor-progression-free survival and overall survival were observed in histotripsy-treated animals compared to controls.
Qu et al. [43]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency 1–2 cycle pulse duration 100 Hz PRF 50 pulses/point 0.4 to 0.7 mm spacing Estimated p‒ 30 MPa	Liver SQ Hepa 1-6 in C57BL/6 mice Melanoma SQ B16GP33 in C57BL/6 mice	Histotripsy promoted local and systemic anti-tumor immune effects and improved the efficacy of checkpoint inhibition immunotherapy.
Worlikar et al. [7]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency 1–2 cycle pulse duration 100 Hz PRF 50 pulses/point 0.5–0.7 mm lateral, elevational 0.75–0.9 mm axial spacing Estimated p‒ 30 MPa	Liver OT N1S1 in Sprague Dawley rats	Histotripsy was used successfully for treating orthotopic liver tumors noninvasively. Tumor burden reduction and improved tumor-progression-free survival were observed in histotripsy-treated animals (even when the tumor was ablated partially) compared to controls.
Worlikar et al. [44]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency 1–2 cycle pulse duration 100 Hz PRF Scan velocity ∼6 mm/s, Estimated p‒ 30 MPa	Liver OT McA-RH7777 in Sprague Dawley rats	Anti-tumor immune effects and improved tumor-progression-free survival were observed in histotripsy-treated animals compared to controls.
Pepple et al. [45]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency 1–2 cycle pulse duration 100 Hz PRF 50 pulses/point Estimated p‒ 30 MPa	Liver SQ Hepa 1-6 in C57BL/6 mice Melanoma SQ B16F10 in C57BL/6 mice	Histotripsy promoted necroptotic immunogenic cell death of ablated tumor and ferroptotic cancer cell death in abscopal, untreated tumor. Histotripsy homogenate was used as a tumor vaccine with the ability to inhibit tumor growth.
Singh et al. [46]	Boiling Histotripsy 1.5 MHz transducer frequency 1 Hz PRF 1% duty cycle Acoustic power: 450 W 10 s exposure	Melanoma SQ B16F10 in C57BL/6 mice	Histotripsy augmented checkpoint inhibition immunotherapy in poorly immunogenic tumors.
Choi et al. [48]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency 1.5 cycle pulse duration 1 Hz PRF 100 pulses single point Estimated p‒ 32 MPa	Brain OT GL261 in C57BL/6 albino mice	Histotripsy was used successfully to target orthotopic brain tumors noninvasively using a stereotactic MR-guided system.
Choi et al. [49]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency 5 Hz PRF 15 pulses/point 0.25 mm spacing Estimated p‒ 36 MPa	Brain OT GL261 in C57BL/6 mice	The radiological and pathological characterization of the temporal response following histotripsy ablation was described.
Gerhardson et al. [50]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency 1–2 cycle pulse duration 1 Hz PRF 50 pulses single point Estimated p‒ 36 MPa	Brain OT GL261 in C57BL/6 albino mice	Histotripsy promoted immunomodulatory effects in murine brain tumors.
Duclos et al. [51]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency 1 cycle pulse duration 5 Hz PRF 5, 20 or 200 pulses at a single point, or 5 or 10–20 pulses/point at multiple treatment points, 0.25 mm spacing Estimated p‒ 30 MPa	Brain OT GL261 and LL/2-Luc2 in C57BL/6 mice	A dosimetry study demonstrated that lower doses were able to achieve tumor destruction with minimal bleeding effects as compared to higher doses.
Iwanicki et al. [52]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency 2 cycle pulse duration 50 Hz PRF 0.33 mm/s scan velocity Estimated p‒ 35 MPa	Brain OT Neuroblastoma NGP-Luciferase in NCr mice	Histotripsy resulted in effective tumor ablation, promoted apoptotic cell death, impacted tumor vasculature, and reduced hypoxia.
Eranki et al. [53]	Boiling Histotripsy 1.5 MHz transducer frequency 1 Hz PRF 13.33 ms pulse duration p + 85 MPa and p‒ 14 MPa 15s exposure	Brain SQ Neuro2a in A/J mice	Histotripsy resulted in abscopal effects on untreated tumor, promoted immune sensitization of poorly immunogenic tumors to checkpoint inhibition immunotherapy, and improved survival outcomes.
Hendricks-Wenger et al. [54]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency <2 cycle pulse duration 250 Hz PRF 250 pulses/point 0.75 mm transverse 1.25 mm axial spacing	Pancreas SQ Pan02 SQ in C57BL/6 mice	Histotripsy promoted anti-tumor immune response and resulted in improved tumor-free and overall survival as compared to controls.
Imran et al. [55]	Intrinsic Threshold Histotripsy 500 kHz transducer frequency 1 cycle pulse duration 500 Hz PRF 1000 pulses/point 3.5 mm axial, 1.5 mm lateral/elevational spacing Estimated p‒ 31.2 MPa	Pancreas OT Panc1 in RAG2/IL2RG-deficient immunocompromised pigs	Histotripsy was used successfully for targeting orthotopic pancreatic tumors in a porcine model.
Mouratidis et al. [56]	Boiling Histotripsy 1.5 MHz transducer frequency 10 ms pulse duration 1 Hz PRF p + 32 MPa and p‒ 17 MPa Duty cycle: 1% 25s exposure Acoustic power: 132 W	Pancreas SQ KPC cells (KrasG12D/+; Trp53R172H/+; Pdx-1-Cre) in C57BL/6 mice	Histotripsy promoted anti-tumor immune effects and improved the efficacy of checkpoint inhibition immunotherapy.
Hendricks-Wenger et al. [58]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency <2 cycle pulse duration RencaRCC and 4T1: 250 Hz PRF 250 pulses/point 1.5 mm axial and 0.5 mm lateral spacing Cholangiocarcinoma: 500 Hz PRF 500 pulses/point 0.75 mm axial and 0.25 mm lateral spacing Estimated p‒ ∼32 MPa	Renal SQ RencaRCC in BALB/cCr mice Breast SQ 4T1 in BALB/C mice Liver SQ Cholangiocarcinoma patient-derived xenograft in NSG mice	Histotripsy was used successfully to target subcutaneous tumors; a higher dose was necessary to effectively ablate stiffer tumors.
Ashar et al. [59]	Boiling Histotripsy 1 MHz transducer frequency 5 Hz PRF Duty cycle: 1% 20–25s exposure Acoustic power: 450–600 W	Spontaneously arising mammary mass, papillary adenocarcinoma, soft tissue sarcoma, lipoma, and low-grade mast cell tumor in canine patients	Partial histotripsy temporarily inhibited tumor growth and induced immune response. However, tumor progression occurred eventually.
Nam et al. [60]	Boiling Histotripsy 1.5 MHz transducer frequency 10 ms pulse duration 1 Hz PRF p + 90 MPa and p‒ 17 MPa Duty cycle: 1% 25 to 50 pulses/point	Breast SQ 4T1 in BALB/C mice Colorectal SQ CT26 in BALB/C mice	Histotripsy promoted anti-tumor immune effects and improved the efficacy of checkpoint inhibition immunotherapy.
Ruger et al. [61]	Intrinsic Threshold Histotripsy 500 kHz transducer frequency 1 cycle pulse duration 500 Hz PRF 1000 pulses/point 3.5 mm axial 1.5 mm lateral/ elevational spacing Estimated p‒ 26.57 ± 3.82 MPa	Spontaneously arising osteosarcoma and chondrosarcoma in canine patients	Histotripsy of osteosarcoma with an increased dose resulted in more extensive tissue destruction compared to a lower dose from a previous study. The radiological and pathological characterization of post-treatment tumor was described.
Ruger et al. [62]	Intrinsic Threshold Histotripsy 500 kHz transducer frequency 1 cycle pulse duration 500 Hz PRF 500 pulses/point 3.5 mm axial 1.5 mm lateral/ elevational spacing Estimated p‒ 22.60 ± 7.21 MPa	Spontaneously arising soft tissue sarcoma in canine patients	Histotripsy promoted immune infiltration in the tumors. Treatment was well tolerated with no significant adverse events.
Ruger et al. [63]	Intrinsic Threshold Histotripsy 500 kHz transducer frequency 1 cycle pulse duration 500 Hz PRF 500 pulses/point 3.5 mm axial 1.5 mm lateral/ elevational spacing Estimated p‒ 29.59 ± 8.17 MPa	Spontaneously arising osteosarcoma and chondrosarcoma in canine patients	Histotripsy treatment was well tolerated with no significant adverse events in this pilot study.
Ruger et al. [64]	Intrinsic Threshold Histotripsy 1 MHz transducer frequency 1 cycle pulse duration 500 Hz PRF 500 pulses/point, 1.25 mm axial 0.75 mm lateral/ elevational Estimated p‒ 29.59 ± 6.08 MPa	Spontaneously arising soft tissue sarcoma in feline patients	Histotripsy induced mild immune infiltration and was well tolerated with no significant adverse events.
Styn et al. [65] Styn et al. [66]	Shock-scattering Histotripsy 1 MHz transducer frequency 3 cycle pulse duration 300 Hz PRF Estimated p‒ ∼20 MPa	Renal OT VX2 cells in New Zealand rabbit kidney	Histotripsy was used successfully for treating orthotopic renal tumors noninvasively. There was no statistically significant increase in pulmonary metastasis development in treated animals as compared to controls.
Schade et al. [67] Brisbane et al. [68]	Boiling Histotripsy 1.5 MHz transducer frequency 10 ms pulse duration 1 Hz PRF Duty cycle: 1% p+ 90–100 MPa and p‒ 17–20 MPa Electric power: 525–575 W 10 pulses/point	Renal OT De novo tumors in Eker rats	Histotripsy was used successfully for treating orthotopic renal tumors noninvasively. Histotripsy induced inflammatory immune response. The radiological and histological characterization of tumor response was reported.
Schade et al. [69]	Shock-scattering Histotripsy 750 kHz transducer frequency 5 cycle pulse duration 500 Hz PRF Duty cycle: 1%	Prostate OT ACE-1 tumors in canine	The feasibility of targeting orthotopic prostate tumors with histotripsy was demonstrated in canines.
Chevillet et al. [70]	Boiling Histotripsy 1.5 MHz transducer frequency 10 ms pulse duration 1 Hz PRF p+ 90 MPa and p‒ 17 MPa Duty cycle: 1% 30s exposure	Prostate SQ MatLyLu cells in Copenhagen rats	Histotripsy induced an increase in tumor-derived microRNA in the plasma within hours post-treatment.
Hoogenboom et al. [71]	Boiling Histotripsy 3 MHz transducer frequency 5 ms pulse duration 1 Hz PRF p+ 90 MPa and p‒ 17 MPa Duty cycle: 0.5% 150 pulses/point Acoustic power: 155–160 W	Thymoma SQ EL4 in C57Bl/6NCrl mice	The radiological and pathological characterization of histotripsy-treated tumors was described.
Hoogenboom et al. [72]	Boiling Histotripsy 3 MHz transducer frequency 5 ms pulse duration Duty cycle: 1% 100–200 pulses/point Acoustic power: 155–160 W	Lymphoma SQ EL4 in C57Bl/6NCrl mice Melanoma SQ B16OVA in C57Bl/6NCrl mice Neuroblastoma SQ 9464D in C57Bl/6NCrl mice	Increased histotripsy dose was required to achieve ablation in structurally stiffer tumors. Optimal histotripsy dose should be tailored to tissue properties.
Bijgaart et al. [73]	Boiling Histotripsy 3 MHz transducer frequency 5 ms pulse duration Duty cycle: 1% 100–200 pulses/point Acoustic power: 155–160 W	Lymphoma SQ EG7-OVA in C57BL/6NCrL mice	Histotripsy induced distinct RNA, DNA, and protein profiles of the ablated material, resulting in anti-tumor immune response.

OT, orthotopic tumors; PRF, pulse repetition frequency; SQ, subcutaneous tumors.

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Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.