Investigating cell death responses associated with histotripsy ablation of canine osteosarcoma

Abstract

Background

Osteosarcoma (OS) is the most frequently occurring primary bone tumor in dogs and people and innovative treatment options are profoundly needed. Histotripsy is an emerging tumor ablation modality, and it is essential for the clinical translation of histotripsy to gain knowledge about the outcome of nonablated tumor cells that could remain postablation. The objective of this study was to characterize the cell death genetic signature and proliferation response of canine OS cells post a near complete histotripsy ablation (96% ± 1.5) and to evaluate genetic cell death signatures associated with histotripsy ablation and OS in vivo.

Methods

In the current study, we ablated three canine OS cell lines with a histotripsy dose that resulted in near complete ablation to allow for a viable tumor cell population for downstream analyses. To assess the in vivo cell death genetic signature, we characterized cell death genetic signature in histotripsy-ablated canine OS tumors collected 24-h postablation.

Results

Differential gene expression changes observed in the 4% viable D17 and D418 cells, and histotripsy-ablated OS tumor samples, but not in Abrams cells, were associated with immunogenic cell death (ICD). The 4% viable OS cells demonstrated significantly reduced proliferation, compared to control OS cells, in vitro.

Conclusion

Histotripsy ablation of OS cell lines leads to direct and potentially indirect cell death as evident by, reduced proliferation in remaining viable OS cells and cell death genetic signatures suggestive of ICD both in vitro and in vivo.

Keywords:

• Focused ultrasound
• tumor ablation
• bone cancer
• cell death
• comparative oncology

Acknowledgments

The authors would like to acknowledge Vlaisavljevich lab members Alex Simon, Hannah Covell, and former member Alissa Hendricks-Wenger for their efforts in equipment and experimental design for the in vitro histotripsy systems used in this study. Lauren Ruger was supported by an ICTAS Doctoral Scholarship from the Virginia Tech Institute for Critical Technology and Applied Science. Jessica Gannon was supported by an NSF Graduate Research Fellowship.

Authors’ contributions

A.H. – Primary contributor to the composition of the manuscript, lead the execution of all experiments, and performed data analysis.

E.R.V- Assisted in histotripsy equipment design, in vitro experiments, and contributed to manuscript writing.

M.P.- Assisted with in vitro experiments. Contributed to manuscript revision.

J.G. and L.R.- Assisted in histotripsy equipment design and experimental design, and trained A. H. to perform in-vitro treatments for the current study. Contributed to manuscript revision.

I.C.A- Contributed to study design and manuscript revision.

E.V.- Oversaw the engineering of the histotripsy equipment and study design. Contributed to manuscript writing and revising.

J.T – Oversaw study design and contributed to manuscript writing and revising.

All authors reviewed the manuscript.

Disclosure statement

Eli Vlaisavljevich has an ongoing research partnership and financial relationship with HistoSonics, Inc. No other authors have a conflict of interest to report. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Data availability

The data that support the findings of this study are available from the corresponding author, [J.L.T.], upon reasonable request.

Additional information

Funding

NIH funding (Project ID 1R21EB030182-01)(J.T.) and NIH NCI (R01CA269811) (I.C.A), American Kennel Club (Canine Health Foundation No. 02773)(J.T.) and Focused Ultrasound Foundation (FUSF-RAP-823R1)(J.T.) helped support the design and reporting of this study.