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...

In vitro cultures of endothelial cells are a widely used model system of the collective behavior of endothelial cells during vasculogenesis and angiogenesis. When seeded in an extracellular matrix, endothelial cells can form blood vessel-like structures, including vascular networks and sprouts. Endothelial morphogenesis depends on a large number of chemical and mechanical factors, including the compliancy of the extracellular matrix, the available growth factors, the adhesion of cells to the extracellular matrix, cell-cell signaling, etc. Although various computational models have been proposed to explain the role of each of these biochemical and biomechanical effects, the understanding of the mechanisms underlying in vitro angiogenesis is still incomplete. Most explanations focus on predicting the whole vascular network or sprout from the underlying cell behavior, and do not check if the same model also correctly captures the intermediate scale: the pairwise cell-cell interactions or single cell responses to ECM mechanics. Here we show, using a hybrid cellular Potts and finite element computational model, that a single set of biologically plausible rules describing (a) the contractile forces that endothelial cells exert on the ECM, (b) the resulting strains in the extracellular matrix, and (c) the cellular response to the strains, suffices for reproducing the behavior of individual endothelial cells and the interactions of endothelial cell pairs in compliant matrices. With the same set of rules, the model also reproduces network formation from scattered cells, and sprouting from endothelial spheroids. Combining the present mechanical model with aspects of previously proposed mechanical and chemical models may lead to a more complete understanding of in vitro angiogenesis.

Alternative splicing of proteins gives rise to different isoforms that play a crucial role in regulating several cellular processes. Notably, splicing profiles are altered in several cancer types, and these profiles are believed to be involved in driving the oncogenic process. Although the importance of alternative splicing alterations occurring during cancer is increasingly appreciated, the underlying regulatory mechanisms remain poorly understood. In this study, we use both biochemical and physical tools coupled with engineered models, patient samples, and a murine model to investigate the role of the mechanical properties of the tumor microenvironment in regulating the production of the extra domain-B (EDB) splice variant of fibronectin (FN), a hallmark of tumor angiogenesis. Specifically, we show that the amount of EDB-FN produced by endothelial cells increases with matrix stiffness both in vitro and within mouse mammary tumors. Matrix stiffness regulates splicing through the activation of serine/arginine rich (SR) proteins, the splicing factors involved in the production of FN isoforms. Activation of the SR proteins by matrix stiffness and the subsequent production of EDB-FN are dependent on intracellular contractility and PI3K-AKT signaling. Notably, matrix stiffness-mediated splicing is not limited to EDB-FN, but also affects splicing in the production of PKC βII and the VEGF 165b splice variant. Together, these results demonstrate that the mechanical properties of the microenvironment regulate alternative splicing and establish a previously unidentified mechanism by which cells can adapt to their microenvironment.alternative splicing | extracellular matrix | matrix stiffness | angiogenesis | cancer progression D ifferential expression of protein isoforms through alternative splicing is a key element to generating protein diversity and can result in widely different cell phenotypes and behaviors (1-3). Interestingly, tumors exhibit several major differences in protein isoform expression patterns compared with healthy tissue (2, 4), and some of these changes are thought to favor oncogenesis (1). Notably, splicing events are regulated at the pre-mRNA level by splicing regulatory factors that include a family of serine/arginine rich (SR) proteins (5). Elevated SR protein levels have been associated with cancer (5); however, the physiological cues that drive splicing are not well defined.Among the various alternatively spliced proteins present in tumors, the splice variant of fibronectin (FN) that includes the extra domain-B (EDB) type III repeat has been of particular interest in the cancer community because inclusion of the EDB fragment has been proposed as a way to identify and target tumor vasculature (6). EDB-FN is produced by endothelial cells (ECs) in tumors and may favor angiogenesis (7-9). Splicing of FN is regulated by several SR proteins, including SRp40 and SRp20, in several different cell types (10-12). Although the presence of EDB-FN is fairly unique to tumor vasculature, the mechanisms governin...

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.

Cells receive mechanical cues from their extracellular matrix (ECM), which direct migration, differentiation, apoptosis, and in some cases, the transition to a cancerous phenotype. As a result, there has been significant research to develop methods to tune the mechanical properties of the ECM and understand cell-ECM dynamics more deeply. Here, we show that ionizing radiation can reduce the stiffness of an ex vivo tumor and an in vitro collagen matrix. When non-irradiated cancer cells were seeded in the irradiated matrix, adhesion, spreading, and migration were reduced. These data have ramifications for both in vitro and in vivo systems. In vitro , these data suggest that irradiation may be a method that could be used to create matrices with tailored mechanical properties. In vivo , these suggest that therapeutic doses of radiation may alter tissue mechanics directly.

Angiogenesis, the process where new blood vessels form from existing vasculature, is essential for the successful integration of most tissue-engineered constructs and is dysregulated in many diseases, including cancer. To be functional, the newly formed vasculature must have similar structure and integrity as existing blood vessels, both of which are dependent upon mechanical and chemical cues from the surrounding extracellular matrix (ECM). ECM stiffness has emerged as a critical extracellular parameter that can modulate capillary network formation and barrier integrity. Moreover, matrix stiffness can alter how endothelial cells respond to soluble, angiogenic factors released by stromal cells, such as vascular endothelial growth factor (VEGF). In this review, we will discuss how matrix stiffness can affect the formation and structure of angiogenic vessels, and we will highlight the role of this work in the development of therapeutics to treat angiogenesis in cancer. Knowledge of the governing parameters for vessel formation is critical to the intelligent design of materials made to foster blood vessel growth for tissueengineering applications and pharmaceuticals designed to intervene with newly formed vasculature in diseased tissue.

Drug development is often hindered by the failure of preclinical models to accurately assess and predict the efficacy and safety of drug candidates. Body-on-a-chip (BOC) microfluidic devices, a subset of microphysiological systems (MPS), are being created to better predict human responses to drugs. Each BOC is designed with separate organ chambers interconnected with microfluidic channels mimicking blood recirculation. Here, we describe the design of the first pumpless, unidirectional, multiorgan system and apply this design concept for testing anticancer drug treatments.HCT-116 colon cancer spheroids, HepG2/C3A hepatocytes, and HL-60 promyeloblasts were embedded in collagen hydrogels and cultured within compartments representing "colon tumor", "liver," and "bone marrow" tissue, respectively. Operating on a pumpless platform, the microfluidic channel design provides unidirectional perfusion at physiologically realistic ratios to multiple channels simultaneously. The metabolism-dependent toxic effect of Tegafur, an oral prodrug of 5-fluorouracil, combined with uracil was examined in each cell type. Tegafur-uracil treatment induced substantial cell death in HCT-116 cells and this cytotoxic response was reduced for multicellular spheroids compared to single cells, likely due to diffusionlimited drug penetration. Additionally, off-target toxicity was detected by HL-60 cells, which demonstrate that such systems can provide useful information on doselimiting side effects. Collectively, this microscale cell culture analog is a valuable physiologically-based pharmacokinetic drug screening platform that may be used to support cancer drug development.

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