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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session C

Poster F7 - Interrogating the Cellular Impact of Sonodynamic Therapy on Glioma Cells

Friday, October 25, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Justin Vinh

Undergraduate Research Assistant
University of Virginia
Sterling, Virginia, United States
Ryan Erny (he/him/his)

Undergraduate Research Assistant
University of Virginia, Virginia, United States

Co-Author(s)

AT

Andrew Thede

Graduate Research Assistant
University of Virginia, United States
MD

Matthew DeWitt

Senior Scientist
University of Virginia, United States

Primary Investigator(s)

Natasha D. Sheybani, PhD (she/her/hers)

Assistant Professor of Biomedical Engineering, Radiology & Medical Imaging (by courtesy), and Neurosurgery (by courtesy), Research Director of UVA Focused Ultrasound Cancer Immunotherapy Center
University of Virginia
Charlottesville, Virginia, United States

Introduction: Glioblastoma (GBM) is one of the most prevalent and deadliest types of primary brain cancers in adults, possessing a 5-year survival rate of only 5.8%. One novel method of treating GBM is sonodynamic therapy (SDT), which utilizes focused ultrasound in conjunction with a sonosensitizer to elicit a cytotoxic effect. This effect is made possible through the preferential uptake of the sonosensitizer 5-aminolevulinic acid (5-ALA) by tumor cells and induction of metabolite protoporphyrin IX (PpIX). PpIX experiences sonoluminescence when exposed to ultrasound and induces the production of highly destructive reactive oxygen species, hypothesized to be the main driver of the cytotoxic effect (Figure 1A). Existing literature surrounding SDT largely lacks well-conserved reporting of parameters. Given the highly variable nature of in vitro SDT experimental procedures, it is essential to confirm and validate their selection. This study sets out to establish a high-throughput multimodal platform for deploying SDT in vitro and systematically assessing critical parameters including 5-ALA dosing, acoustic exposure conditions, incubation period, and experimental setup in 2D and 3D GBM culture settings. Additionally, this study aims to examine the effects of SDT on extracellular vesicles (EVs) release post-sonication. Given the role of EVs in cell signaling, their characterization after exposure to SDT can provide fundamental insight into biological mechanisms underpinning this therapy as well as yield insights that may be actionable for adjunct therapy selection or biomarker discovery of translational relevance.

Materials and

Methods: Murine (GL261) and human (G28) glioblastoma cell lines were cultured in 2D monolayer and 3D neurosphere format, respectively. Cells were seeded in 24-well plates 72 hrs prior to sonication then dosed with 1 mM 5-ALA 4 hrs prior to sonication. A custom-built ultrasound system comprised of a 1.1 MHz single-element unfocused transducer with 1 cm aperture was used to perform SDT regimens. A motorized XY staging mechanism maneuvered specific wells to the treatment area above the transducer (Figure 1B). The well plate was sealed, submerged, and kept in the dark for the duration of treatment. SDT exposure conditions included 10-40% duty cycle, 100-120 mV power, and 5 min sonication duration (Figure 1D, E, F, G). Viability was assessed via bioluminescence assay at 0.5 and 24 hrs post-sonication. Isolation of extracellular vesicles (EVs) was conducted using ExoQuick-TC Ultra, with size and concentration analysis conducted via ZetaView nanoparticle tracking analysis.

Results, Conclusions, and Discussions: Results. The window of PpIX accumulation was experimentally defined to select 5-ALA dose and timing for SDT studies. These parameters were determined to be 1.0 mM and 4-8 hour incubation period across both cell lines (Figure 1C, H). In 2D culture, SDT duty cycle and power were both determined to influence cell viability response. Increases in these parameters evoked a general trend in decreased viability relative to untreated control (Figure 1D, F). Needle thermocouple measurements reflected a duty cycle-dependent temperature rise of < 2°C and < 4° C at 100 mV and 120 mV, respectively (Figure 1E, G). EV concentration under SDT and FUS-only exposure conditions were observed to trend higher than untreated control.

Discussion & Conclusions. Cell viability post-SDT was consistently less than untreated control wells. However, viability decreases from SDT protocols at higher exposure, reaching up to a power of 120 mV and/or 40% duty cycle, were not significantly different from their FUS-only counterparts (Fig. 1D, F), suggesting potential influence of secondary mechanical effects since thermocouple measurements only reflected negligible to moderate temperature rises. Ongoing studies are continuing to deconvolve this parameter space and fortify EV analyses, as well as extend SDT insights from 2D to 3D culture settings.

Acknowledgements (Optional): We, the authors, would like to express our deepest gratitude to Dr. Natasha Sheybani for her guidance and support of the research project as well as to Andrew Thede and Dr. Matthew DeWitt for their technical contributions to our project.