Histotripsy induces apoptosis and reduces hypoxia in a neuroblastoma xenograft model

Abstract

Background

Neuroblastoma (NB) is the most common extracranial solid tumor of childhood, and high-risk disease is resistant to intensive treatment. Histotripsy is a focused ultrasound therapy under development for tissue ablation via bubble activity. The goal of this study was to assess outcomes of histotripsy ablation in a xenograft model of high-risk NB.

Methods

Female NCr nude mice received NGP-luciferase cells intrarenally. Under ultrasound image guidance, histotripsy pulses were applied over a distance of 4–6 mm within the tumors. Bioluminescence indicative of tumor viability was quantified before, immediately after, and 24 h after histotripsy exposure. Tumors were immunostained to assess apoptosis (TUNEL), endothelium (endomucin), pericytes (αSMA), hypoxia (pimonidazole), vascular endothelial growth factor A (VEGFA), and platelet-derived growth factor-B (PDGF-B). The apoptotic cytokine TNFα and its downstream effector cleaved caspase-3 (c-casp-3) were assessed with SDS-PAGE.

Results

Histotripsy induced a 50% reduction in bioluminescence compared to untreated controls, with an absence of nuclei in the treatment core surrounded by a dense rim of TUNEL-positive cells. Tumor regions not targeted by histotripsy also showed an increase in TUNEL staining density. Increased apoptosis in histotripsy samples was consistent with increases in TNFα and c-casp-3 relative to controls. Treated tumors exhibited a decrease in hypoxia, VEGF, PDGF-B, and pericyte coverage of vasculature compared to control samples. Further, increases in vasodilation were found in histotripsy-treated specimens.

Conclusions

In addition to ablative effects, histotripsy was found to drive tumor apoptosis through intrinsic pathways, altering blood vessel architecture, and reducing hypoxia.

GRAPHICAL ABSTRACT

Keywords:

• Histotripsy
• neuroblastoma
• hypoxia
• VEGF
• PDGFB
• apoptosis

Acknowledgments

We thank The University of Chicago Integrated Light Microscopy Core, especially Shirley Bond and Christine Labno, for their assistance with slide scanning and image analysis, which receives financial support from the Cancer Center Support Grant (P30CA014599). RRID: SCR_019197.

Classification

major: Cell Biology, minor: applied Physical Sciences.

Author contributions

Conception and design: HSL, BKB.

Development of methodology: BKB, HSL, IJI, WLL, FFG.

Acquisition of data: HSL, BKB, IJI, WLL, FFG, SR, VGP, CSC.

Analysis and interpretation of data: HSL, BKB, IJI, WLL, FFG, NR, JJK.

Writing, review, and/or revision of the manuscript: HSL, BKB, AM, CC, WL, IJI, JJK.

Administrative, technical, or material support: HSL, BKB, IJI, WLL, FFG.

Disclosure statement

None of the authors have competing interests.

Data availability statement

All data and protocols are available: 10.6084/m9.figshare.22557934.

Additional information

Funding

This work was supported by Focused Ultrasound Foundation; Feis Family Fellowship; Sorkin Fellowship. Funding for this project was provided by the Pediatric Cancer Foundation, The University of Chicago Human Tissue Resource Center, the Feis Family, the American Cancer Society (Grant # RSG-21-171-01-ET) and National Cancer Institute (Grant # R01 CA235756).