L8 - TRAIL-mediated mechanotransduction in cancer cells and immune cell activation enabled by a multiplex, high-throughput system

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 known as a form of mechanotransduction, the process by which mechanical stimuli are translated to biochemical cues. Physiological FSS ranges from 0.5 dyn/cm2 to values over 1000 dyn/cm^2 as cells briefly experience 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 become sensitized to TRAIL-mediated apoptosis through activation of Piezo1, a mechanosensitive ion channel (MSC) [2]. Additionally, immune cells such as T cells and dendritic cells are exposed to biophysical forces in the circulatory system, and less is known regarding how mechanical forces affect their function and communication. Studies to examine the effects of FSS on CTCs and therapeutic targets are limited using current techniques due to singe channel fluid systems and restricted parameter space. Therefore, we have developed a higher throughput approach based on the VIAFLO96, a commercial, multichannel, automated pipettor to expand these capabilities [3]. We achieved this through investigating the response of tumor cells to a wide range of FSS durations while treated with TRAIL (Fig. 1A). A second model system was employed to examine the cellular response of murine bone marrow-derived dendritic cells (BMDCs) to FSS exposure using the VIAFLO96 system, to advance previous studies which exposed BMDCs to low-intensity FSS (0.2 – 5.0 dyn/cm^2) ex vivo [4].

Materials and

Methods: : The shear stress (τ) and velocity (Vx) were computed in Matlab for the compatible long pipette tips to represent a cell traversing the length of the narrowing region in fluid flow at the VIAFLO96 operating speeds . The highest average FSS permitted using these tips is 8.79 dyn/cm^2. To increase the FSS 94-fold higher than the unmodified standard tips, 22 G needles were interfaced with the multipipetter to increase the average FSS to 290 dyn/cm^2 at the maximum mixing speed (295 µL/s). The number of mixing (shearing) cycles was varied to evaluate the relationship between shear-induced TRAIL sensitization in weakly (LNCaP) and highly (PC3) metastatic prostate cancer (PCa) cells treated with 500 ng/mL TRAIL. Cell death was assessed using Annexin-V/ Propidium iodide (AV/PI) staining (Fig. 1B), in which cells AV/PI-positive are considered late-stage apoptotic. Mitochondrial depolarization was examined via JC-1 staining (Fig. 1C). To study the mechanism of TRAIL-sensitization, cells were pre-treated with GsMTx-4 (10 µM) to inhibit Piezo1. Control experiments were conducted to confirm the validity of these newly established methodologies, by comparing the efficacy of the 22 G needles to the unmodified tips (Fig. 1D). TRAIL sensitization was measured by comparing the viability of the untreated condition to the TRAIL-treated conditions. Our second model system used BMDCs isolated from healthy, female BALB/c mice to develop semi-automated protocols to intracellularly process samples for flow cytometry analysis, while studying immune cell activation via FSS. FlowJo software was used to analyze flow cytometry data.

Results, Conclusions, and Discussions:: The apoptosis and mitochondrial depolarization increased as the number of shearing cycles increased, significantly enhancing the TRAIL-mediated apoptosis for the TRAIL+FSS condition (Fig. 1B,C) for both cell lines. This indicates that apoptosis is occurring intrinsically, supported by an increase in TRAIL sensitization for LNCaPs and PC3s with increasing FSS exposure. The LNCaPs showed similar results and were more prone to cell death with increased FSS exposure. The treatment of PC3 cells with GsMTx-4, the only known pharmacological inhibitor of Piezo1, decreased TRAIL-sensitization compared to TRAIL+FSS exposure, due to a decreased TRAIL-mediated apoptosis. Together, these results demonstrate that MSC activation is likely driving the observed FSS-induced apoptosis. Control experiments confirmed that the 22G needles more effectively induced TRAIL sensitization in PC3 cells, compared to unmodified pipette tips while maintaining consistent results (Fig. 1D). These findings also established that the VIALINK scripts that we developed and used to semi-automatically process and stain the samples for flow cytometry analysis perform comparatively to processing samples downstream manually. To our knowledge, this is the first study that uses an automated multichannel fluidic system to examine TRAIL sensitization on PCa cells over FSS durations not previously explored.

With the second model system, we demonstrated how 290 dyn/cm^2 of FSS enhanced the expression of activation and co-stimulatory markers, such as phosphorylated-nuclear factor-kappa B (phospho-NF-kB) and CD80, respectively, in primary BMDCs. This is critical since phospho-NF-kB modulates DC maturation and function, such as the regulation of inflammatory responses by driving T cell priming through cytokine release [3]. Additionally, the MHC-II expression after FSS exposure was 2.4-fold higher than previous reports that exposed BMDCs to IL-4 as a stimulant [4]. Studying DC response to mechanical stimuli is critical since DCs encounter mechanical strain while residing on surfaces in the bloodstream [5]. These observations demonstrate the potential to activate DCs ex vivo to enhance immune cell response in cancer therapies.

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 and/or References (Optional):: This research was funded by NIH Grant R01 CA256054 and the NSF-GRFP.

References: [1] JM Hope, Journal of Cell Sci, 2021, 134, 1-12; [2] JM Hope, Cell Death & Dis, 2019, 10, 1-15; [3] AR Fabiano, Comm. Bio., 2024, 7, 674. [4] JA Dombroski, Immunobiology, 2023, 228, 152744; [4] W Wang, Med Sci Monit, 2016, 22, 244–250; [5] JS Lewis, Biomaterials, 2013, 34, 9063–9070.