Lymphedema-associated fibroblasts are a TGF-β1 activated myofibroblast subpopulation related to fibrosis and stage progression in patients and a murine microsurgical model

The driver of secondary lymphedema (SL) progression is chronic inflammation, which promotes fibrosis. Despite advances in preclinical research, a specific effector cell subpopulation as a biomarker for therapy response or stage progression is still missing for SL.Whole skin samples of 35 murine subjects of a microsurgical-induced SL model and 12 patients with SL were collected and their fibroblasts were isolated. These lymphedema-derived fibroblasts (LAF) were cultured in a collagen I-poly-D-Lysine 3D hydrogel to mimic skin conditions. Fibroblasts from non-lymphedema skin were used as negative control and TGF-β-stimulated fibroblasts were used to recreate profibrotic myofibroblasts. Quantitative immunocytofluorescence confocal microscopy analysis and invasion functional assays were performed in all subpopulations and statistically compared.In contrast to normal skin fibroblasts, LAF exhibit α-SMA-positive stress fibers and a reduced number of tight junctions in 3D hydrogel conditions. The switch from normal E-cadherin high phenotype to an N-cadherin high-E-cadherin low morphology suggests epithelial to mesenchymal transition for expansion and proliferation. This pathological behavior of LAF was confirmed by live cell imaging analysis of invasion assays. The significant activation of markers of the TGFBR2-Smad pathway and collagen synthesis (HSP-47) in LAF supports EMT phenotypic changes and previous findings relating to TGF-β1 and fibrosis with lymphedema.A characteristic SL myofibroblast subpopulation was identified and translationally related to fibrosis and TGF-β1-associated stage progression. This SL-related subpopulation was termed lymphedema-associated fibroblasts. A comprehensive stage-related characterization is required to validate LAF as a reliable biomarker for SL disease progression.