טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentMazzawi Nasma
SubjectThe Effect of Low-Intensity Ultrasound on Motility and
Morphology of Living Cells
DepartmentDepartment of Biomedical Engineering
Supervisors Professor Emeritus Eitan Kimmel
Dr. Ilan Bruchim


Abstract

Low-intensity ultrasound (LIUS) application is known to induce non-thermal effects ranging from cell necrosis to more delicate alterations of cells and their membranes. Reversible LIUS effects include permeability enhancement for drug delivery, blood-brain barrier opening, and DNA transfer. However, we have yet to use the majority of these modalities in clinical practice. This is due to the lack in comprehension of the biophysical mechanism underlying ultrasound-cell interaction in tissues. Here we present a study on ultrasound-cell interaction while focusing on cell morphology and kinetics. The cells' morphological and kinetic traits are affected by signaling modulation and manifest the impact of internal and external forces applied to cells. The membrane-bound MET tyrosine kinase receptor, and its ligand Hepatocytes- Growth-Factor/Scatter-Factor (HGF/SF-MET) activate cellular signaling pathways which promote proliferation, motility, and metastasis in cancer cells. We hypothesize that there is an interplay between HGF/SF-MET-activation and LIUS, and that LIUS has a direct effect on cell membranes and cellular mechano-transduction processes. This is relevant to LIUS modulating HGF/SF-MET-induced motility and metastasis, which accounts for 90% of cancer deaths today. We conducted in-vitro experiments and measured cells' morpho-kinetic parameters (MKPs) using live-cell time-lapse imaging and single-cell analysis. We applied LIUS with an amplitude pressure of 200kPa and 0.962 kHz frequency in a continuous mode. Results demonstrate that LIUS inhibits both basal and HGF/SF-MET-induced motility. LIUS decreased the average velocity of motile cells by approximately 85%, and modulated cells' morphological parameters such as surface area and sphericity. Also, LIUS presented long-term effects on cells' morpho-kinetics. Cells post-LIUS were characterized by significantly lower values of kinetic parameters in comparison to baseline. A cluster analysis, performed on 36 MKPs acquired post-LIUS exposure, was performed by Hierarchical Agglomerative Clustering method to classify cells of different treatments by similarity.  HGF/SF-MET-activated cells presented different morpho-kinetic characteristics in comparison to cells exposed to LIUS. On a sub-cellular level, LIUS induced membrane alterations. MKPs are correlated with membrane morphological and mechanical changes. We claim that membrane alterations determine cells' MKPs. In drug-related experiments of liposomal drug DOXIL, LIUS mediated an immediate 10-25% increase in drug release in comparison to baseline. Therefore, we claim that LIUS effects on mechano-transduction processes and drug release are the outcome of the interaction between this mechanical force and phospholipidic membranes, of either living cells or artificial cells (liposomes). Passive cavitation detection experiments revealed that cavitation is involved in ultrasound-cell interaction as their activity was detected only in the presence of cells. This may imply that cavitation is involved in the inhibition of cell motility through morpho-kinetic modifications and cell membrane alterations. We posit that membrane-related cavitation inhibits cell motility presumably by membrane-tension application. 

In conclusion, our study is the first to unveil the ability of LIUS to modify mechano-transduction processes and inhibit motility. We presented the hidden potential of LIUS to act as a therapeutic force for damaged cells, such as in metastatic diseases. Our results suggest that membrane-related cavitation contours ultrasound-cell interaction and plays a role in the biophysical mechanism of ultrasound in cells and tissues.