Tumor microvasculature tends to be malformed, more permeable, and more tortuous than vessels in healthy tissue, effects that have been largely attributed to up-regulated VEGF expression. However, tumor tissue tends to stiffen during solid tumor progression, and tissue stiffness is known to alter cell behaviors including proliferation, migration, and cell-cell adhesion, which are all requisite for angiogenesis. Using in vitro, in vivo, and ex ovo models, we investigated the effects of matrix stiffness on vessel growth and integrity during angiogenesis. Our data indicate that angiogenic outgrowth, invasion, and neovessel branching increase with matrix cross-linking. These effects are caused by increased matrix stiffness independent of matrix density, because increased matrix density results in decreased angiogenesis. Notably, matrix stiffness up-regulates matrix metalloproteinase (MMP) activity, and inhibiting MMPs significantly reduces angiogenic outgrowth in stiffer crosslinked gels. To investigate the functional significance of altered endothelial cell behavior in response to matrix stiffness, we measured endothelial cell barrier function on substrates mimicking the stiffness of healthy and tumor tissue. Our data indicate that barrier function is impaired and the localization of vascular endothelial cadherin is altered as function of matrix stiffness. These results demonstrate that matrix stiffness, separately from matrix density, can alter vascular growth and integrity, mimicking the changes that exist in tumor vasculature. These data suggest that therapeutically targeting tumor stiffness or the endothelial cell response to tumor stiffening may help restore vessel structure, minimize metastasis, and aid in drug delivery.tumor stiffness | endothelial cells | vascular permeability | glycation | extracellular matrix T he ingrowth of newly sprouted blood vessels is necessary for solid tumor growth, and tumor vasculature is typically malformed, leakier, and more tortuous than the vasculature of normal tissues (1-3). Generally, aberrant tumor vasculature is considered to be caused by up-regulated VEGF expression resulting in chaotic vascular growth and failure to establish mature, well-regulated networks (4, 5). Here, we propose a different hypothesis, namely that extracellular matrix (ECM) mechanical properties also contribute to the aberrant vascular phenotype seen in tumors.Solid tumor tissue is typically stiffer than native, healthy tissue (1, 6). Increased ECM stiffness within tumors is caused primarily by both increased collagen deposition and increased cross-linking within the tumor stroma (7). Increased ECM density and crosslinking are associated with poor prognosis in a number of cancers (8, 9). Many studies have investigated the role of matrix density on angiogenesis and, in both collagen and fibrin matrices, have shown that angiogenesis decreases with increasing matrix concentration (10-13). Increased matrix density appears to act as a physical barrier that restricts cell migration, and cells rely on matr...

Communication between the inner cell mass (ICM) and the trophoblast layer of the blastocyst is known to occur, but its functional consequences on early developmental events is unclear. Here we demonstrate that embryonic stem (ES) cells derived from the ICM generate and shed microvesicles (MVs), a major class of extracellular vesicles (EVs), which influence trophoblast behaviour during the implantation process. The MV cargo proteins laminin and fibronectin interact with integrins along the surfaces of the trophoblasts, triggering the activation of two signalling kinases, JNK and FAK, and stimulating trophoblast migration. We further show that injecting MVs isolated from ES cells into blastocysts results in an increase in their implantation efficiency. Thus, these findings highlight a unique mechanism by which ES cells communicate with trophoblasts within the blastocyst to increase their ability to migrate into the uterus, thereby promoting one of the earliest and most important steps during pregnancy.

One of the key steps during tumour metastasis is tumour cell migration and invasion, which require actin cytoskeletal reorganization. Among the critical actin cytoskeletal protrusion structures are the filopodia, which act like cell sensory organs to communicate with the extracellular microenvironment and participate in fundamental cell functions such as cell adhesion, spreading and migration in the three-dimensional environment. Fascin is the main actin-bundling protein in filopodia. Using high-throughput screening, here we identify and characterize small molecules that inhibit the actin-bundling activity of fascin. Focusing on one such inhibitor, we demonstrate that it specifically blocks filopodial formation, tumour cell migration and invasion in vitro, and metastasis in vivo. Hence, target-specific anti-fascin agents have a therapeutic potential for cancer treatment.

The ability of primary tumor cells to invade into adjacent tissues, followed by the formation of local or distant metastasis, is a lethal hallmark of cancer. Recently, locomoting clusters of tumor cells have been identified in numerous cancers and associated with increased invasiveness and metastatic potential. However, how the collective behaviors of cancer cells are coordinated and their contribution to cancer invasion remain unclear. Here we show that collective invasion of breast cancer cells is regulated by the energetic statuses of leader and follower cells. Using a combination of in vitro spheroid and ex vivo organoid invasion models, we found that cancer cells dynamically rearrange leader and follower positions during collective invasion. Cancer cells invade cooperatively in denser collagen matrices by accelerating leader–follower switching thus decreasing leader cell lifetime. Leader cells exhibit higher glucose uptake than follower cells. Moreover, their energy levels, as revealed by the intracellular ATP/ADP ratio, must exceed a threshold to invade. Forward invasion of the leader cell gradually depletes its available energy, eventually leading to leader–follower transition. Our computational model based on intracellular energy homeostasis successfully recapitulated the dependence of leader cell lifetime on collagen density. Experiments further supported model predictions that decreasing the cellular energy level by glucose starvation decreases leader cell lifetime whereas increasing the cellular energy level by AMP-activated kinase (AMPK) activation does the opposite. These findings highlight coordinated invasion and its metabolic regulation as potential therapeutic targets of cancer.

Cells expend energy to migrate. Metastatic cancer cell energy levels were investigated as a function of collagen architecture. In more migration-permissive environments or when migration is pharmacologically inhibited, cells reduce ATP:ADP levels. Changes in intracellular ATP:ADP levels during migration were associated with changes in cell speed. The data suggest that cells tune their energy production and utilization relative to their migration.