Stricturing Crohn’s Disease Single-Cell RNA Sequencing Reveals Fibroblast Heterogeneity and Intercellular Interactions

Background: Fibroblasts play a key role in stricture formation in Crohn's disease (CD), but understanding it's pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation. Methods: We performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 138 tissue samples and 409,001 cells. We validated Cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models. Results: Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and upregulated, and its pro-fibrotic function was validated by NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and two animal models of experimental colitis. Conclusion: A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and opens potential therapeutic developments. ### Competing Interest Statement P.K.M., Q.T.N., J.L., S.Z., G.A.W., J.C., S.L., J.W., R.M., P.K., T.P., S.H., N.N., M.D., S.T., S.R.S, A.I.I have no conflict of interest. I.O.G. received funding from UCB, Celgene, Gossamer and Pliant. S.D.H. was consultant to Shionogi and Takeda. S.M.C., S.L., T.F., S.A., K.B., K.H., R.M., K.D., T.W., and K.M.K. are employees of Pfizer, Inc and may hold stock equity. C.P. was a visiting graduate scholar at Pfizer, Inc. P.K., M.D., S.T. are employees of Pliant Inc. C.F. received speaker fees from UCB, Genentech, Sandoz, Janssen and he is consultant for Athos Therapeutics, Inc. F.R. was consultant to AbbVie, Adnovate, Agomab, Allergan, Arena, Astra-Zeneca, Boehringer-Ingelheim, Celgene/BMS, CDISC, Celsius, Cowen, Ferring, Galapagos, Galmed, Genentech, Gilead, Gossamer, Granite, Guidepoint, Helmsley, Horizon Therapeutics, Image Analysis Limited, Index Pharma, Landos, Jannsen, Koutif, Mestag, Metacrine, Mopac, Morphic, Organovo, Origo, Pfizer, Pliant, Prometheus Biosciences, Receptos, RedX, Roche, Samsung, Sanofi, Surmodics, Surrozen, Takeda, Techlab, Teva, Theravance, Thetis, UCB, Ysios, 89Bio.