CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer [5920]

Cancer-associated fibroblasts (CAFs) are abundantly present in the microenvironment of virtually all tumors and strongly impact tumor progression. Despite increasing insight into their function and heterogeneity, little is known regarding the origin of CAFs. Understanding the origin of CAF heterogeneity is needed to develop successful CAF-based targeted therapies. Through various transplantation studies in mice we determined that CAFs in both invasive lobular breast cancer and triple negative breast cancer originate from mammary tissue-resident normal fibroblasts (NFs). Single-cell transcriptomics, in vivo tracing and in vitro studies revealed the transition of CD26+ and CD26- NF populations into inflammatory CAFs (iCAFs) and myofibroblastic CAFs (myCAFs), respectively. In vitro functional assays showed that CD26+ NFs transition into pro-tumorigenic iCAFs which recruit myeloid cells in a CXCL12-dependent manner and enhance tumor cell invasion via matrix-metalloproteinase (MMP) activity. Together, our data show that CD26+ and CD26- NFs transform into distinct CAF subpopulations in breast cancer. Cdh1F/F;PtenF/F (Eptn) mice (8 weeks old) were intraductally injected with lenti-virus expressing Cre-recombinase to induce tumor formation. Injected mammary glands with tumors were isolated 6, 12 or 18 weeks after intraductal injection (n=2 mice per time point). Together with PBS injected control mice (T=12 weeks after injection) the tissues were processed into a single cell suspension (see manuscript for protocol) and fibroblasts were isolated by FACS sorting (fibroblasts were defined as dapi-/EpCAM-/CD49f/-CD31-/CD45- cells) and processed fro single cells RNA sequencing using the 10X genomics platform (see manuscript for detailed protocol).