FIGURE 2 from Cancer-associated Fibroblast–specific Expression of the Matricellular Protein CCN1 Coordinates Neovascularization and Stroma Deposition in Melanoma Metastasis

Mice harboring Col1A2-Cre-fibroblast–specific deletion for Ccn1 show impaired metastasis of B16F10 melanoma cells to the lung. A–C, Mice were examined after 14 days of tumor growth. A, Sections of tumor stroma were stained with anti-CCN1 antibody (N = 3, representative images are shown), verifying loss of CCN1 protein expression in mice deleted for CCN1 in fibroblasts. Note CCN1 staining remains in the tumor. Scale bar = 50 µm. B, Representative images of lung sections from wild-type or mice harboring a deletion for Ccn1 in Col1A2-Cre-fibroblasts. Hematoxylin and eosin was used to reveal detect dense metastatic foci (purple). Scale Bar = 300 µm. Total area of the lung section covered by metastases was quantified. Deletion of Ccn1 caused reduced metastasis (N = 6, ***, P < 0.001). C, Reduced CCN2 expression in the stroma did not significantly alter tumor growth. Kruskal–Wallis analysis (N = 8). D, To more accurately define the cells in which CCN1 was deleted, three-week old Col1A2-cre(ER)T/); mTmG mice were injected for 5 consecutive days with tamoxifen to activate Cre recombinase. Six weeks later, skin was isolated, digested with collagenase, and cells expressing GFP were isolated by FACS, and subjected to RNA-seq analysis. Col1A2 lineage fibroblasts were identified as universal fibroblasts based on the gene expression of Pi16, Col15a1, C3, and Cd84 genes, but not papillary or reticular fibroblasts based on the gene expression of Crabp1 and Defb8 or Nexn, Trim63, Actn2, and Hspb7, respectively.