Phenotypically sorted highly and weakly migratory triple negative breast cancer cells exhibit migratory and metastatic commensalism.

BACKGROUND: Intratumor heterogeneity is a well-established hallmark of cancer that impedes cancer research, diagnosis, and treatment. Previously, we phenotypically sorted human breast cancer cells based on migratory potential. When injected into mice, highly migratory cells were weakly metastatic and weakly migratory cells were highly metastatic. The purpose of this study was to determine whether these weakly and highly migratory cells interact with each other in vitro or in vivo. METHODS: To assess the relationship between heterogeneity in cancer cell migration and metastatic fitness, MDA-MB-231 and SUM159PT triple negative breast cancer cells were phenotypically sorted into highly migratory and weakly migratory subpopulations and assayed separately and in a 1:1 mixture in vitro and in vivo for metastatic behaviors. Unpaired, two-tailed Student's t-tests, Mann-Whitney tests, ordinary, one-way ANOVAs, and Kruskal-Wallis H tests were performed as appropriate with p < 0.05 as the cutoff for statistical significance. RESULTS: When highly and weakly migratory cells are co-seeded in mixed spheroids, the weakly migratory cells migrated farther than weakly migratory only spheroids. In mixed spheroids, leader-follower behavior occurred with highly migratory cells leading the weakly migratory cells in migration strands. When cell suspensions of highly migratory, weakly migratory, or a 1:1 mixture of both subpopulations were injected orthotopically into mice, both the mixed cell suspensions and weakly migratory cells showed significant distal metastasis, but the highly migratory cells did not metastasize significantly to any location. Notably, significantly more distal metastasis was observed in mice injected with the 1:1 mixture compared to either subpopulation alone. CONCLUSIONS: This study suggests that weakly migratory cells interact with highly migratory cells in a commensal fashion resulting in increased migration and metastasis. Together, these findings indicate that cancer cell subpopulation migration ability does not correlate with metastatic potential and that cooperation between highly migratory and weakly migratory subpopulations can enhance overall metastatic fitness.

Mitochondrial transfer from cancer-associated fibroblasts increases migration in aggressive breast cancer.

Cancer-associated fibroblasts (CAFs) have distinct roles within the tumor microenvironment, which can impact the mode and efficacy of tumor cell migration. CAFs are known to increase invasion of less-aggressive breast cancer cells through matrix remodeling and leader-follower dynamics. Here, we demonstrate that CAFs communicate with breast cancer cells through the formation of contact-dependent tunneling nanotubes (TNTs), which allow for the exchange of cargo between cell types. CAF mitochondria are an integral cargo component and are sufficient to increase the 3D migration of cancer cells. This cargo transfer results in an increase in mitochondrial ATP production in cancer cells, whereas it has a negligible impact on glycolytic ATP production. Manually increasing mitochondrial oxidative phosphorylation (OXPHOS) by providing extra substrates for OXPHOS fails to enhance cancer cell migration unless glycolysis is maintained at a constant level. Together, these data indicate that tumor-stromal cell crosstalk via TNTs and the associated metabolic symbiosis is a finely controlled mechanism by which tumor cells co-opt their microenvironment to promote cancer progression and may become a potential therapeutic target.

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.

Weakly migratory metastatic breast cancer cells activate fibroblasts via microvesicle-Tg2 to facilitate dissemination and metastasis.

Cancer cell migration is highly heterogeneous, and the migratory capability of cancer cells is thought to be an indicator of metastatic potential. It is becoming clear that a cancer cell does not have to be inherently migratory to metastasize, with weakly migratory cancer cells often found to be highly metastatic. However, the mechanism through which weakly migratory cells escape from the primary tumor remains unclear. Here, utilizing phenotypically sorted highly and weakly migratory human breast cancer cells, we demonstrate that weakly migratory metastatic cells disseminate from the primary tumor via communication with stromal cells. While highly migratory cells are capable of single cell migration, weakly migratory cells rely on cell-cell signaling with fibroblasts to escape the primary tumor. Weakly migratory cells release microvesicles rich in tissue transglutaminase 2 (Tg2) which activate murine fibroblasts and lead weakly migratory cancer cell migration in vitro. These microvesicles also induce tumor stiffening and fibroblast activation in vivo and enhance the metastasis of weakly migratory cells. Our results identify microvesicles and Tg2 as potential therapeutic targets for metastasis and reveal a novel aspect of the metastatic cascade in which weakly migratory cells release microvesicles which activate fibroblasts to enhance cancer cell dissemination.

Link between glucose metabolism and epithelial-to-mesenchymal transition drives triple-negative breast cancer migratory heterogeneity.

Intracellular and environmental cues result in heterogeneous cancer cell populations with different metabolic and migratory behaviors. Although glucose metabolism and epithelial-to-mesenchymal transition have previously been linked, we aim to understand how this relationship fuels cancer cell migration. We show that while glycolysis drives single-cell migration in confining microtracks, fast and slow cells display different migratory sensitivities to glycolysis and oxidative phosphorylation inhibition. Phenotypic sorting of highly and weakly migratory subpopulations (MDA+, MDA-) reveals that more mesenchymal, highly migratory MDA+ preferentially use glycolysis while more epithelial, weakly migratory MDA- utilize mitochondrial respiration. These phenotypes are plastic and MDA+ can be made less glycolytic, mesenchymal, and migratory and MDA- can be made more glycolytic, mesenchymal, and migratory via modulation of glucose metabolism or EMT. These findings reveal an intrinsic link between EMT and glucose metabolism that controls migration. Identifying mechanisms fueling phenotypic heterogeneity is essential to develop targeted metastatic therapeutics.

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.

Cancer cell metabolic plasticity in migration and metastasis.

Metabolic reprogramming is a hallmark of cancer metastasis in which cancer cells manipulate their metabolic profile to meet the dynamic energetic requirements of the tumor microenvironment. Though cancer cell proliferation and migration through the extracellular matrix are key steps of cancer progression, they are not necessarily fueled by the same metabolites and energy production pathways. The two main metabolic pathways cancer cells use to derive energy from glucose, glycolysis and oxidative phosphorylation, are preferentially and plastically utilized by cancer cells depending on both their intrinsic metabolic properties and their surrounding environment. Mechanical factors in the microenvironment, such as collagen density, pore size, and alignment, and biochemical factors, such as oxygen and glucose availability, have been shown to influence both cell migration and glucose metabolism. As cancer cells have been identified as preferentially utilizing glycolysis or oxidative phosphorylation based on heterogeneous intrinsic or extrinsic factors, the relationship between cancer cell metabolism and metastatic potential is of recent interest. Here, we review current in vitro and in vivo findings in the context of cancer cell metabolism during migration and metastasis and extrapolate potential clinical applications of this work that could aid in diagnosing and tracking cancer progression in vivo by monitoring metabolism. We also review current progress in the development of a variety of metabolically targeted anti-metastatic drugs, both in clinical trials and approved for distribution, and highlight potential routes for incorporating our recent understanding of metabolic plasticity into therapeutic directions. By further understanding cancer cell energy production pathways and metabolic plasticity, more effective and successful clinical imaging and therapeutics can be developed to diagnose, target, and inhibit metastasis.

Phenotypic Heterogeneity and Metastasis of Breast Cancer Cells.

Although intratumoral genomic heterogeneity can impede cancer research and treatment, less is known about the effects of phenotypic heterogeneities. To investigate the role of cell migration heterogeneities in metastasis, we phenotypically sorted metastatic breast cancer cells into two subpopulations based on migration ability. Although migration is typically considered to be associated with metastasis, when injected orthotopically in vivo, the weakly migratory subpopulation metastasized significantly more than the highly migratory subpopulation. To investigate the mechanism behind this observation, both subpopulations were assessed at each stage of the metastatic cascade, including dissemination from the primary tumor, survival in the circulation, extravasation, and colonization. Although both subpopulations performed each step successfully, weakly migratory cells presented as circulating tumor cell (CTC) clusters in the circulation, suggesting clustering as one potential mechanism behind the increased metastasis of weakly migratory cells. RNA sequencing revealed weakly migratory subpopulations to be more epithelial and highly migratory subpopulations to be more mesenchymal. Depletion of E-cadherin expression from weakly migratory cells abrogated metastasis. Conversely, induction of E-cadherin expression in highly migratory cells increased metastasis. Clinical patient data and blood samples showed that CTC clustering and E-cadherin expression are both associated with worsened patient outcome. This study demonstrates that deconvolving phenotypic heterogeneities can reveal fundamental insights into metastatic progression. More specifically, these results indicate that migratory ability does not necessarily correlate with metastatic potential and that E-cadherin promotes metastasis in phenotypically sorted breast cancer cell subpopulations by enabling CTC clustering. SIGNIFICANCE: This study employs phenotypic cell sorting for migration to reveal a weakly migratory, highly metastatic breast cancer cell subpopulation regulated by E-cadherin, highlighting the dichotomy between cancer cell migration and metastasis.

A discrete interface in matrix stiffness creates an oscillatory pattern of endothelial monolayer disruption.

Intimal stiffening upregulates endothelial cell contractility, disrupting barrier integrity; however, intimal stiffening is non-uniform. The impact of local changes in intimal stiffness on proximal and distal cell-cell interactions is unknown. To investigate the range at which matrix stiffness heterogeneities impact neighboring endothelial cells within a monolayer, we built a micropillar system with adjacent regions of stiff and compliant matrix. The stiffness interface results in an oscillatory pattern of neutrophil transendothelial migration, symmetrical about the interface and well-fit by a sinusoid function. 'Peaks' of the sinusoid were found to have increased cellular contractility and decreased barrier function relative to 'troughs' of the sinusoid. Pharmacological modulation of contractility was observed to break symmetry, altering the amplitude and wavelength of the sinusoid, indicating that contractility may regulate this effect. This work illuminates a novel biophysical phenomenon of the role of stiffness-mediated cell-matrix interactions on cell-cell interactions at a distance. Additionally, it provides insight into the range at which intimal matrix stiffness heterogeneities will impact endothelial barrier function and potentially contribute to atherogenesis.

Oxygen-Sensing Biomaterial Construct for Clinical Monitoring of Wound Healing.

OBJECTIVE: Oxygen is essential to wound healing; therefore, accurate monitoring can guide clinical decisions. Clinical wound assessment is often subjective, and tools to monitor wound oxygen are typically expensive, indirect, and highly variable. This study demonstrates the utility of a novel, low-cost oxygen-sensing thin film for serial assessment of wound oxygenation. DESIGN: Dual-layer films were fabricated with boron oxygen-sensing nanoparticles (BNPs) impregnated into a chitosan-polycaprolactone layer for direct wound bed contact with a relatively oxygen impermeable calcium alginate surface layer. The BNPs are a dual-emissive difluoroboron ß-diketonate dye incorporated into poly(lactic acid) nanoparticles. Under UV excitation, the BNPs emit fluorescence based on concentration and oxygen-sensitive phosphorescence. The fluorescence/phosphorescence ratio is directly proportional to oxygen concentration. METHODS: A series of in vitro oxygen challenges and in vivo murine and porcine wound healing models were used to validate the utility of the film in sensing wound oxygenation. MAIN RESULTS: In vitro testing demonstrated the oxygen-sensing capability of the BNP film and its ability to shield ambient oxygen to isolate wound oxygen. In vivo testing demonstrated the ability of the film to accurately monitor relative oxygen changes in a murine wound over time, measuring a 22% fluorescence/phosphorescence increase during acute healing. CONCLUSIONS: This study presents a low-cost, noninvasive, direct, and serial oxygen mapping technology to detect spatial differences in wound oxygenation. Clinical use of the films has the potential to monitor wound healing trajectories and guide wound care decisions.

Mechanobiology of microvesicle release, uptake, and microvesicle-mediated activation.

Microvesicles are small, membrane-bound vesicles that are shed from the plasma membrane of cells into the extracellular space. Microvesicles contain a variety of cargo not typically thought to be released from cells, including receptor tyrosine kinases, cytosolic signaling proteins, and microRNAs, which are transferred from donor cells to recipient cells. The transfer of microvesicle cargo can result in the transformation of recipient cells thereby supporting disease progression, including modified fibroblast metabolism, epithelial cell contractility, vascular remodeling, and immune cell inflammatory signaling. Additionally, microvesicles are believed to play prominent roles in cell-cell communication and disease progression as they are detected at elevated concentrations in diseased tissues. As microvesicle uptake by recipient cells can modulate cell function to promote disease progression, understanding the mechanisms and mechanosensitivity of microvesicle release, internalization, and the resulting signaling is crucial to fully comprehend their functions in disease. Here, we review recent advances in the understanding of actomyosin-regulated microvesicle biogenesis, microvesicle uptake via pinocytosis, and the resulting cellular transformation. We discuss the effects of altered cell contractility, mode of cell migration, and extracellular matrix compliance on microvesicle signaling, with direct implications in disease progression and identifying future therapeutic targets.

Cell-Cell Mechanical Communication in Cancer.

Communication between cancer cells enables cancer progression and metastasis. While cell-cell communication in cancer has primarily been examined through chemical mechanisms, recent evidence suggests that mechanical communication through cell-cell junctions and cell-ECM linkages is also an important mediator of cancer progression. Cancer and stromal cells remodel the ECM through a variety of mechanisms, including matrix degradation, cross-linking, deposition, and physical remodeling. Cancer cells sense these mechanical environmental changes through cell-matrix adhesion complexes and subsequently alter their tension between both neighboring cells and the surrounding matrix, thereby altering the force landscape within the microenvironment. This communication not only allows cancer cells to communicate with each other, but allows stromal cells to communicate with cancer cells through matrix remodeling. Here, we review the mechanisms of intercellular force transmission, the subsequent matrix remodeling, and the implications of this mechanical communication on cancer progression.

Matrix stiffness regulates microvesicle-induced fibroblast activation.

Extracellular vesicles released by cancer cells have recently been implicated in the differentiation of stromal cells to their activated, cancer-supporting states. Microvesicles, a subset of extracellular vesicles released from the plasma membrane of cancer cells, contain biologically active cargo, including DNA, mRNA, and miRNA, which are transferred to recipient cells and induce a phenotypic change in behavior. While it is known that microvesicles can alter recipient cell phenotype, little is known about how the physical properties of the tumor microenvironment affect fibroblast response to microvesicles. Here, we utilized cancer cell-derived microvesicles and synthetic substrates designed to mimic the stiffness of the tumor and tumor stroma to investigate the effects of microvesicles on fibroblast phenotype as a function of the mechanical properties of the microenvironment. We show that microvesicles released by highly malignant breast cancer cells cause an increase in fibroblast spreading, α-smooth muscle actin expression, proliferation, cell-generated traction force, and collagen gel compaction. Notably, our data indicate that these phenotypic changes occur only on stiff matrices mimicking the stiffness of the tumor periphery and are dependent on the cell type from which the microvesicles are shed. Overall, these results show that the effects of cancer cell-derived microvesicles on fibroblast activation are regulated by the physical properties of the microenvironment, and these data suggest that microvesicles may have a more robust effect on fibroblasts located at the tumor periphery to influence cancer progression.

Matrix Stiffness Enhances VEGFR-2 Internalization, Signaling, and Proliferation in Endothelial Cells.

Vascular endothelial growth factor (VEGF) can mediate endothelial cell migration, proliferation, and angiogenesis. During cancer progression, VEGF production is often increased to stimulate the growth of new blood vessels to supply growing tumors with the additional oxygen and nutrients they require. Extracellular matrix stiffening also occurs during tumor progression, however, the crosstalk between tumor mechanics and VEGF signaling remains poorly understood. Here, we show that matrix stiffness heightens downstream endothelial cell response to VEGF by altering VEGF receptor-2 (VEGFR-2) internalization, and this effect is influenced by cell confluency. In sub-confluent endothelial monolayers, VEGFR-2 levels, but not VEGFR-2 phosphorylation, are influenced by matrix rigidity. Interestingly, more compliant matrices correlated with increased expression and clustering of VEGFR-2; however, stiffer matrices induced increased VEGFR-2 internalization. These effects are most likely due to actin-mediated contractility, as inhibiting ROCK on stiff substrates increased VEGFR-2 clustering and decreased internalization. Additionally, increasing matrix stiffness elevates ERK 1/2 phosphorylation, resulting in increased cell proliferation. Moreover, cells on stiff matrices generate more actin stress fibers than on compliant substrates, and the addition of VEGF stimulates an increase in fiber formation regardless of stiffness. In contrast, once endothelial cells reached confluency, stiffness-enhanced VEGF signaling was no longer observed. Together, these data show a complex effect of VEGF and matrix mechanics on VEGF-induced signaling, receptor dynamics, and cell proliferation that is mediated by cell confluency.