Physical changes in skin are among the most visible signs of aging. We found that young dermal fibroblasts secrete high levels of extracellular matrix (ECM) constituents, including proteoglycans, glycoproteins and cartilage-linking proteins. The most abundantly secreted was HAPLN1, a hyaluronic and proteoglycan link protein. HAPLN1 was lost in aged fibroblasts, resulting in a more aligned ECM that promoted metastasis of melanoma cells. Reconstituting HAPLN1 inhibited metastasis in an aged microenvironment, in 3D skin reconstruction models, and in vivo. Intriguingly, aged fibroblast-derived matrices had the opposite effects on the migration of T-cells, inhibiting their motility. HAPLN1 treatment of aged fibroblasts restored motility of mononuclear immune cells, while impeding that of polymorphonuclear immune cells, which in turn affected Treg recruitment. These data suggest while age-related physical changes in the ECM can promote tumor cell motility, they may adversely impact the motility of some immune cells, resulting in an overall change in the immune microenvironment. Understanding the physical changes in aging skin may provide avenues for more effective therapy for older melanoma patients.

BackgroundAlterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions.We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer.MethodsWe generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses.ResultsWe observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated.ConclusionCancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and β1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking β1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions.

Desmoplasia, a fibrotic mass including cancer-associated fibroblasts (CAFs) and self-sustaining extracellular matrix (D-ECM), is a puzzling feature of pancreatic ductal adenocarcinoma (PDACs). Conflicting studies have identified tumor-restricting and tumor-promoting roles of PDAC-associated desmoplasia, suggesting that individual CAF/D-ECM protein constituents have distinguishable tumorigenic and tumor-repressive functions. Using 3D culture of normal pancreatic versus PDAC-associated human fibroblasts, we identified a CAF/D-ECM phenotype that correlates with improved patient outcomes, and that includes CAFs enriched in plasma membrane-localized, active α5β1-integrin. Mechanistically, we established that TGFβ is required for D-ECM production but dispensable for D-ECM-induced naïve fibroblast-to-CAF activation, which depends on αvβ5-integrin redistribution of pFAK-independent active α5β1-integrin to assorted endosomes. Importantly, the development of a simultaneous multi-channel immunofluorescence approach and new algorithms for computational batch-analysis and their application to a human PDAC panel, indicated that stromal localization and levels of active SMAD2/3 and α5β1-integrin distinguish patient-protective from patient-detrimental desmoplasia and foretell tumor recurrences, suggesting a useful new prognostic tool.DOI: http://dx.doi.org/10.7554/eLife.20600.001

For decades tumors have been recognized as “wounds that do not heal.” Besides the commonalities that tumors and wounded tissues share, the process of wound healing also portrays similar characteristics with chronic fibrosis. In this review, we suggest a tight interrelationship, which is governed as a concurrence of cellular and microenvironmental reactivity among wound healing, chronic fibrosis, and cancer development/progression (i.e., the WHFC triad). It is clear that the same cell types, as well as soluble and matrix elements that drive wound healing (including regeneration) via distinct signaling pathways, also fuel chronic fibrosis and tumor progression. Hence, here we review the relationship between fibrosis and cancer through the lens of wound healing.

Fibroblasts secrete and organize extracellular matrix (ECM), which provides structural support for their adhesion, migration, and tissue organization, besides regulating cellular functions such as growth and survival. Cell-to-matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer, and other diseases. This unit describes a method for preparing a three-dimensional matrix derived from fibroblastic cells; the matrix is three-dimensional, cell and debris free, and attached to a two-dimensional culture surface. Cell adhesion and spreading are normal on these matrices. This matrix can also be compressed into a two-dimensional matrix and solubilized to study the matrix biochemically. Culturing fibroblasts on traditional two-dimensional (2-D) substrates induces an artificial polarity between lower and upper surfaces of these normally nonpolar cells. Not surprisingly, fibroblast morphology and migration differ once suspended in three-dimensional (3-D) collagen gels (Friedl and Brocker, 2000). However, the molecular composition of collagen gels does not mimic the natural fibroblast (i.e., mesenchymal) microenvironment. Fibroblasts secrete and organize ECM, which provides structural support for their adhesion, migration, and tissue organization, in addition to regulating cellular functions such as growth and survival (Buck and Horwitz, 1987; Hay, 1991; Hynes, 1999; Geiger et al., 2001). Cell-to-matrix interactions are vital for vertebrate development. Disorders in these processes have been associated with fibrosis, developmental malformations, cancer (i.e., desmoplastic tumor microenvironment), and other diseases (Rybinski et al., 2014). This unit describes methods for generating tissue culture surfaces coated with a fibroblast-derived 3-D ECM produced and deposited by both established and primary fibroblasts. The matrices closely resemble in vivo mesenchymal matrices and are composed mainly of fibronectin fibrillar lattices. Utilizing in vivo-like 3-D matrices as substrates allows the acquisition of information that is physiologically relevant to cell-matrix interactions, structure, function, and signaling, which differ from data obtained by culturing cells on conventional 2-D substrates in vitro (Cukierman et al., 2001). These protocols were initially derived from methods described in UNIT 10.4, which were modified to obtain fibroblast-derived 3-D matrices and to characterize cellular responses to them. The basic approach is to allow fibroblasts to produce their own 3-D matrix (see Basic Protocol). For this purpose, fibroblasts are plated and maintained in culture in a confluent state. After 5 to 9 days, unextracted 3-D matrix cultures can be sorted into normal or activated (i.e., myofibroblastic, fibrotic or desmoplastic) phenotypes (see Support Protocol 1) or matrices are denuded of cells, and cellular remnants are removed. Such extraction results in an intact fibroblast-derived 3-D matrix that is free of cellular deb...