Gkretsi, Vasiliki

As extracellular matrix (ECM) nano-characteristics play a crucial role in cell behavior, including cancer development and metastasis, several ECM in vitro models have been used in order to study cells behavior under different biochemical and mechanical conditions. Among the ECM constituents, collagen (especially collagen type I) has been extensively used as an essential component of ECM models, since it is one of the most abundant ECM protein. Use of three-dimensional (3D) collagen gels provides the advantage of allowing the cells to grow in a 3D environment that bears strong similarities to their natural, in vivo setting. Thus, the ability to form collagen gels with tunable stiffness and well defined naturally occurring nano-characteristics is crucial for these studies. Atomic Force Microscopy (AFM) is a unique tool that is ideal for the complete characterization of such models, in terms of morphology and mechanical properties without destroying the collagen fiber structure. In this protocol, the development and the AFM nano-scale characterization of 3D collagen type I gels is presented. The protocol includes: • The formation of 3D collagen type I gels with tunable stiffness• The preparation of histological sections from collagen gels• The AFM-based morphological and mechanical nano-characterization of the gels

Atomic force microscope (AFM) is a powerful and invaluable tool for imaging and probing the mechanical properties of biological samples at the nanometric scale. The importance of nano-scale characterization and nanomechanics of soft biological tissues is becoming widely appreciated, and AFM offers unique advantages in this direction. In this chapter, we describe the procedure to collect data sets (imaging and mechanical properties measurement) of collagen gels and tumor tissues. We provide step-by-step instructions throughout the procedure, from sample preparation to cantilever calibration, data acquisition, analysis, and visualization, using two commercial AFM systems (PicoPlus and Cypher ES) and software that accompanied the AFM systems and/or are freeware available (WSxM, AtomicJ). Our protocols are written specifically for these two systems and the mentioned software; however, most of the general concepts can be readily translated to other AFM systems and software.

In many solid tumours a desmoplastic reaction takes place, which results in tumour tissue stiffening due to the extensive production of extracellular matrix (ECM) proteins, such as collagen, by stromal cells, mainly fibroblasts (FBs) and cancer-associated fibroblasts (CAFs). In this study, we investigated the effect of collagen stiffness on pancreatic FBs and CAFs, particularly on specific cytoskeleton properties and gene expression involved in tumour invasion. We found that cells become stiffer when they are cultured on stiff substrates and express higher levels of alpha-smooth muscle actin (a-SMA). Also, it was confirmed that on stiff substrates, CAFs are softer than FBs, while on soft substrates they have comparable Young's moduli. Furthermore, the number of spread FBs and CAFs was higher in stiffer substrates, which was also confirmed by Ras-related C3 botulinum toxin substrate 1 (RAC1) mRNA expression, which mediates cell spreading. Although stress fibres in FBs become more oriented on stiff substrates, CAFs have oriented stress fibres regardless of substrate stiffness. Subsequently, we demonstrated that cells' invasion has a differential response to stiffness, which was associated with regulation of Ras homologue family member (RhoA) and Rho-associated, coiled-coil containing protein kinase 1 (ROCK-1) mRNA expression. Overall, our results demonstrate that collagen stiffness modulates FBs and CAFs cytoskeleton remodelling and alters their invasion properties.