Deconstructing the tumour microenvironment: the role of fibronectin

[摘要] Cancer is a worldwide issue affecting millions and the need for understanding specific mechanisms linked to its progression has never been higher. One of the biggest challenges in cancer research is the complexity of the tumour microenvironment (TME) which not only consists of cancerous cells, but auxiliary lymphocytes, macrophages, blood vessels and the extracellular matrix (ECM). There is therefore a need to unravel the impact of these specific components on cancer cells by deconstructing their environment. In this study, we aim to understand fundamental cell-ECM interactions by breaking down the complex TME to its building blocks: the cancer cell and the ECM. We use poly(alkyl acrylates) material surfaces which have been shown to direct the organisation of fibronectin (FN), a key ECM protein, upon adsorption to study cell behaviour such as adhesion, growth, migration and drug resistance. Here we show that poly(methyl acrylate) (PMA) and poly(ethyl acrylate) PEA are able to organise FN into globular and fibrillar conformations respectively upon adsorption, thus exposing or concealing specific integrin binding domains such as the RGD cell binding domain and the PHSRN synergy sequence. With these conditions, we assess: cell adhesion through studying attachment, focal adhesion formation and single cell traction on the surface of the polymers; cell migration by looking at the speed of gap closure in wound healing assays; drug resistance by studying cytotoxicity of a well-established anticancer drug in docetaxel as well as PND-1186 (VS-4718) which is a novel drug currently in phase 1 clinical trials; and intracellular signalling by quantifying protein expressions of focal adhesion kinase (FAK). Furthermore, we conduct a preliminary study of cancer cells in a highly tuneable PEG-based (poly ethylene glycol) 3D system functionalised with the RGD cell binding domain. We analyse hydrogel stability, cell viability and cell invasion. We demonstrate that upon adsorption to the polymers the fibrillar and globular forms of FN lead to the PHSRN synergy sequence being more exposed on PEA compared to PMA respectively. This is shown to have significant impact on cell anchorage, mediated primarily by the RDG domain of FN via integrin αvβ3, and cell motility which is mediated by both the RGD and PHSRN sequences via integrin α5β1. We also demonstrate that although this 2D model provides essential information for cell-ECM interactions, it does not take into account the 3D environment. We show that cells are able to interact with the proposed PEG-based hydrogel and that it can be fine-tuned by altering gel stiffness and functional components independently. Overall, the methods and systems used in this study have allowed for a better understanding of the material-protein and the cell-protein interfaces and how they affect cell behaviour in regard to adhesion, migration and invasion.

[发布机构] University:University of Glasgow;Department:School of Engineering

[关键词] Cancer, fibronectin, migration, focal adhesion, extracellular matrix.