The lack of centrosymmetry that necessarily accompanies this gives collagen-l the optical properties required for SHG. The peptide bonds linking together amino acids in the chains have their own dipole which, when amplified along the helix length of collagen-I, gives the fibrillar structure a permanent dipole moment ( Fig. 2A). Collagen-I is a triple helix, made up of three α-helical chains, with these individual helices self-assembling into fibrils and larger-scale fibres. SHG is a microscopy technique that is highly responsive to fibrillar collagen ( Chen et al., 2012). The collagen topography can be analysed through existing as well as newly developed methods to correlate specific changes in ECM composition with changes in mechanical properties such as stiffness. An optical signal for collagen-I can be obtained through SHG imaging with high specificity and without the need for immunostaining. This remodelling can also be quantified by specifically imaging collagen-I, an abundant fibrous ECM component, using second harmonic generation (SHG) alongside multiphoton microscopy (MPM) ( Acerbi et al., 2015 Campagnola and Loew, 2003 Raub et al., 2010). This method of obtaining a Young's modulus allows localised mechanical differences due to ECM remodelling to be detected that would not be obtained through larger-scale rheology studies.
A glass micro-sphere attached to a cantilever indents the sample and is deflected in a manner determined by the sample stiffness, measured through deflection of a laser ( Alexander et al., 1989). AFM indentation can be used to obtain the Young's modulus of samples through force spectroscopy. In this work, we used atomic force microscopy (AFM) indentation, high resolution optical imaging, and custom made algorithms to provide a platform for the study of 3D matrix remodelling by cells. Assessing these quantitative changes in the ECM will provide a better understanding of the remodelling processes. Collagen alignment in the tumour periphery is used as a prognostic marker for survival in several cancers including breast cancer ( Conklin et al., 2011), and it is known that highly aligned fibroblast derived matrices promote cancer cell invasion ( Goetz et al., 2011). In pancreatic ductal adenocarcinoma (PDAC), the strong fibrosis in the stromal region around the tumour is mediated via ECM remodelling and orchestrated by pancreatic stellate cells (PSCs) ( Apte et al., 2011 Apte and Wilson, 2012 Olsen et al., 2011). Additionally, in cancer, ECM rigidity promotes breast cancer progression via oncogenic signalling in epithelial cells ( Levental et al., 2009), and tumour-associated fibroblasts remodel the ECM via Rho-dependent cytoskeleton contraction to facilitate cancer cell invasion ( Calvo et al., 2013 Gaggioli et al., 2007 Goetz et al., 2011). We use PSCs to implement our methodology and demonstrate that PSC matrix remodelling capabilities depend on their contractile machinery and β1 integrin-mediated cell-ECM attachment.ģD remodelling of the extracellular matrix (ECM), which involves changes in ECM rigidity and organisation, is integral to several biological processes, such as wound healing ( Darby et al., 2014 Reinke and Sorg, 2012), fibrosis ( Duscher et al., 2014 Ho et al., 2014), and embryogenesis, where mechanical forces dictate tissue organisation ( Krieg et al., 2008).
Pancreatic stellate cells (PSCs) are the key effectors of the stromal fibrosis associated to pancreatic cancer. Monitoring and quantification of collagen-I structure in remodelled matrices, through designated algorithms, show that 3D matrices can be used to correlate remodelling with increased ECM stiffness observed in fibrosis. We present an integrated methodology where cell-ECM interactions can be investigated in 3D environments via ECM remodelling.
Until recently, most cellular studies have been conducted on 2D environments where mechanical cues significantly differ from physiologically relevant 3D environments, impacting cellular behaviour and masking the interpretation of cellular function in health and disease. Extracellular matrix (ECM) remodelling is integral to numerous physiological and pathological processes in biology, such as embryogenesis, wound healing, fibrosis and cancer.
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