(M-515) Multiplex Method to Investigate Circulating Tumor Cell Mechanotransduction Under Shear Stress

Introduction:: Less than 0.01% of circulating tumor cells (CTCs) successfully metastasize, which is governed in-part by cellular responses to fluid shear stress (FSS) in the bloodstream [1]. This is denoted as mechanotransduction, in which mechanical stimuli are translated to biochemical cues. Physiological FSS ranges from 0.5 dyn/cm² to values over 1000 dyn/cm² as briefly occurs in arterial bifurcations and the heart [1]. The King Lab previously demonstrated that breast, prostate, and colon cancer cells exposed to FSS using cone-and-plate viscometers became sensitized to TRAIL-mediated apoptosis through activation of Piezo1, a mechanosensitive ion channel (MSC) [2]. Studies to examine the effects of FSS on CTCs and therapeutic targets are limited using current techniques due to a vast parameter space and single channel fluid systems. Therefore, we have developed a higher throughput approach based on a commercial multichannel, automated pipettor (Fig 1A).


Materials and Methods:: The average FSS that the multichannel device (Integra Biosciences) produces at ten different mixing speeds, was modeled theoretically. The shear stress (τ) and velocity (V_x) were computed for the compatible long pipette tips to represent a cell traversing the length of the pipette tip in fluid flow (Fig 1B). The highest average FSS this device permits using these tips is 8.79 dyn/cm². To increase the FSS, small bore needles were interfaced with the multipipetter system to increase the average FSS to 290 dyn/cm² at the maximum mixing speed of 295 µL/s. The number of mixing (shearing) cycles was varied to establish a correlation between the apoptosis in LNCaP and PC3 prostate cancer cells with the duration of the automated FSS exposure, to investigate effects on TRAIL-sensitization. TRAIL concentrations were maintained at 500 ng/mL. Cell death was assessed using Annexin-V/ Propidium iodide (AV/PI) staining (Fig 1C-E), in which representative flow plots for PC3 cells are shown in Figure 1D, with the populations denoted as shown in Figure 1C. Mitochondrial depolarization was examined via JC-1 staining (Fig 1F). GsMTx-4, an MSC inhibitor, was used at 10 µM to confirm that Piezo1 activation via mechanotransduction contributed to increased TRAIL-sensitization (Fig 1H), determined from AV/PI staining.


Results, Conclusions, and Discussions:: The apoptosis and mitochondrial depolarization of PC3 cells increased as the number of shearing cycles increased, significantly enhancing the TRAIL-mediated apoptosis for the TRAIL+Sh condition (Fig 1E-F). This indicates that apoptosis is occurring intrinsically, supported by an increase in TRAIL sensitization for LNCaPs and PC3s with increasing FSS exposure (Fig 1G). The LNCaPs showed similar results and were more prone to cell death with increased FSS exposure (data not shown). Treatment of PC3 cells with GsMTx-4 decreased TRAIL-sensitization compared to TRAIL+FSS exposure (Fig 1H), due to a decreased TRAIL-mediated apoptosis. Together, these results demonstrate that MSC activation is likely driving the observed FSS-induced apoptosis.

FSS exposure increased the effects of the reduced cancer cell viability mediated by the addition of TRAIL. This is the first study to our knowledge that uses an automated multichannel fluidic system to examine TRAIL sensitization on prostate cancer cells. Future experiments will examine cultured cell aggregates that resemble clinical CTC samples. This automated device presents a more high-throughput method for researchers to mimic physiological conditions and further study the effects governing the mechanics of CTC apoptosis.


Acknowledgements (Optional): : This research was funded by NIH Grant 1R01CA256054-01A1 and the NSFGRFP.


References (Optional): : [1] JM Hope, Journal of Cell Sci, 2021, 134, 1-12.
[2] JM Hope, Cell Death & Dis, 2019, 10, 1-15.