Discovery of a Linked Constellation of Gene Expression Revealed by Local Editing of Fibroblasts in Tumors

Fibroblasts play critical roles in regulating cellular relationships during tissue homeostasis, immunity, and tumor biology at multiple sites. However, tools to perturb fibroblasts at just one site in vivo are limited, restricting our understanding of how these cellular relationships develop on a local level. We optimized local gene editing of fibroblasts in multiple mouse tumor models to investigate how locally restricted fibroblast perturbations affect the cellular tumor microenvironment (TME). By knocking out surface receptors Osmr, Tgfbr2, or Il1r1 on cancer-associated fibroblasts (CAFs), we uncover that TGFBR2 signaling loss uniquely induces the emergence of a Col18a1hi CAF cell state that is distinct from previously described fibroblast states and is associated with worse survival in human PDAC patients. Further application of a local as well as combinatorial gene knockout technology in CAFs reveals a circuit in which these Col18a1hi CAFs reshape the TME by recruiting Siglec-Fhi neutrophils via Cxcl5 expression; and that the Col18a1hi CAF cell state is further dependent on fibroblast TNFR1 and canonical Wnt signaling. Together, a fast, affordable, and modular engineering method is demonstrated, allowing discovery of a modified fibroblast identify, and through this the network details of a local inter-cellular circuitry in a tumor. Overall design: live cells from s.c. grown pancreatic tumors were sorted by FACS after AAV-mediated knockout in cancer-associated fibroblasts and then analyzed using scRNA-seq (10x Genomics FLEX)