Mapping and tracing Grem1+ stromal cells in an ApcMin/+ mouse utilizing cryopreserved intestinal sections prepared via modified Swiss-roll technique

Grem1 + cancer-associated fibroblasts (CAFs) are crucial in colorectal cancer (CRC) development, yet technical challenges have limited understanding of their origins, spatiotemporal distribution, and potential roles. Here, we devised a custom mold, optimizing the gut Swiss-roll technique to create a single cryopreserved slide for comprehensive staining. Our integrated approach uncovered a marked increase in Grem1 + CAFs within Apc Min/+ mouse tumors at 12 weeks, compared to normal mucosa. Subsequent lineage tracing in Grem1-CreER T2 ; R26-LSL-tdTomato ; Apc Min/+ mice revealed that most Grem1 + CAFs infiltrating the tumor core originated from Grem1 + intestinal reticular stem cells (iRSCs). A minor subset of Grem1 + CAFs, located in the submucosa, retained characteristics of Grem1 + intestinal sub-epithelial myofibroblasts (ISEMFs). Altogether, CAFs derived from Grem1 + iRSCs may serve as a principal stromal cell type driving early-stage CRC progression, while Grem1 + ISEMFs contribute less from a more distant location. Hence, targeting Grem1 + CAFs presents an early and promising therapeutic strategy in CRC.