Unraveling a new ECM stiffening function of the axon guidance molecule Netrin-1

State of the art The extracellular matrix (ECM) is a complex and dynamic network composed of core structural proteins and matrisome-associated components. Under physiological conditions, ECM homeostasis contributes to the maintenance of natural tissue stiffness. However, disruption of this balance can lead to abnormal stiffening, a hallmark observed in various pathological conditions, including fibrosis and cancer. Our laboratory investigates the role of the dependence receptor ligand Netrin-1 (NTN1), a neuronal guidance protein predominantly expressed during early developmental stages. Intriguingly, NTN1 is frequently re-expressed in tumors, where its presence has been correlated with increased ECM stiffness. As such, our research aims to elucidate the role of NTN1 in ECM remodelling and stiffening. Methodology and resultsUsing atomic force microscopy (AFM) on inert Matrigel matrices, we observed a marked increase in stiffness upon supplementation with recombinant Netrin-1 (rNTN1). This stiffening effect was reversed by NP137, a monoclonal antibody targeting NTN1. Structural analyses via immunofluorescence staining for laminin-111 and rNTN1, alongside scanning electron microscopy (SEM), revealed a significant reduction in pore size in the presence of rNTN1. Complementary in silico analysis using PEPPI software indicated strong interactions between NTN1 and key ECM components such as Nidogen and Laminin. PerspectivesTo validate these predicted interactions, we performed biolayer interferometry with various ECM proteins. Furthermore, 3D cultures models of MCF10A normal mammary epithelial cells exposed to NTN1 displayed a phenotypic transition toward a more mesenchymal and invasive state. Interestingly, knockdown of NTN1 receptors did not prevent this phenotypic shift, suggesting a novel receptor-independent function for NTN1 in promoting cell invasiveness through direct modulation of ECM stiffness. Ongoing structural analyses are being conducted on fibroblast-derived matrices, with and without endogenous Netrin-1 expression, to evaluate its potential remodeling effects on collagen IV and fibronectin networks. In parallel, these matrices are utilized in functional assays to investigate how Netrin-1 ECM impact, influences cellular behaviours and transcriptional activity, using a range of experimental approaches.