Boudreault, F. | Search bioRxiv

bioRxiv2025

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

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.

bioengineering↗

bioRxiv2024

Nano-scale viscometry reveals an inherent mucus defect in cystic fibrosis

Abnormally viscous and thick mucus is a hallmark of cystic fibrosis (CF). How the genetic defect causes abnormal mucus in CF remains unanswered and a question of paramount interest. Mucus is produced by hydration of gel-forming mucin macromolecules that are stored in secretory granules prior to release. Current understanding of mucin/mucus structure before and after secretion remains limited and contradictory models exist. Here we used a molecular viscometer and fluorescence lifetime imaging of primary epithelial cells (Normal and CF) to measure nanometer-scale viscosity. We found significantly elevated intraluminal nanoviscosity in a population of CF mucin granules, indicating an intrinsic, pre-secretory, mucin defect. Validation experiments showed that high nanoviscosity in cellular environments is mainly due to the low mobility of water that hydrates macromolecules. Nanoviscosity influences protein conformational dynamics and function. Its elevation along the protein secretory pathway indicates molecular overcrowding and is expected to alter mucins post-translational processing, hydration, and mucus rheology after release. The nanoviscosity of extracellular CF mucus was elevated compared to non-CF mucus. Remarkably, it was higher after secretion than in granules, which suggests mucins have a weakly-ordered state in granules and adopt a highly-ordered, nematic crystalline structure extracellularly. This challenges the classical view of mucus as a porous agarose-like gel and suggests an alternative model for mucin organization before and after secretion. Our study also suggests that endoplasmic reticulum stress due to molecular overcrowding contributes to mucus pathogenesis in CF cells. It encourages the development of therapeutics that target pre-secretory mechanisms in CF and other muco-obstructive lung diseases.

cell biology↗