Fig. 5

SPINK1 induces profound reprogramming of cancer cell expression and causes phenotypic alteration. a Heatmap depicting differentially expressed transcripts in PC3 cells after a 3-day culture with SPINK1⁺ CM from PSC27 cells. Contrasting the control group (vector), 2671 and 4285 genes were upregulated and downregulated, respectively, in cells treated with PSC27^SPINK1 CM. b Statistics of transcripts differentially expressed (fold change either ≥2 or ≤0.5, with p < 0.05) in PC3 and DU145 cells upon SPINK stimulation, and classified into typical categories according to functional annotations mapped by Genecode (V27). c Venn diagram indicating the overlap of 465 transcripts upregulated in PCa cells upon treatment with SPINK1⁺ CM from PSC27 (2518 and 3170 genes with unique annotations for PC3 and DU145, respectively). d Heatmap showing the top 38 upregulated transcripts by both PCa cell lines, sorted according to their expression fold changes in PC3. e Pie chart displaying the biological processes associated with 2671 transcripts upregulated by stromal SPINK1 in PC3 cells. f Column chart manifesting the expression sites of 2671 transcripts upregulated in PC3 cells after SPINK1 stimulation, with percentage and log10 (P value) per specific site indicated on the left and right Y axis, respectively. Data derived from by the FunRich program. g Heatmap of gene expression signatures associated with phenotypic changes including epithelial–mesenchymal transition (EMT)/cancer stem cell (CSC)/angiogenesis (ANG) after SPINK1 stimulation of PC3 cells. Data were acquired from qRT-PCR assays. h Immunoblot assessment of protein level expression of phenotype-associated markers displayed in (g). GAPDH, loading control. i Representative phase contrast images for morphological changes observed in PC3 and DU145 cells, upon in vitro culture for 3 days with SPINK1⁺ CM from PSC27 cells. Scale bars, 20 μm. j Immunofluorescence staining of CDH1 (E-cadherin) and vimentin expressed in PC3 cells treated with PSC27 subline CM. Scale bars, 20 μm. k Representative fluorescence images for in vitro tube formation assay to assess angiogenesis of PCa cells placed on the polymerized matrigel. Scale bars, 100 μm. Data of (g–k) are mean ± SD and representative of 3 independent experiments, with 3 technical replicates performed per cell-based assay