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...

Tumor cell invasion through the stromal extracellular matrix (ECM) is a key feature of cancer metastasis, and understanding the cellular mechanisms of invasive migration is critical to the development of effective diagnostic and therapeutic strategies. Since cancer cell migration is highly adaptable to physiochemical properties of the ECM, it is critical to define these migration mechanisms in a context-specific manner. Although extensive work has characterized cancer cell migration in two- and three-dimensional (3D) matrix environments, the migration program employed by cells to move through native and cell-derived microtracks within the stromal ECM remains unclear. We previously reported the development of an in vitro model of patterned type I collagen microtracks that enable matrix metalloproteinase-independent microtrack migration. Here we show that collagen microtracks closely resemble channel-like gaps in native mammary stroma ECM and examine the extracellular and intracellular mechanisms underlying microtrack migration. Cell-matrix mechanocoupling, while critical for migration through 3D matrix, is not necessary for microtrack migration. Instead, cytoskeletal dynamics, including actin polymerization, cortical tension, and microtubule turnover, enable persistent, polarized migration through physiological microtracks. These results indicate that tumor cells employ context-specific mechanisms to migrate and suggest that selective targeting of cytoskeletal dynamics, but not adhesion, proteolysis, or cell traction forces, may effectively inhibit cancer cell migration through preformed matrix microtracks within the tumor stroma.

As cancer progresses, cells must adapt to a new and stiffer environment, which can ultimately alter how normal cells within the tumor behave. In turn, these cells are known to further aid tumor progression. Therefore, there is potentially a unique avenue to better understand metastatic potential through single-cell biophysical assays performed on patient-derived cells. Here, we perform biophysical characterization of primary human fibroblastic cells obtained from mammary carcinoma and normal contralateral tissue. Through a series of tissue dissociation, differential centrifugation and trypsinization steps, we isolate an adherent fibroblastic population viable for biomechanical testing. 2D TFM and 3D migration measurements in a collagen matrix show that fibroblasts obtained from patient tumors generate more traction forces and display improved migration potential than their counterparts from normal tissue. Moreover, through the use of an embedded spheroid model, we confirmed the extracellular matrix (ECM) remodeling behavior of primary cells isolated from carcinoma. Overall, correlating biophysical characterization of normal- and carcinoma-derived samples from individual patient along with patient outcome may become a powerful approach to further our comprehension of metastasis and ultimately design drug targets on a patient-specific basis.

Purpose: Clinical changes are best evaluated with standardized, validated outcomes, including both patient-reported outcome measures and surgeon-reported outcome measures (PROMs and SROMs). The purpose of this study was to describe the spectrum of outcome measures used in pediatric orthopaedic publications over the past 10 years and to determine the proportion that are in fact age-appropriate, validated, and appropriately applied in terms of condition and population. Methods: The Journal of Bone and Joint Surgery, The Bone and Joint Journal, Journal of Pediatric Orthopaedics A and B, and Journal of Children’s Orthopaedics were systematically searched for studies including children aged 18 and below, over a 10-year period from January 2005 to December 2014. Economic evaluations, letters, editorials, review articles, and clinical guidelines were excluded. SROMs and PROMs used were extracted, as were details on subject age and condition for which they were used. Each outcome scale was assessed for validity, and the proportion of scales used appropriately was calculated. Cochrane-Armitage test of trend was used to determine changes in PROM and SROM utilization over the study period. Results: A total of 4614 articles were identified, of which 2251 met inclusion and exclusion criteria. In total, 259 (11.5%) of studies used a PROM, whereas 326 (14.5%) used a SROM. A total of 230 different outcome scales were identified; 115 were patient reported and 115 were surgeon reported. However, only 18.7% of SROMs and 38.3% of PROMs were applied to an age and disease-appropriate demographic. Overall, there was a significant increase in the overall utilization of PROMs during the study period (P=0.004), but no corresponding increase in pediatric-validated PROMs (P=0.164). SROM utilization did not significantly change over the study period (P=0.337). Conclusions: Within the field of pediatric orthopaedics, an expansive variety of outcome scales are used, many of which have not been validated in children. Improved uniformity in reporting of outcomes and use of disease and age-validated outcomes scales is essential to improve multicenter research collaboration and data quality to generate appropriate evidence-based conclusions and treatment strategies in pediatric orthopaedics. Level of Evidence: Level IV—systematic review.

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