Focused Ultrasound Stimulates ER Localized Mechanosensitive PANNEXIN-1 to Mediate Intracellular Calcium Release in Invasive Cancer Cells.

Focused ultrasound (FUS) is a rapidly developing stimulus technology with the potential to uncover novel mechanosensory dependent cellular processes. Since it is non-invasive, it holds great promise for future therapeutic applications in patients used either alone or as a complement to boost existing treatments. For example, FUS stimulation causes invasive but not non-invasive cancer cell lines to exhibit marked activation of calcium signaling pathways. Here, we identify the membrane channel PANNEXIN1 (PANX1) as a mediator for activation of calcium signaling in invasive cancer cells. Knockdown of PANX1 decreases calcium signaling in invasive cells, while PANX1 overexpression enhances calcium elevations in non-invasive cancer cells. We demonstrate that FUS may directly stimulate mechanosensory PANX1 localized in endoplasmic reticulum to evoke calcium release from internal stores. This process does not depend on mechanosensory stimulus transduction through an intact cytoskeleton and does not depend on plasma membrane localized PANX1. Plasma membrane localized PANX1, however, plays a different role in mediating the spread of intercellular calcium waves via ATP release. Additionally, we show that FUS stimulation evokes cytokine/chemokine release from invasive cancer cells, suggesting that FUS could be an important new adjuvant treatment to improve cancer immunotherapy.

Non-contact high-frequency ultrasound microbeam stimulation for studying mechanotransduction in human umbilical vein endothelial cells.

We describe how contactless high-frequency ultrasound microbeam stimulation (HFUMS) is capable of eliciting cytoplasmic calcium (Ca(2+)) elevation in human umbilical vein endothelial cells. The cellular mechanotransduction process, which includes cell sensing and adaptation to the mechanical micro-environment, has been studied extensively in recent years. A variety of tools for mechanical stimulation have been developed to produce cellular responses. We developed a novel tool, a highly focused ultrasound microbeam, for non-contact cell stimulation at a microscale. This tool, at 200 MHz, was applied to human umbilical vein endothelial cells to investigate its potential to elicit an elevation in cytoplasmic Ca(2+) levels. It was found that the response was dose dependent, and moreover, extracellular Ca(2+) and cytoplasmic Ca(2+) stores were involved in the Ca(2+) elevation. These results suggest that high-frequency ultrasound microbeam stimulation is potentially a novel non-contact tool for studying cellular mechanotransduction if the acoustic pressures at such high frequencies can be quantified.

Development of a dual-modal tissue diagnostic system combining time-resolved fluorescence spectroscopy and ultrasonic backscatter microscopy.

We report a tissue diagnostic system which combines two complementary techniques of time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) and ultrasonic backscatter microscopy (UBM). TR-LIFS evaluates the biochemical composition of tissue, while UBM provides tissue microanatomy and enables localization of the region of diagnostic interest. The TR-LIFS component consists of an optical fiber-based time-domain apparatus including a spectrometer, gated multichannel plate photomultiplier, and fast digitizer. It records the fluorescence with high sensitivity (nM concentration range) and time resolution as low as 300 ps. The UBM system consists of a transducer, pulser, receiving circuit, and positioning stage. The transducer used here is 45 MHz, unfocused, with axial and lateral resolutions 38 and 200 microm. Validation of the hybrid system and ultrasonic and spectroscopic data coregistration were conducted both in vitro (tissue phantom) and ex vivo (atherosclerotic tissue specimens of human aorta). Standard histopathological analysis of tissue samples was used to validate the UBM-TRLIFS data. Current results have demonstrated that spatially correlated UBM and TR-LIFS data provide complementary characterization of both morphology (necrotic core and calcium deposits) and biochemistry (collagen, elastin, and lipid features) of the atherosclerotic plaques at the same location. Thus, a combination of fluorescence spectroscopy with ultrasound imaging would allow for better identification of features associated with tissue pathologies. Current design and performance of the hybrid system suggests potential applications in clinical diagnosis of atherosclerotic plaque.

Alumina/epoxy nanocomposite matching layers for high-frequency ultrasound transducer application.

Mismatch of acoustic impedance at the interface between a piezoelectric transducer and the medium to be probed will substantially reduce the amount of ultrasound energy being transmitted into the medium. Therefore, matching layer is a critical component of an ultrasonic transducer. A spin-coating process was used to fabricate alumina/polymer nanocomposite films with alumina volume fractions ranging from 14 to 32%. The particle size of alumina is in the range of 10 to 40 nm. The thicknesses of the matching layer can be controlled by the spinning speed and the concentration of solution. Acoustic impedances of these nanocomposite matching layers are in the range of 2.8 to 5.1 MRayls with different alumina contents, which meet the matching layer requirement. The attenuation of a nanocomposite matching layer with smooth surface is about 15 dB/mm at 40 MHz. The pulse-echo spectrum and frequency spectrum of a high-frequency transducer using this nanocomposite matching layer are reported.

Acoustic properties of alumina colloidal/polymer nano-composite film on silicon.

Alumina colloidal/polymer composite films on silicon substrates have been successfully fabricated using the sol-gel method, in which the crystallite sizes of alumina are between 20 and 50 nm. The density and ultrasonic phase velocities in these films with different alumina ratios from 14% to 32% were measured at the desired operating frequency. We have proved that the density, acoustic phase velocities, and hence the acoustic impedance of the nano-composite films increase with the alumina content, which gives us another option of tailoring the acoustic impedance of the nano-composite film for making the matching layer of high-frequency medical ultrasonic transducers.