Single cell level analysis of ATP release kinetics and cell fate following ultrasound targeted microbubble cavitation using microscopy techniques

Amate, M.; Tan, J. J.; Boudreault, F.; Grygorczyk, R.; Gervais, T.; Yu, F.

doi:10.1101/2025.02.02.636151

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bioRxiv · 10.1101/2025.02.02.636151

Single cell level analysis of ATP release kinetics and cell fate following ultrasound targeted microbubble cavitation using microscopy techniques

Amate, M.·Tan, J. J.·Boudreault, F.·Grygorczyk, R.·Gervais, T.·Yu, F.

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Abstract

It is known that ultrasound-targeted microbubble cavitation (UTMC) can induce vasodilation. This image guided spatially targeted approach is called provascular therapy when used as a radiotherapy sensitizer in radiation oncology. Extracellular adenosine-5-triphosphate (eATP), which plays an important role in vascular tone regulation, is released by cells following UTMC, possibly through sonoporation (formation of temporary and non-deadly pores in the cell membrane) and/or cell death. Herein, we were interested in quantifying UTMC-mediated ATP released in vitro using a microfluidics-based model and study its relationship with UTMC-mediated cell fate to better understand and improve UTMC mediated bioeffects. Lipid microbubbles (MB, Definity(R)), luciferin-luciferase (LL - for eATP quantification), and propidium iodide (PI - poration tracer) were flown over HUVEC cells cultured in a microfluidic device. Ultrasounds at 1 MHz, varying in pressure (300, 400 kPa) and length (10, 100, 1000 cycles) were applied to the chip. The LL chemiluminescent signal after the ultrasound pulse was acquired with an EMCCD camera to characterize ATP release kinetics. Then, a viability assay was performed with calcein-AM. An in-house MATLAB program pairing eATP kinetics with PI/calcein data was used to classify cells into three categories (sonoporated, dead, and untreated). Within the testing conditions, a single UTMC pulse caused between 4% and 55% PI-positive (PI+) cells in the ultrasound-treated area. Amongst PI+ cells, we generally found more dead cells than sonoporated cells, except for milder pulses (300 kPa; 10 and 100 cycles). The analysis of individual responses of ATP release demonstrated that dead cells released more ATP (up to 22.4 {+/-} 12.2 fmol/cell) than sonoporated cells (6.8 {+/-} 3.4 fmol/cell) and at a faster release rate which peaked at 4s. This study showed that sonoporation plays a significant role in UTMC-mediated ATP release, advancing our understanding of UTMCs potential use as a radiosensitizer in solid tumors.

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Amate, M., Tan, J. J., Boudreault, F., Grygorczyk, R., Gervais, T., Yu, F.. 2025-02-10. Single cell level analysis of ATP release kinetics and cell fate following ultrasound targeted microbubble cavitation using microscopy techniques. https://doi.org/10.1101/2025.02.02.636151

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