Fig. 3.

α-SMA^high CAF-secreted osteopontin increases tumor growth. A nCounter PanCancer Immune Profiling panel analysis of gene expression in patients with α-SMA^high versus α-SMA^low CAFs (top up- and downregulated genes are presented) and B in α-SMA^high versus α-SMA^low BC; genes with logFC > 1 were considered upregulated, and genes with logFC < −1 were considered downregulated. C OPN concentration in α-SMA^high versus α-SMA^low CAFs measured in CAF medium by ELISA. Graph presents data from three independent experiments (n = 2 technical replicates), and error bars show standard deviation. Unpaired Student’s t-test test was applied. D Representative staining for OPN in CAFs in TME of breast cancer samples, magnification 20×. E Representative pictures of MCF7 cells growing in 3D-Matrigel cultures treated with conditioned medium from α-SMA^low, α-SMA^high CAFs, BJ, and DMEM with or without addition of OPN-neutralizing antibodies. F Quantification of colony area fold change. Colony area was determined with ImageJ software. The values presented are mean ± SD from three independent experiments (n = 2 technical replicates). Unpaired Student’s t-test test was applied, ***p < 0.0001 calculated versus control (DMEM). G Ki67 percentage in patients with different OPN staining intensity—negative to moderate (neg-to-mod) and strong, evaluated immunohistochemically. Mann–Whitney U test was applied