Fibrosis is defined as the excessive accumulation of extracellular matrix (ECM) components as a result of a failed tissue-repair process. Fibrosis can occur in any organ and is responsible for nearly 45% of all deaths in the industrialized world. In muscle, fibrosis is one of the main complications in many myopathies. In this project, we will use as paradigms COL6-related (COL6-RD), LAMA2-related (LAMA2-RD) and Duchenne (DMD) muscular dystrophies which, although clinically and genetically different, are all characterized by the presence of fibrosis that provokes the disruption of muscle architecture, and ultimately its functional failure. Fibro-adipogenic progenitors (FAPs), the main cell actors of fibrosis, are the major ECM collagen-producing cells within the stromal tissue environment. While they have been extensively described in mouse, very few studies have analysed their profile in human dystrophic/fibrotic conditions and a comprehensive study comparing different muscle pathologies is lacking. While there is an urgent need for strategies to ameliorate the efficacy of therapeutic options, in particular based on gene therapy, which have been shown to be less efficient in fibrotic tissue due to the increased extracellular microenvironment reducing the accessibility of viral vectors to their target, today there is no efficient treatment to cure muscle fibrosis. Data on human samples to define muscle fibrosis are therefore essential. The goal of the FibroDys project is to compare in parallel three different fibrotic muscle diseases using already available human samples to investigate which molecular pathways and cellular actors are unique and which ones are shared among these diseases, with the final goal to pave the way towards appropriate and effective therapeutic avenues. Three specific aims are therefore developed. A first aim based on high-dimensional omics analyses on human biopsies will characterise the heterogeneity of the resident cell populations and their interactions in the intricate ECM fibrotic muscle (snRNAseq, Imaging Mass Cytometry) and identify the ECM molecular components altered in each fibrotic muscle (proteomic approach). A second aim will study the secretome and proteome of FAPs and the cross-talk between FAPs and ECM/muscle cells, revealing the exact contribution of FAPs from each pathology to the secretome in muscle fibrosis. This aim will also dissect how ECM proteins influence FAPs behaviour and how ECM produced by FAPs impact muscle differentiation (co-culture experiments). A third aim will screen an anti-fibrotic drugs library (Medium-Throughput screening) to identify a list of compounds active on muscle FAPs secretion and proliferation, and a list of signalling pathways implicated in FAPs-mediated fibrosis. This list will be further refined and validated using a pseudo-3D in vitro system (scar-in-a-jar system) and in vivo models (xenotransplantation of human FAPs in immunodeficient mice) to identify potentially shared or disease-specific therapeutic candidates.