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.

Activation of Dendritic Cells Isolated from the Blood of Patients with Prostate Cancer by Ex Vivo Fluid Shear Stress Stimulation.

Prostate cancer is one of the most common cancers among men in the United States and a leading cause of cancer-related death in men. Treatment options for patients with advanced prostate cancer include hormone therapies, chemotherapies, radioligand therapies, and immunotherapies. Provenge (sipuleucel-T) is an autologous cancer-vaccine-based immunotherapy approved for men with asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). Administration of sipuleucel-T involves leukapheresis of patient blood to isolate antigen-presenting cells (APCs), including dendritic cells (DCs), and subsequent incubation of isolated APCs with both an antigen, prostatic acid phosphatase (PAP), and granulocyte macrophage-colony stimulating factor (GM-CSF) before their infusion back into the patient. Although sipuleucel-T has been shown to improve overall survival, other meaningful outcomes, such as prostate-specific antigen (PSA) levels and radiographic response, are inconsistent. This lack of robust response may be due to limited ex vivo activation of DCs using current protocols. Earlier studies have shown that many cell types can be activated ex vivo by external forces such as fluid shear stress (FSS). We hypothesize that novel fluid shear stress technologies and methods can be used to improve ex vivo efficacy of prostate cancer DC activation in prostate cancer. Herein, we report a new protocol for activating DCs from patients with prostate cancer using ex vivo fluid shear stress. Ultimately, the goal of these studies is to improve DC activation to expand the efficacy of therapies such as sipuleucel-T. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample collection and DC isolation Basic Protocol 2: Determination and application of fluid shear stress Basic Protocol 3: Flow cytometry analysis of DCs after FSS stimulation.

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.

Fluid shear stress enhances T cell activation through Piezo1.

BACKGROUND: T cell activation is a mechanical process as much as it is a biochemical process. In this study, we used a cone-and-plate viscometer system to treat Jurkat and primary human T cells with fluid shear stress (FSS) to enhance the activation of the T cells through mechanical means. RESULTS: The FSS treatment of T cells in combination with soluble and bead-bound CD3/CD28 antibodies increased the activation of signaling proteins essential for T cell activation, such as zeta-chain-associated protein kinase-70 (ZAP70), nuclear factor of activated T cells (NFAT), nuclear factor kappa B (NF-κB), and AP-1 (activator protein 1). The FSS treatment also enhanced the expression of the cytokines tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), and interferon gamma (IFN-γ), which are necessary for sustained T cell activation and function. The enhanced activation of T cells by FSS was calcium dependent. The calcium signaling was controlled by the mechanosensitive ion channel Piezo1, as GsMTx-4 and Piezo1 knockout reduced ZAP70 phosphorylation by FSS. CONCLUSIONS: These results demonstrate an intriguing new dynamic to T cell activation, as the circulatory system consists of different magnitudes of FSS and could have a proinflammatory role in T cell function. The results also identify a potential pathophysiological relationship between T cell activation and FSS, as hypertension is a disease characterized by abnormal blood flow and is correlated with multiple autoimmune diseases.

Channeling the Force: Piezo1 Mechanotransduction in Cancer Metastasis.

Cancer metastasis is one of the leading causes of death worldwide, motivating research into identifying new methods of preventing cancer metastasis. Recently there has been increasing interest in understanding how cancer cells transduce mechanical forces into biochemical signals, as metastasis is a process that consists of a wide range of physical forces. For instance, the circulatory system through which disseminating cancer cells must transit is an environment characterized by variable fluid shear stress due to blood flow. Cancer cells and other cells can transduce physical stimuli into biochemical responses using the mechanosensitive ion channel Piezo1, which is activated by membrane deformations that occur when cells are exposed to physical forces. When active, Piezo1 opens, allowing for calcium flux into the cell. Calcium, as a ubiquitous second-messenger cation, is associated with many signaling pathways involved in cancer metastasis, such as angiogenesis, cell migration, intravasation, and proliferation. In this review, we discuss the roles of Piezo1 in each stage of cancer metastasis in addition to its roles in immune cell activation and cancer cell death.

Prophylactic Cancer Vaccines Engineered to Elicit Specific Adaptive Immune Response.

Vaccines have been used to prevent and eradicate different diseases for over 200 years, and new vaccine technologies have the potential to prevent many common illnesses. Cancer, despite many advances in therapeutics, is still the second leading causes of death in the United States. Prophylactic, or preventative, cancer vaccines have the potential to reduce cancer prevalence by initiating a specific immune response that will target cancer before it can develop. Cancer vaccines can include many different components, such as peptides and carbohydrates, and be fabricated for delivery using a variety of means including through incorporation of stabilizing chemicals like polyethylene glycol (PEG) and pan-DR helper T-lymphocyte epitope (PADRE), fusion with antigen-presenting cells (APCs), microneedle patches, and liposomal encapsulation. There are currently five cancer vaccines used in the clinic, protecting against either human papillomavirus (HPV) or hepatitis B virus (HBV), and preventing several different types of cancer including cervical and oral cancer. Prophylactic cancer vaccines can promote three different types of adaptive responses: humoral (B cell, or antibody-mediated), cellular (T cell) or a combination of the two types. Each vaccine has its advantages and challenges at eliciting an adaptive immune response, but these prophylactic cancer vaccines in development have the potential to prevent or delay tumor development, and reduce the incidence of many common cancers.

Circulating prostate cancer cells have differential resistance to fluid shear stress-induced cell death.

Circulating tumor cells (CTCs) are exposed to fluid shear stress (FSS) of greater than 1000 dyn/cm2 (100 Pa) in circulation. Normally, CTCs that are exposed to FSS of this magnitude die. However, some CTCs develop resistance to this FSS, allowing them to colonize distant organs. We explored how prostate CTCs can resist cell death in response to forces of this magnitude. The DU145, PC3 and LNCaP human prostate cancer cell lines were used to represent cells of different metastatic origins. The cell lines were briefly treated with an average FSS of 3950 dyn/cm2 (395 Pa) using a 30 G needle and a syringe pump. DU145 cells had no change in cell viability, PC3 cells had some cell death and LNCaP cells exhibited significant cell death. These cell death responses correlated with increased cell membrane damage, less efficient membrane repair and increased stiffness. Additionally, FSS treatment prevented the LNCaP FSS-sensitive cell line from forming a growing tumor in vivo This suggests that these properties play a role in FSS resistance and could represent potential targets for disrupting blood-borne metastasis.

Fabrication and Characterization of Tumor Nano-Lysate as a Preventative Vaccine for Breast Cancer.

Breast cancer is the most common cancer among women in the United States, with late stages associated with the lowest survival rates. The latest stage, defined as metastasis, accounts for 90% of all cancer-related deaths. There is a strong need to develop antimetastatic therapies. TRAIL, or TNF-related apoptosis inducing ligand, has been used as an antimetastatic therapy in the past, and conjugating TRAIL to nanoscale liposomes has been shown to enhance its targeting efficacy. When circulating tumor cells (CTCs) released during metastasis are exposed to TRAIL-conjugated liposomes and physiologically relevant fluid shear stress, this results in rapid cancer cell destruction into cell fragments. We sought to artificially recreate this phenomenon using probe sonication to mechanically disrupt cancer cells and characterized the resulting cell fragments, termed "tumor nano-lysate", with respect to size, charge, morphology, and composition. Furthermore, an in vivo pilot study was performed to investigate the efficacy of tumor nano-lysate as a preventative vaccine for breast cancer in an immunocompetent mouse model.

Engineering of Exosomes to Target Cancer Metastasis.

As a nanoscale subset of extracellular vehicles, exosomes represent a new pathway of intercellular communication by delivering cargos such as proteins and nucleic acids to recipient cells. Importantly, it has been well documented that exosome-mediated delivery of such cargo is involved in many pathological processes such as tumor progression, cancer metastasis, and development of drug resistance. Innately biocompatible and possessing ideal structural properties, exosomes offer distinct advantages for drug delivery over artificial nanoscale drug carriers. In this review, we summarize recent progress in methods for engineering exosomes including isolation techniques and exogenous cargo encapsulation, with a focus on applications of engineered exosomes to target cancer metastasis.