Mapping and tracing Grem1 stromal cells in an 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 mouse tumors at 12 weeks, compared to normal mucosa. Subsequent lineage tracing in ; ; 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.