Modelling TGFßR and Hh pathway regulation of prognostic matrisome molecules in ovarian cancer.

In a multi-level "deconstruction" of omental metastases, we previously identified a prognostic matrisome gene expression signature in high-grade serous ovarian cancer (HGSOC) and twelve other malignancies. Here, our aim was to understand how six of these extracellular matrix (ECM) molecules, COL11A1, cartilage oligomeric matrix protein, FN1, versican, cathepsin B, and COL1A1, are upregulated in cancer. Using biopsies, we identified significant associations between TGFßR activity, Hedgehog (Hh) signaling, and these ECM molecules and studied the associations in mono-, co-, and tri-culture. Activated omental fibroblasts (OFs) produced more matrix than malignant cells, directed by TGFßR and Hh signaling cross talk. We "reconstructed" omental metastases in tri-cultures of HGSOC cells, OFs, and adipocytes. This combination was sufficient to generate all six ECM proteins and the matrisome expression signature. TGFßR and Hh inhibitor combinations attenuated fibroblast activation and gel and ECM remodeling in these models. The tri-culture model reproduces key features of omental metastases and allows study of diseased-associated ECM.

Deconstruction of a Metastatic Tumor Microenvironment Reveals a Common Matrix Response in Human Cancers.

We have profiled, for the first time, an evolving human metastatic microenvironment by measuring gene expression, matrisome proteomics, cytokine and chemokine levels, cellularity, extracellular matrix organization, and biomechanical properties, all on the same sample. Using biopsies of high-grade serous ovarian cancer metastases that ranged from minimal to extensive disease, we show how nonmalignant cell densities and cytokine networks evolve with disease progression. Multivariate integration of the different components allowed us to define, for the first time, gene and protein profiles that predict extent of disease and tissue stiffness, while also revealing the complexity and dynamic nature of matrisome remodeling during development of metastases. Although we studied a single metastatic site from one human malignancy, a pattern of expression of 22 matrisome genes distinguished patients with a shorter overall survival in ovarian and 12 other primary solid cancers, suggesting that there may be a common matrix response to human cancer.Significance: Conducting multilevel analysis with data integration on biopsies with a range of disease involvement identifies important features of the evolving tumor microenvironment. The data suggest that despite the large spectrum of genomic alterations, some human malignancies may have a common and potentially targetable matrix response that influences the course of disease. Cancer Discov; 8(3); 304-19. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 253.

Differential nuclear expression of Yap in basal epithelial cells across the cornea and substrates of differing stiffness.

Corneal epithelium is maintained throughout life by well-orchestrated proliferation of limbal epithelial stem cells, followed by migration and maturation centripetally across the ocular surface. The present study sets out to explore the role tissue stiffness (compliance) may have in directing both differentiation and centripetal migration of limbal epithelial stem cells during homeostasis. For that, we analysed the localization of the Yes-associated protein (Yap), a transcriptional co-activator previously shown to mediate cellular response and mechanical stimuli. Using both models of ocular surface compliance and normal bovine corneas we evaluated the nuclear/cytoplasmic expression ratio of Yap. Expression levels within corneal epithelial cells were compared in situ between the limbus and central cornea, and in vitro between limbal epithelial stem cells expanded upon biomimetic collagen gels of increasing stiffness. Nuclear expression of Yap was shown to increase within the expanded cells upon substrates of increasing stiffness. Subsequently, Yap was used as a novel molecular probe to investigate the mechanical microenvironment within a normal ocular surface. The in situ localization of Yap was predominantly cytoplasmic within basal limbal epithelial cells and nuclear within basal central corneal epithelial cells. Furthermore, nuclear p63 expression was not co-localized with Yap in basal limbal epithelial cells. In conclusion, the current investigation provides new insights into the relationship between Yap and distinct cell populations across the ocular surface indicating that cells experience a different mechanical environment between the limbus and central cornea. A new hypothesis is put forward, in which centripetal differences in substrate stiffness drives the migration and differentiation of limbal epithelial stem cells, thus controlling corneal epithelium homeostasis.

The bioactivity of composite Fmoc-RGDS-collagen gels.

The incorporation of small bioactive peptide motifs within robust hydrogels constitutes a facile procedure to chemically functionalise cell and tissue scaffolds. In this study, a novel approach to utilise Fmoc-linked peptide amphiphiles comprising the bio-functional cell-adhesion RGDS motif within biomimetic collagen gels was developed. The composite scaffolds thus created were shown to maintain the mechanical properties of the collagen gel while presenting additional bio-activity. In particular, these materials enhanced the adhesion and proliferation of viable human corneal stromal fibroblasts by 300% compared to non-functionalised gels. Furthermore, the incorporation of Fmoc-RGDS nanostructures within the collagen matrix significantly suppressed gel shrinkage resulting from the contractile action of encapsulated fibroblasts once activated by serum proteins. These mechanical and biological properties demonstrate that the incorporation of peptide amphiphiles provides a suitable and easy method to circumvent specific biomaterial limitations, such as cell-derived shrinkage, for improved performance in tissue engineering and regenerative medicine applications.

Collagen stimulating effect of peptide amphiphile C16-KTTKS on human fibroblasts.

The collagen production of human dermal and corneal fibroblasts in contact with solutions of the peptide amphiphile (PA) C16-KTTKS is investigated and related to its self-assembly into nanotape structures. This PA is used in antiwrinkle cosmeceutical applications (trade name Matrixyl). We prove that C16-KTTKS stimulates collagen production in a concentration-dependent manner close to the critical aggregation concentration determined from pyrene fluorescence spectroscopy. This suggests that self-assembly and the stimulation of collagen production are inter-related.

Ex vivo expansion of limbal stem cells is affected by substrate properties.

Limbal epithelial stem cells play a key role in the maintenance and regulation of the corneal surface. Damage or destruction of these cells results in vascularisation and corneal opacity. Subsequent limbal stem cell transplantation requires an ex vivo expansion step and preserving cells in an undifferentiated state remains vital. In this report we seek to control the phenotype of limbal epithelial stem cells by the novel application of compressed collagen substrates. We have characterised the mechanical and surface properties of conventional collagen gels using shear rheology and scanning electron microscopy. In doing so, we provide evidence to show that compressive load can improve the stiffness of collagen substrates. In addition Western blotting and immunohistochemistry display increased cytokeratin 3 (CK3) protein expression relating to limbal epithelial cell differentiation on stiff collagen substrates. Such gels with an elastic modulus of 2900 Pa supported a significantly higher number of cells than less stiff collagen gels (3 Pa). These findings have substantial influence in the development of ocular surface constructs or experimental models particularly in the fields of stem cell research, tissue engineering and regenerative medicine.