Functional Assay of Cancer Cell Invasion Potential Based on Mechanotransduction of Focused Ultrasound

Abstract

Cancer cells undergo a number of biophysical changes as they transform from an indolent to an aggressive state. These changes, which include altered mechanical and electrical properties, can reveal important diagnostic information about disease status. Here, we introduce a high-throughput, functional technique for assessing cancer cell invasion potential, which works by probing for the mechanically excitable phenotype exhibited by invasive cancer cells. Cells are labeled with fluorescent calcium dye and imaged during stimulation with low-intensity focused ultrasound, a non-contact mechanical stimulus. We show that cells located at the focus of the stimulus exhibit calcium elevation for invasive prostate (PC-3 and DU-145) and bladder (T24/83) cancer cell lines, but not for non-invasive cell lines (BPH-1, PNT1A, and RT112/84). In invasive cells, ultrasound stimulation initiates a calcium wave that propagates from the cells at the transducer focus to other cells, over distances greater than 1 mm. We demonstrate that this wave is mediated by extracellular signaling molecules and can be abolished through inhibition of transient receptor potential channels and inositol trisphosphate receptors, implicating these proteins in the mechanotransduction process. If validated clinically, our technology could provide a means to assess tumor invasion potential in cytology specimens, which is not currently possible. It may therefore have applications in diseases such as bladder cancer, where cytologic diagnosis of tumor invasion could improve clinical decision-making.

Focused ultrasound stimulates ER localized mechanosensitive PANNEXIN-1 to mediate intracellular calcium release in invasive cancer cells

“…FUS stimulation caused invasive, but not noninvasive cancer cell lines, to exhibit marked calcium signaling suggesting a novel means to determine the invasion potential. We validated this using a Matrigel invasion assay, demonstrating that the degree of invasion correlates well with the degree of FUSdependent Ca 2+ signaling (Hwang et al, 2013;Weitz et al, 2017). FUS stimuli evoke widespread Ca 2+ oscillatory dynamics in several invasive cancer cell lines (breast MDA-MB-231, prostate PC-3 and bladder T24/83), but not in noninvasive cells of the same cancer type (MCF-7, BPH-1, and RT112/84) suggesting that this is a general property of invasive cells (Hwang et al, 2013;Weitz et al, 2017).…”

“…A custom microscope system was used to image cellular fluorescence while performing simultaneous ultrasonic stimulation as described previously (Hwang et al, 2013;Weitz et al, 2017). Petri dishes or plates containing cells were placed on an inverted epifluorescence microscope (Olympus IX70), and the ultrasound transducer was lowered into the external buffer solution.…”

“…FUS stimulation of invasive cells also results in a time dependent propagation of an extracellular calcium wave spreading away from cells located at the transducer focus. The mechanism(s) for extracellular calcium wave propagation is unclear, it does not depend on ultrasonic surface waves or gap junctions (Weitz et al, 2017). Additional pharmacological studies in invasive cancer cells suggested the involvement of IP3 receptors (IP3Rs) or TRP channels (Weitz et al, 2017), as suggested by others (Bootman et al, 2002;Diver et al, 2001;Xu et al, 2005).…”

“…The mechanism(s) for extracellular calcium wave propagation is unclear, it does not depend on ultrasonic surface waves or gap junctions (Weitz et al, 2017). Additional pharmacological studies in invasive cancer cells suggested the involvement of IP3 receptors (IP3Rs) or TRP channels (Weitz et al, 2017), as suggested by others (Bootman et al, 2002;Diver et al, 2001;Xu et al, 2005). Nonfocused US also stimulates calcium signaling mediated by the Piezo1 mechanosensitive ion channel directly couple to microbubbles (Pan et al, 2018).…”

“…Calcium signaling, however is known to be critical in these processes. To develop a functional assay of cancer cell invasion potential, we recently used FUS stimulation to probe the altered calcium signaling pathways exhibited by invasive cancer cells (Hwang et al, 2013;Weitz et al, 2017). FUS stimulation caused invasive, but not noninvasive cancer cell lines, to exhibit marked calcium signaling suggesting a novel means to determine the invasion potential.…”