|Program and Abstracts
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Therapeutic Modulation of an Embryonic Fibroblast Lineage
Graham G. Walmsley, BA, Michael S. Hu, MD, Zeshaan N. Maan, MD, Yuval Rinkevich, PhD, Michael Januszyk, MD, Dominic Duscher, MD, Alexander Whittam, BA, David Atashroo, MD, Ruth Tevlin, MD, Elizabeth Zielins, MD, Peter H. Lorenz, MD, Irving L. Weissman, MD, Geoffrey C. Gurtner, MD, Michael T. Longaker, MD.
Stanford University, Stanford, CA, USA.
Effective treatment of fibrosis depends upon a mechanistic understanding of its pathogenesis. Fibroblasts are central to the fibrotic response across a range of pathologic states. Unfortunately, few fibroblast lineages have been identified and fibroblast heterogeneity remains poorly characterized. Using a new murine model, we reveal the presence of multiple embryonic lineages of dermal fibroblasts within the dorsal skin of mice and identify a highly fibrogenic lineage defined by embryonic expression of Engrailed-1 (En1). Targeted inhibition of this scar-forming lineage allowed for therapeutic modulation of the fibrotic response.
En1-derived fibroblasts were traced by crossing En1Cre transgenic mice with ROSA26mTmG mice. Flow cytometry allowed for the isolation of En1-derived fibroblasts from wild type mice on the basis of highly expressed surface molecules. Transplantation methodologies in conjunction with small molecule-based inhibition functionally corroborated these surface markers in the context of wound healing and cancer stroma formation. Lineage-specific cell ablation in mice (En1Cre; R26mTmG; R26tm1(HBEGF)Awai) expressing simian diphtheria toxin receptor selectively in lineage-derived cells allowed for therapeutic modulation of scar and cancer stroma formation. Finally, a small molecule was used to assess the effect of inhibiting En1-derived fibroblasts during wound healing in adult wild type mice.
Engrailed-1 derived fibroblasts were found to be responsible for the majority of connective tissue deposition during dermal development, wound healing, radiation-induced fibrosis, and cancer stroma formation in the dorsal skin. Lineage-specific cell ablation using transgenic-mediated expression of the simian diphtheria toxin receptor and localized administration of diphtheria toxin (DT) led to significantly reduced scar formation (*p<0.01) following excisional wounding and significantly reduced melanoma growth (*p<0.01). Tensile strength testing of DT-treated and control wounds revealed that although scar formation was significantly reduced in DT-treated as compared to control wounds, tensile strength was not significantly affected. Furthermore, we identified CD26/DPP4 as a surface marker that allows for the isolation of this fibrogenic scar-forming lineage and demonstrated that small molecule-based inhibition of CD26/DPP4 leads to significantly reduced scar formation (*p<0.001) in a humanized mouse model of excisional wound healing.
We have identified multiple lineages of fibroblasts in the dorsal skin. Among these, we have characterized a single lineage responsible for the majority of connective tissue deposition during wound healing, radiation-induced fibrosis, and cancer stroma formation. We further demonstrate that targeted inhibition of this lineage results in reduced scar formation with no effect on the structural integrity of the healed skin. These results hold promise for the development of therapeutic approaches to fibrotic disease, wound healing, and cancer progression.
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