Extracellular Matrix Tension Research Articles

MECHANICAL REGULATION OF MATRIX REMODELLING IN TENDON WOUND HEALING

Extracellular matrix (ECM) mechanical cues guide healing in tendons. Yet, the molecular mechanisms orchestrating the healing processes remain elusive. Appropriate tissue tension is essential for tendon homeostasis and tissue health. By mapping the attainment of tensional homeostasis, we aim to understand how ECM tension regulates healing. We hypothesize that diseased tendon returns to homeostasis only after the cells reach a mechanically gated exit from wound healing.We engineered a 3D mechano-culture system to create tendon-like constructs by embedding patient-derived tendon cells into a collagen I hydrogel. Casting the hydrogel between posts anchored in silicone allowed adjusting the post stiffness. Under this static mechanical stimulation, cells remodel the (unorganized) collagen representing wound healing mechanisms. We quantified tissue-level forces using post deflection measurements. Secreted ECM was visualized by metabolic labelling with non-canonical amino acids, click chemistry and confocal microscopy. We blocked cell-mediated actin-myosin contractility using a ROCK inhibitor (Y27632) to explore the involvement of the Rho/ROCK pathway in tension regulation.Tissue tension forces reached the same homeostatic level at day 21 independent of post compliance (p = 0.9456). While minimal matrix was synthesized in early phases of tissue formation (d3-d5), cell-deposited ECM was present in later stages (d7-d9). More ECM was deposited by tendon constructs cultured on compliant (1Nm) compared to rigid posts (p = 0.0017). Matrix synthesized by constructs cultured on compliant posts was less aligned (greater fiber dispersion, p = 0.0021). ROCK inhibition significantly decreased tissue-level tensional forces (p < 0.0001).Our results indicate that tendon cells balance matrix remodeling and synthesis during tissue repair to reach an intrinsically defined “mechanostat setpoint” guiding tension-mediated exit from wound healing towards homeostasis. We are identifying specific molecular mechanosensors governing tension-regulated healing in tendon and investigate the Rho/ROCK system as their possible downstream pathway.

Myofibroblast repair mechanisms post-inflammatory response: a fibrotic perspective.

Fibrosis is a complex chronic disease characterized by a persistent repair response. Its pathogenesis is poorly understood but it is typically the result of chronic inflammation and maintained with the required activity of transforming growth factor-β (TGFβ) and extracellular matrix (ECM) tension, both of which drive fibroblasts to transition into a myofibroblast phenotype. As the effector cells of repair, myofibroblasts migrate to the site of injury to deposit excessive amounts of matrix proteins and stimulate high levels of contraction. Myofibroblast activity is a decisive factor in whether a tissue is properly repaired by controlled wound healing or rendered fibrotic by deregulated repair. Extensive studies have documented the various contributing factors to an abrogated repair response. Though these fibrotic factors are known, very little is understood about the opposing antifibrotic molecules that assist in a successful repair, such as prostaglandin E2 (PGE2) and ECM retraction. The following review will discuss the general development of fibrosis through the transformation of myofibroblasts, focusing primarily on the prominent profibrotic pathways of TGFβ and ECM tension and antifibrotic pathways of PGE2 and ECM retraction. The idea is to understand the ways in which the cell, after an injury and inflammatory response, normally controls its repair mechanisms through its homeostatic regulators so as to mimic them therapeutically to control abnormal pathways.

Abstract S2-06: Extracellular matrix stiffness modulates tissue inflammation to promote breast tumor aggression

Abstract Tumor progression is influenced by the dynamic interplay between the genetically-modified epithelium and the associated microenvironment. We previously showed that the extra-cellular matrix (ECM) progressively stiffens as mammary tumors evolve, and that stiffening the ECM promotes malignant transformation while inhibiting ECM stiffening reduces tumor progression (Levental et al., 2009; reviewed in Butcher et al., 2009). Yet breast cancers also exhibit elevated influx of inflammatory cells and tissue inflammation promotes tumor progression by fostering angiogenesis and enhancing tumor cell growth and motility (Ruffell et al., 2011). Tumor-associated macrophages additionally stimulate stromal fibroblasts, implying that tissue inflammation could promote malignancy by stiffening the ECM. To address this possibility, we explored the relationship between ECM tension and macrophages in human breast cancer and during mammary tumor development in transgenic mice. Using fresh and archived tissue samples, we quantified a progressive increase in ECM remodeling and stiffening as human breast tumors and mouse mammary cancers developed. We found that the ECM associated with triple negative breast cancers was almost twice as stiff as the ECM adjacent to ER+/PR+ tumors and that ECM stiffness correlated significantly with increased numbers of infiltrating activated macrophages. Consistently, we established a positive correlation between tumor progression, elevated mechanosignaling, ECM stiffness and tissue inflammation in both human and mouse tissue. However surprisingly, when macrophages were depleted from the mammary glands of MMTV-PyMT tumors, ECM tension did not change. Instead we found that inhibiting lysyl oxidase activity to decrease collagen cross-linking and ECM stiffness reduced the activation state of the macrophages within the mammary glands of 14-week old mice. Consistently, in vitro studies showed that ECM tension potentiates the expression of pro-inflammatory chemokines and increases levels of phosphoStat3 and that ECM tension directly modifies macrophage polarity. These findings suggest that ECM tension may promote malignancy either by directly regulating macrophage activity or indirectly through enhancing expression of pro-inflammatory chemokines. Further studies are now underway to explore these findings and to clarify their impact on tumor progression and response to therapy. Acknowledgements: supported by W81XWH-05-1-0330 and R01 CA138818-01A1 to VMW, 1U01 ES019458-01 to VMW and ZW, and P50 CA 58207 to JG, VW, SH and LC, U54CA143836-01 to JL and VW, and Susan G. Komen for the Cure PSF12230246 to IA. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S2-06.

Dynamic Changes of Collagen Ⅳ During Cancer Invasion and Migration: Pulse Mode

Cancer invasion and metastasis remain two root causes of mortality. This process involves alterations of tumor microenvironment, particularly the remodeling of extracellular matrix(ECM), characterized by collagen Ⅳ uncoiling, degradation, fragments deposition and cross-linking. This study was aimed to reveal the cancer invasion mode based on dynamic changes of collagen Ⅳ by novel quantum dots(QDs) imaging technology. Cancer tissues of hepatocellular carcinoma, gastric cancer, breast cancer and cervical carcinoma were collected and stained by traditional immunohistochemistry and novel QDs-based imaging technology. Several key molecules representing of cancer cells and tumor microenvironment were studied, including Ki67, representing of proliferation of cancer cells, macrophages, representing of monocytes infiltration and collagen Ⅳ, representing of tumor stroma remodeling during cancer invasion and migration. During cancer invasion and migration, collagen Ⅳ had structural and functional changes. In different cancer tissues, 4 types of collagen Ⅳ could be observed, consisting of linear, irregular, fragmented and disappeared. However, several common features were evident during the dynamic process of cancer progression. First, collagen Ⅳ at basement membrane increased, presenting an irregular sheath surrounding cancer nests; then, collagen Ⅳ was degraded to form invasion fronts at several sites accompanied with linear re-deposition of collagen Ⅳ and increased macrophages in ECM. Second, cancer cells escaped from large cancer nests to seed in ECM. And also the dynamic changes of collagen Ⅳ were accompanied with the recruitment of macrophages, together to regulate and affect biological behaviors of cancer cells. A series changes caused by dynamic changes of collagen Ⅳ provide a proper tumor microenvironment for cancer invasion and migration. In this dysfunctional tumor microenvironment, tumor mass becomes increasingly harder with tumor stroma stiffening causing high ECM stress. With cancer cells proliferation, tension becomes increasingly higher, till reaching a critical point, where large tumor nests "burst", releasing many tiny seeding nests and reducing the central ECM tension. The resulting seeding tumor nests repeat the same process of tumor mass growth →prominent central hypoxia→collagen Ⅳ cross-linking and deposition→ECM remodeling→ECM stress buildup→tumor nest bursts, leading to accelerated cancer progression in this rich "soil" of dysfunctional tumor microenvironment, presenting a "pulse mode".

159 The Role of the Biophysical Properties of the Glycocalyx and Extracellular Matrix Tension in Enhancing Glioma Cell Invasion

159 The Role of the Biophysical Properties of the Glycocalyx and Extracellular Matrix Tension in Enhancing Glioma Cell Invasion

P1-03-01: ECM Stiffness and Breast Tumor Histology and Treatment Phenotype.

Abstract Currently, the extent of local surgery and radiation for breast cancer is not guided by biological or anatomical principles, and the optimal volume of treatment is not defined. Local tumor progression is strongly influenced by a dynamic interplay between the genetically-modified epithelium and the associated cellular and non-cellular stroma (Butcher, 2009; Levental, 2009). The extracellular matrix (ECM) is modified in breast tumors and data indicate that the ECM stiffens progressively as mammary tumors evolve and enhancing ECM stiffness promotes mammary tumorigenesis while inhibiting ECM stiffening reduces tumor progression. However, the degree and extent of local stromal changes, and their clinical impact in breast cancer evolution and treatment remain unclear. This has led us to predict that the desmoplastic stroma surrounding breast tumors reflects tumor phenotype and behavior and can be characterized by biophysical metrics of the ECM stroma and molecular signatures of the breast epithelium. Accordingly, we hypothesize that these features may be integrated into a stromal phenotype that can be developed as a predictive tool. To test this concept we used a rigorous biomechanical and biochemical analysis of mastectomy specimens from women with various stages and histology phenotypes of treated and non-treated breast cancer to explore the relationship between ECM stiffness, mechanotransduction and tumor behavior. The goal of the study was to characterize stromal changes as a function of tumor stage and histology phenotype as well as before and after chemotherapy by measuring biophysical and topological features of the ECM and biochemical and molecular signatures of the mammary epithelium. The objective was to determine if there is a significant association with tumor histology phenotype that could be integrated to generate a stromal phenotype. Egan sections of were acquired from breast tissue containing benign and invasive ER positive and negative carcinoma and from treated and non-treated patients. All tissues were subjected to IHC histological and mechanosignaling analysis (H&E; 397FAK; Lysyl Oxidase; phosphomyosin) and biomechanical assessment (Atomic Force Microscopy; Structured Illumination Polarized Imaging; Two Photon Imaging). Preliminary data were consistent with prior in vitro and in vivo studies and showed that there was a significant increase in ECM stiffness as the tissues transitioned from normal to an invasive lesion with the highest stiffness being located at the tumor edge (∼2-4 folds greater). Intriguingly, we observed that ER negative tumors were substantially stiffer than ER positive tumors (50% increase in upper 10-percentile) and that there were tracks of ECM stiffness that correlated with orientated parallel collagen fibers and ECM birefringence. Moreover, we quantified a significant decrease in ECM stiffness following treatment (40% lower) with the most striking reduction in ECM tension being noted in ER negative patient tissues who demonstrated the most robust response. This study should lead to a deeper understanding of the nature of breast cancer stroma and its role in tumor phenotype and response to therapy. Supported by: NCI SPORE P50 CA058207 to VMW, CP, & SH; U01 ES019458-02 to ZW; NCI R01 CA138818-01A1 to VMW; & U54 CA143836-01 to VMW & JL. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-03-01.

Choreographing Metastasis to the Tune of LTBP

Latent Transforming Growth Factor beta (TGFβ) Binding Proteins (LTBPs) are chaperones and determinants of TGFβ isoform-specific secretion. They belong to the LTBP/Fibrillin family and form integral components of the fibronectin and microfibrillar extracellular matrix (ECM). LTBPs serve as master regulators of TGFβ bioavailability, functioning to incorporate and spatially pattern latent TGFβ at regular intervals within the ECM, and actively participate in integrin-mediated stretch activation of TGFβ in vivo. In so doing they create a highly patterned sensory system where local changes in ECM tension can be detected and transduced into focal signals. The physiological role of LTBPs in the mammary gland remains largely unstudied, however both loss and gain of LTBP expression is found in breast cancers and breast cancer cell lines. Importantly, elevated LTBP1 levels appear in two gene signatures predictive of enhanced metastatic behavior. LTBP may promote metastasis by providing the bridge between structural and signaling components of the epithelial to mesenchymal transition (EMT).

Corneal fibroblasts respond rapidly to changes in local mechanical stress.

To investigate the response of corneal fibroblasts to local changes in extracellular matrix (ECM) tension. Rabbit and human corneal fibroblasts were plated inside fibrillar collagen matrices. After 18 to 72 hours, a glass microneedle was inserted into the ECM and either pushed toward a cell to reduce local tension, or pulled away to increase tension. Time-lapse differential interference contrast (DIC) imaging was performed both before and after needle micromanipulation. ECM displacements were quantified, and strain maps were generated by finite element modeling. In some experiments, cells were treated with the Rho-kinase inhibitor Y-27632 either 30 minutes before, or 1 hour after they were pushed with the microneedle. Changes in focal adhesion organization were also evaluated in a subset of cells expressing green fluorescent protein (GFP)-zyxin, by simultaneous fluorescent and DIC imaging. Pulling on the ECM resulted in initial cell elongation, followed by disengagement and retraction of pseudopodia. In contrast, pushing the ECM toward a cell induced rapid shortening (contraction), presumably since existing cellular forces were no longer counterbalanced by ECM tension. Pseudopodial extension (spreading) was then observed at both ends of the cell. The ECM was pulled inward during this secondary spreading, and rapid turnover of focal adhesions was observed along extending pseudopodia. Preincubation with Y-27632 or cytochalasin D blocked both the initial contractile and secondary spreading responses. Overall, the data suggest that corneal fibroblasts actively respond to increases or decreases in local matrix stress in an attempt to maintain tensional homeostasis (constant tension), and that this response may be mediated by Rho and/or Rac.