Figure 4. GREM1⁺ CAFs are myofibroblastic CAFs, which are distinct from ISLR⁺ CAFs in human and mouse CRC.

(A, B) Dual smFISH for Grem1 and Islr in an azoxymethane/dextran sodium sulfate mouse model of CRC. (A) Representative pictures. Red, green, and yellow arrowheads denote Grem1⁺Islr⁻, Grem1⁻Islr⁺, and Grem1⁺Islr⁺ CAFs, respectively. (B) Semiquantification of the ratio of double-positive (Grem1⁺Islr⁺) cells in Grem1⁺ cells and Islr⁺ cells. Four high-power fields (400×)/mouse, 4 mice. (C, D) Grem1 smFISH or Islr smFISH followed by αSMA IF in azoxymethane/dextran sodium sulfate tumors. (C) Representative pictures. Yellow and green arrowheads indicate double-positive cells (Grem1⁺αSMA⁺ cells or Islr⁺αSMA⁺ cells) and Islr⁺αSMA⁻ cells, respectively. (D) αSMA positivity in Grem1⁺cells and Islr⁺ cells. Four high-power fields/mouse, 4 mice each. (E–G) Dual smFISH for GREM1 and ISLR followed by αSMA IF in human CRC. (E) Representative pictures. Red and green arrowheads denote GREM1⁺αSMA⁺ cells and ISLR⁺αSMA⁻ cells, respectively. (F) Semiquantification of the ratio of double-positive (GREM1⁺ISLR⁺) cells in GREM1⁺ cells and ISLR⁺ cells. (G) αSMA positivity in GREM1⁺ cells and ISLR⁺ cells; 4–6 high-power fields/patient, 5 patients. (H, I) GREM1⁺ CAFs are spatially distinct from ISLR⁺ CAFs in human desmoplastic rectal cancer. (H) Representative pictures of GREM1 and ISLR ISH on human desmoplastic rectal cancer samples. Red and green arrowheads denote GREM1 and ISLR expression, respectively. (I) Quantification of the minimum distance between GREM1 or ISLR ISH signals and the closest tumor cells; n = 38,396 (GREM1) and 18,028 DAB⁺ signals (ISLR) from 3 low-power fields (100×)/patient, 7 patients each. (J) YH2 cells were stimulated with a vehicle, recombinant TGF-β1, or recombinant TGFβ1 + Galunisertib for 24 hours, followed by qRT-PCR (n = 3). Mean ± SEM. Mann-Whitney U test (D, G, and I) and 1-way ANOVA with Tukey post hoc multiple comparisons (J). The boxed areas are magnified in the adjacent panels (A, C, and E). Scale bars, 50 μm (A, C, and E) and 250 μm (H). ****P < .0001, **P = .0013, *P = .0122. AOM, azoxymethane; DMSO, dimethyl sulfoxide; DSS, dextran sodium sulfate; M, mol/L.