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.

Piezo1 Mechano-Activation Is Augmented by Resveratrol and Differs between Colorectal Cancer Cells of Primary and Metastatic Origin.

Cancer cells must survive aberrant fluid shear stress (FSS) in the circulation to metastasize. Herein, we investigate the role that FSS has on colorectal cancer cell apoptosis, proliferation, membrane damage, calcium influx, and therapeutic sensitization. We tested this using SW480 (primary tumor) and SW620 cells (lymph node metastasis) derived from the same patient. The cells were exposed to either shear pulses, modeling millisecond intervals of high FSS seen in regions of turbulent flow, or sustained shear to model average magnitudes experienced by circulating tumor cells. SW480 cells were significantly more sensitive to FSS-induced death than their metastatic counterparts. Shear pulses caused significant cell membrane damage, while constant shear decreased cell proliferation and increased the expression of CD133. To investigate the role of mechanosensitive ion channels, we treated cells with the Piezo1 agonist Yoda1, which increased intracellular calcium. Pretreatment with resveratrol further increased the calcium influx via the lipid-raft colocalization of Piezo1. However, minimal changes in apoptosis were observed due to calcium saturation, as predicted via a computational model of apoptosis. Furthermore, SW480 cells had increased levels of Piezo1, calcium influx, and TRAIL-mediated apoptosis compared to SW620 cells, highlighting differences in the mechano-activation of metastatic cells, which may be a necessary element for successful dissemination in vivo.

TRAIL-conjugated liposomes that bind natural killer cells to induce colorectal cancer cell apoptosis.

Natural killer (NK) cell functionality is a strong indicator of favorable prognosis in cancer patients, making NK cells an appealing therapeutic target to prevent lymph node dissemination. We engineered liposomes that are conjugated with anti-CD335 antibodies for NK cell targeting, and the apoptotic ligand TRAIL to kill cancer cells. Liposomes were made using a thin film hydration method followed by extrusion to approximately 100 nm in diameter and conjugation of proteins via thiol-maleimide click chemistry. TRAIL/anti-CD335 liposomes successfully bound to isolated NK cells. Once piggybacked to the surface of NK cells, these "Super Natural Killer Cells" were able to more effectively kill oxaliplatin-resistant SW620 cells and metastatic COLO205 colorectal cancer cells via TRAIL-mediated apoptosis compared to NK cells alone. Importantly, Super NK cells were more effective under physiological levels of fluid shear stress found in the lymphatics. Liposome biodistribution after intravenous administration confirmed the sustained presence of liposomes within the spleen and tumor draining mesenteric lymph nodes for at least 4 days. These results demonstrate the enhanced apoptotic effects of NK cells armored with liposomal TRAIL against clinically relevant colorectal cancer cells, providing the groundwork for in vivo treatment studies in mouse models of colorectal cancer metastasis.

Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by "piggybacking" on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

Matrix stiffness induces epithelial-to-mesenchymal transition via Piezo1-regulated calcium flux in prostate cancer cells.

Cells utilize calcium channels as one of the main signaling mechanisms to sense changes in the extracellular space and convert these changes to intracellular signals. Calcium regulates several key signaling networks, such as the induction of EMT. The current study expands on the understanding of how EMT is controlled via the mechanosensitive calcium channel Piezo1 in cancerous cells, which senses changes in the extracellular matrix stiffness. We model the biophysical environment of healthy and cancerous prostate tissue using polyacrylamide gels of different stiffnesses. Significant increases in calcium steady-state concentration, vimentin expression, and aspect ratio, and decreases in E-cadherin expression were observed by increasing matrix stiffness and also after treatment with Yoda1, a chemical agonist of Piezo1. Overall, this study concludes that Piezo1-regulated calcium flux plays a role in prostate cancer cell metastatic potential by sensing changes in ECM stiffness and modulating EMT markers.

Matrix stiffness enhances cancer-macrophage interactions and M2-like macrophage accumulation in the breast tumor microenvironment.

The role of intratumor heterogeneity is becoming increasingly apparent in part due to expansion in single cell technologies. Clinically, tumor heterogeneity poses several obstacles to effective cancer therapy dealing with biomarker variability and treatment responses. Matrix stiffening is known to occur during tumor progression and contribute to pathogenesis in several cancer hallmarks, including tumor angiogenesis and metastasis. However, the effects of matrix stiffening on intratumor heterogeneity have not been thoroughly studied. In this study, we applied single-cell RNA sequencing to investigate the differences in the transcriptional landscapes between stiff and compliant MMTV-PyMT mouse mammary tumors. We found similar compositions of cancer and stromal subpopulations in compliant and stiff tumors but differential intercellular communication and a significantly higher concentration of tumor-promoting, M2-like macrophages in the stiffer tumor microenvironments. Interestingly, we found that cancer cells seeded on stiffer substrates recruited more macrophages. Furthermore, elevated matrix stiffness increased Colony Stimulating Factor 1 (CSF-1) expression in breast cancer cells and reduction of CSF-1 expression on stiffer substrates reduced macrophage recruitment. Thus, our results demonstrate that tissue phenotypes were conserved between stiff and compliant tumors but matrix stiffening altered cell-cell interactions which may be responsible for shifting the phenotypic balance of macrophages residing in the tumor microenvironment towards a pro-tumor progression M2 phenotype. STATEMENT OF SIGNIFICANCE: Cells within tumors are highly heterogeneous, posing challenges with treatment and recurrence. While increased tissue stiffness can promote several hallmarks of cancer, its effects on tumor heterogeneity are unclear. We used single-cell RNA sequencing to investigate the differences in the transcriptional landscapes between stiff and compliant MMTV-PyMT mouse mammary tumors. We found similar compositions of cancer and stromal subpopulations in compliant and stiff tumors but differential intercellular communication and a significantly higher concentration of tumor-promoting, M2-like macrophages in the stiffer tumor microenvironments. Using a biomaterial-based platform, we found that cancer cells seeded on stiffer substrates recruited more macrophages, supporting our in vivo findings. Together, our results demonstrate a key role of matrix stiffness in affecting cell-cell communication and macrophage recruitment.

A syngeneic MC38 orthotopic mouse model of colorectal cancer metastasis.

While subcutaneous tumor models remain the standard for studying drug efficacy in vivo, these tumors rarely metastasize and lack physiological relevance due to differences in the tumor microenvironment, vascularization, immune landscape, and physiological cues associated with the organ of interest. Orthotopic tumors, grown from the organ corresponding with the cancer type, provide a more translational approach to study disease progression and drug efficacy. Utilization of a syngeneic mouse model allows for a complete immune landscape, key for adaptive immunotherapy studies. MC38 and CT26 cells are commonly used murine colorectal cancer cell lines with clinically relevant mutations. While CT26 cells have been orthotopically implanted with high fidelity, successful engraftment of orthotopic MC38 tumors varies greatly between studies. Thus, we have developed a detailed protocol for MC38 orthotopic tumor inoculation via intracecal injection. Nine C57BL/6 mice were injected with 2 × 106 cells into the cecal wall and sacrificed after 7 weeks. Survival after surgery was 100%, and one mouse died before the 7-week study end point from tumor burden and metastatic spread. We observed a successful tumor engraftment rate of 67%. Half of mice presenting with tumors were found to have macroscopic metastatic lesions in clinically relevant foci, including the mesenteric lymph nodes, liver, and peritoneum. These mice also presented with very large tumors and an enlarged spleen. The other half of the mice presented with small, localized tumors that did not metastasize. Herein, we describe tips specific for the intracecal injection of MC38 cells to improve the engraftment rate consistency in this model.

Rafting Down the Metastatic Cascade: The Role of Lipid Rafts in Cancer Metastasis, Cell Death, and Clinical Outcomes.

Lipid rafts are tightly packed, cholesterol- and sphingolipid-enriched microdomains within the plasma membrane that play important roles in many pathophysiologic processes. Rafts have been strongly implicated as master regulators of signal transduction in cancer, where raft compartmentalization can promote transmembrane receptor oligomerization, shield proteins from enzymatic degradation, and act as scaffolds to enhance intracellular signaling cascades. Cancer cells have been found to exploit these mechanisms to initiate oncogenic signaling and promote tumor progression. This review highlights the roles of lipid rafts within the metastatic cascade, specifically within tumor angiogenesis, cell adhesion, migration, epithelial-to-mesenchymal transition, and transendothelial migration. In addition, the interplay between lipid rafts and different modes of cancer cell death, including necrosis, apoptosis, and anoikis, will be described. The clinical role of lipid raft-specific proteins, caveolin and flotillin, in assessing patient prognosis and evaluating metastatic potential of various cancers will be presented. Collectively, elucidation of the complex roles of lipid rafts and raft components within the metastatic cascade may be instrumental for therapeutic discovery to curb prometastatic processes.

Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization.

Colorectal cancer (CRC) remains a leading cause of cancer death, and its mortality is associated with metastasis and chemoresistance. We demonstrate that oxaliplatin-resistant CRC cells are sensitized to TRAIL-mediated apoptosis. Oxaliplatin-resistant cells exhibited transcriptional downregulation of caspase-10, but this had minimal effects on TRAIL sensitivity following CRISPR-Cas9 deletion of caspase-10 in parental cells. Sensitization effects in oxaliplatin-resistant cells were found to be a result of increased DR4, as well as significantly enhanced DR4 palmitoylation and translocation into lipid rafts. Raft perturbation via nystatin and resveratrol significantly altered DR4/raft colocalization and TRAIL sensitivity. Blood samples from metastatic CRC patients were treated with TRAIL liposomes, and a 57% reduction of viable circulating tumor cells (CTCs) was observed. Increased DR4/lipid raft colocalization in CTCs was found to correspond with increased oxaliplatin resistance and increased efficacy of TRAIL liposomes. To our knowledge, this is the first study to investigate the role of lipid rafts in primary CTCs.

Engineered fluidic systems to understand lymphatic cancer metastasis.

The majority of all cancers metastasize initially through the lymphatic system. Despite this, the mechanisms of lymphogenous metastasis remain poorly understood and understudied compared to hematogenous metastasis. Over the past few decades, microfluidic devices have been used to model pathophysiological processes and drug interactions in numerous contexts. These devices carry many advantages over traditional 2D in vitro systems, allowing for better replication of in vivo microenvironments. This review highlights prominent fluidic devices used to model the stages of cancer metastasis via the lymphatic system, specifically within lymphangiogenesis, vessel permeability, tumor cell chemotaxis, transendothelial migration, lymphatic circulation, and micrometastases within the lymph nodes. In addition, we present perspectives for the future roles that microfluidics might play within these settings and beyond.

Mechanosensitive Ion Channels: TRPV4 and P2X7 in Disseminating Cancer Cells.

Cancer metastasis is the second leading cause of death in the United States. Despite its morbidity, metastasis is an inefficient process that few cells can survive. However, cancer cells can overcome these metastatic barriers via cellular responses to microenvironmental cues, such as through mechanotransduction. This review focuses on the mechanosensitive ion channels TRPV4 and P2X7, and their roles in metastasis, as both channels have been shown to significantly affect tumor cell dissemination. Upon activation, these channels help form tumor neovasculature, promote transendothelial migration, and increase cell motility. Conversely, they have also been linked to forms of cancer cell death dependent upon levels of activation, implying the complex functionality of mechanosensitive ion channels. Understanding the roles of TRPV4, P2X7 and other mechanosensitive ion channels in these processes may reveal new possible drug targets that modify channel function to reduce a tumor's metastatic potential.