Mechanical High-Intensity Focused Ultrasound (Histotripsy) in Dogs With Spontaneously Occurring Soft Tissue Sarcomas

Abstract

Introduction: Histotripsy is a non-invasive focused ultrasound therapy that uses controlled acoustic cavitation to mechanically disintegrate tissue. To date, there are no reports investigating histotripsy for the treatment of soft tissue sarcoma (STS).

Objective: This study aimed to investigate the in vivo feasibility of ablating STS with histotripsy and to characterize the impact of partial histotripsy ablation on the acute immunologic response in canine patients with spontaneous STS.

Methods: A custom 500 kHz histotripsy system was used to treat ten dogs with naturally occurring STS. Four to six days after histotripsy, tumors were surgically resected. Safety was determined by monitoring vital signs during treatment and post-treatment physical examinations, routine lab work, and owners' reports. Ablation was characterized using radiologic and histopathologic analyses. Systemic immunological impact was evaluated by measuring changes in cytokine concentrations, and tumor microenvironment changes were evaluated by characterizing changes in infiltration with tumor-associated macrophages (TAMs) and tumor-infiltrating lymphocytes (TILs) using multiplex immunohistochemistry and differential gene expression.

Results: Results showed histotripsy ablation was achievable and well-tolerated in all ten dogs. Immunological results showed histotripsy induced pro-inflammatory changes in the tumor microenvironment. Conclusion & Significance: Overall, this study demonstrates histotripsy's potential as a precise, non-invasive treatment for STS.

Fig. 1.

Experimental histotripsy set-up. A robotic micro-positioner is connected to an articulating arm supporting the therapy and imaging transducer assembly. The transducer assembly was submerged in a water coupling bowl coupled to the patient’s tumor, and treatment was monitored in real-time using ultrasound imaging.

Fig. 2.

Study workflow. After patient-specific treatment plan development, fur was removed and patients were anesthetized. An automated histotripsy treatment was conducted using custom planning software. Post-treatment images were collected before surgical resection of the tumor.

Fig. 3.

Histotripsy treatment feasibility and safety were measured using CT and US imaging and adverse event monitoring. Representative images from three canine patients show (from left to right) pre-treatment CT scans of STS (circled), post-treatment CT images with clear regions of histotripsy-ablated tissue 1 day and 4 to 6 days after treatment (arrows), bubble clouds during treatment on real-time US imaging, and various degrees of self-limiting cutaneous injury in the histotripsy treatment path after treatment. Darkened skin on Patient #10 is the result of unrelated sun exposure prior to histotripsy treatment.

Fig. 4.

Representative images demonstrating histotripsy ablation of STS for Patient #5. (a) Gross visualization of lesion characterized by extensive tissue necrosis and hemorrhage (arrow). (b-f) H&E stained sections compared (d) untreated (magnification 40x) and (e) treated (magnification 40x) tumor tissues and (b,c,f) interface regions (c – magnification 2x; f – magnification 40x). Clearly delineated boundaries between treated (+) and untreated (*) tumor tissue were observed.

Fig. 5.

Multiplex IHC to investigate macrophage populations following histotripsy. Lower numbers of IBA-1/CD206 double positive cells (orange/red) were distributed throughout untreated tumor samples (a) compared to treated samples (b). When captured, the interface between necrotic and intact tumor cells (c) often showed slightly increased numbers of double positive cells (* = area of necrosis; arrows = interface between necrotic and viable cells; a,b – magnification 40x; c – magnification 20x).

Fig. 6.

Hierarchical clustering of 79 differentially expressed immuno-oncology genes. Increasing orange intensity indicates increased gene expression and increasing blue intensity indicates decreased gene expression, as shown in the scale bar. According to the differential gene expression profile, samples were clustered with patient held as a confounder and arranged with the treated samples at the top of the figure (blue bar) and the untreated samples at the bottom of the figure (yellow bar).