Fig 1: CCL18 expression in bladder cancer tissues. (A) Expression of CCL18 in bladder cancer samples (red box) and adjacent tissues (grey box), *P<0.001. (B) Expression of CCL18 in TNM stage II, III and IV bladder cancer samples in The Cancer Genome Atlas BLCA dataset. (C) Expression of CCL18 in bladder cancer samples, according to the pathological stage, in the GSE31684 dataset. (D) Results of overall survival analysis in patients stratified according to CCL18 mRNA expression (Kaplan-Meier test) in the Human Protein Atlas. (E and F) IHC staining of CCL18 in 30 paired noncancerous and cancerous tissues obtained from patients with bladder cancer (magnification, ×40). (G) Protein expression levels of CCL18 in bladder tumor tissues, as observed by IHC staining. Red arrows indicate CCL18-positive cells in the stroma. Hemalum was used to stain nuclei of cells blue. CCL18 immunoreactivity in stromal cells were located in cytoplasm; no CCL18 staining was detected in the nuclei of cells. BLCA, bladder cancer; CCL18, chemokine (C-C motif) ligand 18; IHC, immunohistochemistry; NT, nontumorous.
Fig 2: Two different subsets of macrophages infiltrated in iCCAphl and iCCApps.a The t-SNE plot showing the subtypes of myeloid cells derived from iCCA peri-tumor and tumor. b Heatmap showing the expression of marker genes in each subtype of myeloid cells. c t-SNE plot of myeloid cells from S100P + SPP1- (red dots) and S100P-SPP1 + (blue dots). d Bar plot showing the proportion of macrophage subsets from S100P + SPP1- and S100P-SPP1+. e, f Scatterplots showing pro-/anti-inflammatory scores (e) and M1/M2 scores (f) for two macrophage subsets. Macro_c1_SPP1, n = 4016 cells; Macro_c2_CCL18, n = 3447 cells. (***P < 0.001; two-sided Wilcoxon rank-sum test; Anti-inflammatory score: P < 2.22e-16; Pro-inflammatory score: P < 2.22e-16; M2 polarization score: P < 2.22e-16; M1 polarization score: P < 2.22e-16). The central mark indicates the median, and the bottom and top edges of the box indicate the first and third quartiles, respectively. The top and bottom whiskers extend the boxes to a maximum of 1.5 times the interquartile range. g, h Representative mIHC images (left) and statistical graphs (right) to show the distribution of CD68+SPP1+CCL18- and CD68+SPP1-CCL18+ macrophages in S100P+SPP1- (g) and S100P-SPP1 + (h), respectively: CK19 (green), S100P (red), SPP1 (purple), CD68 (white), CCL18 (yellow), and DAPI (blue) (S100P + SPP1- n = 68, S100P-SPP1 + n = 112). White arrows (CD68 + SPP1 + CCL18-), yellow arrows (CD68 + SPP1-CCL18+). (***P < 0.001; two-sided Mann–Whitney U-test; CD68 + SPP1 + CCL18- (%): P < 0.0001; CD68 + SPP1-CCL18 + (%): P < 0.0001). Data were presented as median with interquartile range (g and h). Scale bar, 50 µm. Source data are provided as a Source Data file.
Fig 3: PARC gene expression in according to groups.Data of the PARC mRNA expression (fold change) in lung tissue and intercostal arteries according to groups. No significant differences were found between groups. Data are presented as LSM ± SEM. The reported p-value comes from the pair wise comparison with a general linear model using as covariables: gender, pack-years and the presence of diabetes mellitus.
Fig 4: Western blot analysis for PARC.Upper panel: representative membranes of the Western blotting in homogenized lung tissue (A) and intercostal arteries (B). Bands at 37kD and 10kD are consistent with the size of ß-actin and PARC respectively. Lower panel: (A) Band density analysis of 33 lung samples (12 COPD, 11 NOS and 10 NS). Of note, protein content was significantly increased in the COPD group compared to NOS. (B) Band density analysis of 28 intercostal artery samples (12 COPD, 8 NOS and 8 NS). There were no differences in PARC content between groups. Data are presented as LSM ± SEM. The reported p-value comes from the pair wise comparison with a general linear model using as covariables: gender, pack-years and the presence of diabetes mellitus. COPD: Chronic Obstructive Pulmonary Disease; NOS: non-obstructed smokers; NS: never-smokers.
Fig 5: Verification in the clinical samples and gene set enrichment analysis (GSEA). (A) Human carotid artery segments were collected from below (normal control) and at (plaque-containing) the carotid bifurcation. (B) CCL18, CCL4, MMP9 and SPP1 expression in stable plaques (n=15 pair) and ruptured plaques (n=15 pair) were evaluated by qPCR and normalized against the corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. An asterisk represents p<0.05, and two asterisks are shown as p<0.01 when compared with the normal control group. (C) Gene set enrichment analysis (GSEA) plots showing lipid metabolism and inflammatory immune-related gene sets progressively affected advanced-stage AS.
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