L2 - Calcium Responses Induced by Focused Ultrasound Applied to Prostate Cancer Cells

Introduction: : Focused ultrasound (FUS) has emerged as a promising and non-invasive modality with significant translational potential in cancer therapy. By delivering targeted mechanical forces, FUS can activate calcium signaling pathways in cancer cells via mechanosensitive channels such as PANNEXIN1 (PANX1) and Piezo1 [1, 2]. These pathways are critical in regulating cancer cell behavior, including proliferation, migration, and metastasis, making them attractive targets for therapeutic intervention. As part of the growing field of mechanobiology, which explores how mechanical stimuli influence cellular function, FUS offers a noninvasive approach to modulating cancer cell signaling with high spatiotemporal control. Building on this potential, we recently utilized FUS stimulation to probe altered calcium signaling dynamics in cancer cells. Previous work from the King Lab demonstrated that low-intensity pulsed FUS can enhance TRAIL-mediated apoptosis in cancer cells by inducing calcium influx through Piezo1 activation [2]. However, the broader impacts of FUS on calcium signaling remain incompletely understood. In this study, we aim to investigate the mechanobiological responses of prostate cancer PC3 cells to FUS stimulation.

Materials and

Methods: : The diagram of adherent PC3 cells exposed to 1.1 MHz FUS is depicted in Figure 1A. The acoustic pressure at the field of view (FOV) was estimated to be 1.5 MPa. The pulsed FUS stimulation has a 2% duty cycle and repetition frequency of 1000 Hz as shown in Figure 1B. PC3 cells were stained with Fluo-4 AM ester and imaged continuously with an inverted microscope. The quantification of calcium response profiles was performed using ImageJ and MATLAB.

Results, Conclusions, and Discussions:: Applying FUS to PC3 cells resulted in enhanced calcium signaling, as illustrated in Figure 1C. Initially, the lower region of the field of view (FOV) exhibited increased fluorescence intensity, followed by a gradual brightening of the upper regions, suggesting the propagation of calcium signals from the bottom to the top of the FOV. Figure 1D presents the normalized calcium signal across the FOV over time. After an initial brief decrease lasting a few seconds, the calcium levels rose for approximately 5 seconds before gradually returning to the baseline. This propagation of extracellular Ca²⁺ signaling pattern is consistent with the activation of PANNEXIN1 (PANX1) channels, which leads to ATP release and subsequent stimulation of purinergic receptors on neighboring cells. This stimulation can trigger both calcium influx and intracellular calcium release in surrounding cells [1].
FUS exposure induced an enhanced calcium level and a propagation of calcium signals in PC3 cells, possibly mediated by the activation of PANX1 channels. In this study, we used 1.1 MHz FUS to stimulate PC3 cells while investigating their calcium dynamics using a microscope. Although much remains to be understood about mechanotransduction in cancer cells, this study provides a method for probing calcium signaling pathways in response to FUS stimulation. These findings suggest that FUS could serve as a promising strategy to enhance the effectiveness of cancer therapy by understanding the altered calcium signal pathway in cancer cells.

Acknowledgements and/or References (Optional):: This research was funded by the Cancer Prevention and Research Institute of Texas (CPRIT), through grant number RR230029 to M.R.K.