Fig. 1

Tumor cell-derived OPN positively correlated with the degree of tumor stromal fibrosis. (A) Representative immunohistochemical (IHC) staining images (left, scale bars = 20 μm) and the quantification (right) of αSMA and Sirus red-stained images in 4T1^WT and 4T1^{Spp1 − KO} tumor tissues of Balb/c mice (n = 3). Original magnification, ×500. (B) Representative flow cytometric plots (top) and frequency and number (bottom) of CD11b⁺F4/80⁺ macrophages in tumors (n = 5). (C) Representative flow cytometric plots (left) and frequency and number (right) of F4/80⁺Ly6C⁺ cells in tumors (n = 5). (D) Representative flow cytometric plots (left) and frequency (right) of F4/80⁺CD206^high M2 type macrophages in tumors (n = 5). (E) Representative flow cytometric plots (left) and frequency (right) of CD11b⁺Ly6C⁺ and CD11b⁺Ly6G⁺ cells in peripheral blood (n = 5). (F) Mice were inoculated respectively with 4T1, CT26, RM1 and B16-F0. All tumor samples were collected at the point of tumor volume reached 800 mm [3] and fixed for IHC staining. The staining results of αSMA and Sirus red were quantificated and presented as integral optical density (IOD)/Area (n = 3). Original magnification, ×500 (scale bars: 20 μm). (G) Based on transcriptome sequencing data of four tumor cell lines, gene set enrichment analysis was used to screen the potential genes positively associated with tumor fibrosis. (H) Relative mRNA levels of Spp1 in 4T1, CT26, RM1 and B16 cells measured by RT-qPCR (n = 3 biological replicates). Data are presented as means ± SEM, or representative staining profiles or photographs. p values were determined by unpaired two-tailed Student’s t test (A-E), or one-way ANOVA (F, H). Shown are data from a representative of three independent experiments (A–E, H). *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no significance