Multiplex, high-throughput method to study cancer and immune cell mechanotransduction.

Studying cellular mechanoresponses during cancer metastasis is limited by sample variation or complex protocols that current techniques require. Metastasis is governed by mechanotransduction, whereby cells translate external stimuli, such as circulatory fluid shear stress (FSS), into biochemical cues. We present high-throughput, semi-automated methods to expose cells to FSS using the VIAFLO96 multichannel pipetting device custom-fitted with 22 G needles, increasing the maximum FSS 94-fold from the unmodified tips. Specifically, we develop protocols to semi-automatically stain live samples and to fix, permeabilize, and intracellularly process cells for flow cytometry analysis. Our first model system confirmed that the pro-apoptotic effects of TRAIL therapeutics in prostate cancer cells can be enhanced via FSS-induced Piezo1 activation. Our second system implements this multiplex methodology to show that FSS exposure (290 dyn cm-2) increases activation of murine bone marrow-derived dendritic cells. These methodologies greatly improve the mechanobiology workflow, offering a high-throughput, multiplex approach.

Tumor nano-lysate activates dendritic cells to evoke a preventative immune response.

A tumor nano-lysate "TNL" vaccine comprised of sonicated 4T1 cells was developed, characterized and implemented for the prevention of triple-negative breast cancer. This study aimed to gain a better understanding of the immune response behind the success of the vaccine in vivo, through use of ex vivo and in vivo assays. Here, we analyze the activation of various immune cells isolated from healthy mouse spleens and find that antigen-presenting cells (APCs) such as dendritic cells (DCs) are being activated following 24 h incubation with 1:10 mg TNL/mg splenocytes. These cells were further explored to determine the pathway by which activation is occurring, and it was observed that TNL are phagocytosed by DCs to activate NF-kB and c-Fos pathways, resulting in enhanced cytokine release after 24 h. An in vivo temporal analysis was performed in mice to understand the immune response at 1, 3, 7 and 10 days after one 100 µL dose of TNL consisting of 105 sonicated 4T1 cells via cardiac puncture and splenocyte and peripheral blood mononuclear cell (PBMC) analysis. Changes were observed for up to one week. A multiple dose study was performed comparing mice that were vaccinated with one dose of TNL administered every ten days for 3 doses total, as well as a PBS vehicle control. Survival for TNL-vaccinated mice was enhanced compared to the PBS control, and there was an average delay of 10 days in the onset of metastasis. The differences between the groups at the end of the study demonstrate the potential for TNL as a preventative therapeutic.

Fluid shear stress enhances dendritic cell activation.

Ex vivo activation of dendritic cells (DCs) has been widely explored for targeted therapies, although these treatments remain expensive. Reducing treatment costs while enhancing cell activation could help to make immunotherapies more accessible. Cells can be activated by both internal and external forces including fluid shear stress (FSS). FSS activates cells via opening of mechanosensitive ion channels. In this study, dendritic cells were activated by sustained exposure to circulatory levels of fluid shear stress using a cone-and-plate flow device and analyzed for activation markers. After 1 h of shear stress exposure, an increase in cytokine release was present in immortalized cells as well as phosphorylation of the proteins NF-κB and cFos in primary DCs. Changes in DC morphology, metabolism and proliferation were also observed. These compelling new findings point to the potential for using FSS to activate DCs for ex vivo therapeutics.

Chemical Activation and Mechanical Sensitization of Piezo1 Enhance TRAIL-Mediated Apoptosis in Glioblastoma Cells.

Glioblastoma multiforme (GBM), the most common and aggressive type of primary brain tumor, has a mean survival of less than 15 months after standard treatment. Treatment with the current standard of care, temozolomide (TMZ), may be ineffective if damaged tumor cells undergo DNA repair or acquire mutations that inactivate transcription factor p53. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in multiple tumor types, while evading healthy cells, through a transcription-independent mechanism. GBM is particularly resistant to TRAIL, but studies have found that the mechanoreceptor Piezo1 can be activated under static conditions via Yoda1 agonist to induce TRAIL sensitization in other cancer cell lines. This study examines the effects and the mechanism of chemical and mechanical activation of Piezo1, via Yoda1 and fluid shear stress (FSS) stimulation, on TRAIL-mediated apoptosis in GBM cells. Here, we demonstrate that Yoda1 + TRAIL and FSS + TRAIL combination therapies significantly increase apoptosis in two GBM cell lines relative to controls. Further, cells known to be resistant to TMZ were found to have higher levels of Piezo1 expression and were more susceptible to TRAIL sensitization by Piezo1 activation. The combinatory Yoda1 + TRAIL treatment significantly decreased cell viability in TMZ-resistant GBM cells when compared to treatment with both low and high doses of TMZ. The results of this study suggest the potential of a highly specific and minimally invasive approach to overcome TMZ resistance in GBM by sensitizing cancer cells to TRAIL treatment via chemical or mechanical activation of Piezo1.