CD26-negative and CD26-positive tissue-resident fibroblasts contribute to functionally distinct CAF subpopulations in breast cancer [6702]

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. Overall design: CD26- and CD26+ NFs were isolated by FACS from the mammary glands of 8-week old mTmG mice and cultured for one week (2D) before switching the CD26+ NFs to GFP using adeno-Cre. CD26- NFs were expressing Tomato. CD26- and CD26+ NFs were mixed in collagen/BME gels (1:1) and plated alone or in combination with WEPtn (13-MCB-17 or 10-SJK-221)-derived tumor cells for one week. In addition, tumor cells were also plated in collagen/BME gels without fibroblasts as a control. After 7-day culture the gels were dissolved using collagenase and cells were FACS-sorted to separate CD26- NFs (Tomato+), CD26+ NFs (GFP+) and tumor cells (Tomato-/GFP-) and subsequently sequenced.