Fig 2: Single-cell analysis reveals the specific expression of fibroblast activation protein (FAP) in fibroblasts from the public data. (A) Uniform manifold approximation and projection (UMAP) shows the control and idiopathic pulmonary fibrosis (IPF) single-cell populations from study GSE135893. (B) FAP expression level in each cell population using single-cell RNA sequencing data from study GSE135893. (C) UMAP shows the cells that express FAP in healthy donors and patients with IPF from study GSE135893. (D) Expression correlation analysis between FAP and ACTA2, FN1, and COL1A1 (the x-axis and y-axis represent log2 normalized expression level). cDCs = conventional dendritic cells; Ctrl = control; NK = natural killer; tSNE = t-distributed stochastic neighbor embedding.

Fig 3: High FAP expression predicts poor prognosis and promotes cell migration and invasion in NSCLC in vitro. a The FAP mRNA expression levels were significantly upregulated in 24 NSCLC tissues compared to the paired para-cancerous tissues. b FAP mRNA expression levels were significantly upregulated in NSCLC tissues. Data were obtained from the TCGA database. c Boxplot of FAP mRNA expression levels in NSCLC and normal tissues. Data were obtained from the HCMDB database (http://hcmdb.i-sanger.com/index). d FAP upregulation was associated with shorter overall survival (OS) times in NSCLC patients (P < 0.05). Data were obtained from the GEPIA database (http://gepia.cancer-pku.cn). e FAP mRNA and protein expression levels were significantly elevated in NSCLC cell lines. f KEGG analysis reflected that FAP overexpression is related to enrichment of pathways concerning cell metastasis. Data were obtained from the TCGA database. g The relative mRNA and protein expression levels of FTO in H460, H1299, and A549 cells. h–l Quantitative analysis of Transwell and wound healing assay data in H460, H1299, and A549 cells with FAP knockdown and FAP overexpression. m Western blot analysis verified that FAP regulated EMT-related proteins, including Snail, Slug, and N-cadherin, in NSCLC. Data information: Data are shown as the mean ± SDs. In all relevant panels, *P < 0.05; **P < 0.01; ***P < 0.001

Fig 4: MSCs were induced to CAFs by colorectal cancer cells. (A) Western blot showing that the expression of the CAF characteristic proteins α-SMA and FAPA was upregulated after MSCs were co-cultured with LOVO, HCT-116, and HCT-8 cells. (B) Comparison of adipogenic (Oil Red O staining) and osteogenic (alizarin red and alkaline phosphatase staining) differentiation abilities between MSCs and CAFs. Oil Red O staining and alizarin red staining were performed after 12 days of culture. Alkaline phosphatase staining was performed after 5 days of culture. (C) Effect of combined MSC transplantation on primary tumour volume in nude mice. HCT-116 group: HCT-116 5 × 106 cells; MSC + HCT-116 group: HCT-116 5 × 106 cells + MSC 1 × 106 cells. (D) In vivo fluorescence image showing the effect of combined MSC transplantation on tumour metastasis. Two groups of mice were transplanted with HCT-116 5 × 106 cells and HCT-116 5 × 106 cells + MSC 1 × 106 cells. The HCT-116 cell lines carried GFP.

Fig 5: Lung cancer cells induced CAFs activation via IGF2 secretion(A) mRNA levels of IGF2 in cells were detected by qPCR.(B) IGF2 concentrations in cell culture medium were detected by ELISA.(C) NFs were treated with IGF2 (50 and 100 ng/mL) for 24 h. Cell morphology was observed under a microscope.(D) NFs were treated with IGF2. α-SMA and FAP expressions were detected by western blotting.(E) NFs were treated with IGF2. Cell proliferation was examined by CCK-8 kit after 48 h.(F and G) NFs were treated with IGF2. NFs migration was detected by trans-well assay after 24 h.(H) NFs were treated with CM or CM + IGF2 neutralizing antibody. Cell morphology was observed under a microscope.(I) NFs were treated with CM or CM + IGF2 neutralizing antibody. α-SMA and FAP expressions were detected by western blotting.(J) NFs were treated with CM or CM + IGF2 neutralizing antibody, and IGF2 (100 ng/mL). Cell proliferation was examined by CCK-8 kit after 48 h.(K and L) NFs were treated with CM or CM + IGF2 neutralizing antibody. NFs migration was detected by trans-well assay after 24 h.(M) The effect of IGF2 knockdown by RNAi.(N) IGF2 expression was knocked down in lung cancer cells, and then the CM was collected and used to culture NFs. α-SMA and FAP expressions were detected by western blotting.(O and P) IGF2 expression was knocked down in lung cancer cells by RNAi, and then the CM was collected and used to culture NFs. NFs migration was detected by trans-well assay after 24 h. Ab, neutralizing antibody. Scale bar, 100 μm. Data represents the mean ± SD from three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.