Quantitative analysis of 3D extracellular matrix remodelling by pancreatic stellate cells

Abstract

Extracellular matrix (ECM) remodelling is integral to numerous physiological and pathological processes in biology, such as embryogenesis, wound healing, fibrosis and cancer. 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. We present an integrated methodology where cell-ECM interactions can be investigated in 3D environments via ECM remodelling. 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. Pancreatic stellate cells (PSCs) are the key effectors of the stromal fibrosis associated to pancreatic cancer. 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.

Tamoxifen mechanically reprograms the tumor microenvironment via HIF ‐1A and reduces cancer cell survival

“…Untreated control PSCs modified the topology of fibrillar collagen in the ECM substantially differently than their tamoxifen‐treated counterparts. We used the BoneJ plugin for ImageJ to quantify the thickness of fibrillar collagen fibers (Fig B), and a custom‐made algorithm based on fast Fourier transforms (FFT) to quantify the elliptical distribution of the fibrillar collagen network that indicates fiber alignment (Fig C). In this analysis, highly aligned collagen fibers exhibit values closer to 0 (elliptical distribution in Fig C inset), while randomly aligned fibers values close to 1 (circular distribution in Fig C inset).…”

Force-exerting lateral protrusions in fibroblastic cell contraction

“…The parallel fibers used in our method mimic the aligned ECMs produced by cancer associated fibroblasts (CAFs) in vitro and in vivo TACs2/3 fiber configurations known to enable CAF activation 11,61 . The inter-fiber spacing (~10 µm) used in our study mimics those measured by the fibroblastic cell-derived ECMs model and single harmonic generation (SHG) imaging of the patient samples 7,9,11,[61][62][63] . At higher inter-fiber spacing 64,65 , cells attach to single fibers in spindle morphologies with limited instances of twine formation, akin to the behavior reported in elongated spindles in 3D gels 15 .…”

Pancreatic cancer: a mechanobiology approach

“…Furthermore, the periductal collagen in PDAC exhibits architectural and structural differences in terms of linearization, alignment, and thickness, which can serve as an independent prognostic factor but might also facilitate cancer invasion through topographical contact guidance [27,28]. New imaging modalities such as second harmonic generation (SHG) imaging and elastography enable visualisation of cell-matrix interactions in 3D environments (in vitro) [29] and assessment of mechanical properties of tissues (in vivo) [30] and will help advance our understanding how structural, topographical, and mechanical changes of the microenviron ment can impact cancer cell behaviour.…”

“…(i) Second harmonic generation imaging allows label-free visualisation of collagen fibre (green) architecture and alignment when remodelled by stromal cells (red). Panel reproduced from [29]. CC BY 4.0.…”

“…AFM has been also extensively used to assess the mechanics of physiological relevant matrices that recreates different types of tissues. Combining AFM with high resolution microscopy techniques, such as multi-photon SHG facilitates not only to assess the matrix mechanics but also to correlate this with the topological changes in the tissue or matrix architecture, such as the thickness, alignment, and length of the ECM fibres ( figure 4(i)) [29].…”