Fig. 3

a GREM1 (encoding the secreted factor Gremlin-1) is highly expressed on fibroblasts in adenocarcinoma and SCC. Its receptor KDR is highly expressed in endothelial cells of both adenocarcinoma and SCC, and also in malignant cells from adenocarcinoma but not SCC. b Expression of GREM1 in fibroblasts is positively correlated with expression of proliferation and invasiveness related genes in malignant cells in adenocarcinoma (all adjusted p values < 0.05), but not in SCC. c High levels of fibroblasts inferred in adenocarcinoma from TCGA are associated with less favorable overall survival. d–f Treatment of low GREM1-expressing adenocarcinoma cell lines HCC78 and SW1573 with recombinant Gremlin-1 protein resulted in increased number of clones (red), sphere formation in 3-D culture (yellow), and invasion as evaluated by in vitro trans-well migration assays (magenta). g si-RNA knockdown resulted in decreased GREM1 expression in both H1755 and H1792 adenocarcinoma cell lines, which normally express it highly. h Knockdown of GREM1 expression reduced survival in both cell lines that highly express it. i Representative stain for GREM1 RNA shows expression confined to fibroblasts, that spatially colocate preferentially with leading edge of malignant cell nests. Malignant cells are highlighted in green. Black bars show closest malignant cell to each GREM1+ fibroblast. j Western blots showing (left) Gremlin-1 protein levels in CAFs from primary human NSCLC with low vs high GREM1 RNA levels (alpha-Tubulin control also shown), and levels of KDR and pKDR at baseline vs after co-culture with GREM1 low (+) and high (+++) CAFs. k Flow cytometry assessment of KI67 status of malignant cells before and after co-culture with CAFs expressing different Gremlin-1 protein levels