Fig. 4: Direct tumor-fibroblast contact triggers activin A secretion leading to EMT of tumor cells and myofibroblast activation.

a Human cytokine array used conditional medium from transwell (indirect, ID) versus direct (D) co-culture of BxPC-3 with pancreatic stellate cells (PSCs). Cytokine levels in direct co-cultured condition were normalized to indirect co-cultured condition. b Quantitative real-time PCR analyses of CLDN1, VIM, Snail, Twist1, ZEB1, and ZEB2 expression were performed in BxPC-3 cells treated with recombinant human Activin A (50 and 100 ng/ml) compared to untreated cells. Values were presented as mean ± SD (n = 3). c Western blotting analysis of proteins harvested from BxPC-3 cells treated with recombinant human activin A (50 and 100 ng/ml) compared to untreated cells. d Sphere-formation assay of BxPC-3 cells treated with recombinant human activin A compared to untreated cells. Values were presented as mean ± SD (n = 4). e Western blotting analysis of proteins harvested from mono-cultured and direct co-cultured (D) tumor cells (BxPC-3-RFP) treated with IgG (1 μg/ml) or anti-activin A-neutralizing antibody (1 μg/ml, 693604, Biolegend). FACS was performed to collect RFP⁺ tumor cells after 48 h of mono- or co-culture conditions. f Sphere-formation assay of BxPC-3-RFP cells in mono-cultured and direct co-cultured conditions treated with IgG (2 μg/ml) or anti-activin A-neutralizing antibody (1 and 2 μg/ml). Values were presented as mean ± SD (n = 4). g, h 3D spheroid invasion assay of BxPC-3-RFP spheroids in mono-cultured and direct co-cultured conditions treated with IgG (4 μg/ml) or anti-activin A-neutralizing antibody (2 and 4 μg/ml). g Representative images. Scale bar, 200 μm. h Extensive rate of each tumor spheroid was calculated by normalizing RFP⁺ area at 72 h to RFP⁺ area at 1 h (left panel) and by normalizing RFP⁺ maximal diameter at 72 h to RFP⁺ maximal diameter at 1 h (right panel). Values were presented as mean ± SD (n = 9). i, j Subcutaneous injection of BxPC-3 cells and mixed fibroblasts/BxPC-3 cells treated with IgG (1 μg/ml) or anti-activin A-neutralizing antibody (1 μg/ml) every 3 days and collected tumor samples at day 14 after inoculation. Representative images i and quantification results of tumor weight j. Scale bar, 1 cm. Values were presented as mean ± SD (n = 6). k, l Subcutaneous injection of BxPC-3 cells and mixed fibroblasts/BxPC-3 cells treated with recombinant human follistatin 20 ng/ml every 3 days and collected tumor samples at day 14 after inoculation. Representative images k and quantification results of tumor weight l. Scale bar, 1 cm. Values were presented as mean ± SD (n = 5). m, n Quantitative real-time PCR analyses and western blotting analysis of ACTA2 and COL1A1 expression were performed in PSCs treated with recombinant human activin A (50 and 100 ng/ml) compared to untreated cells for 24 h and 48 h, respectively. For qPCR analyses, values were presented as mean ± SD (n = 3). o, p Quantitative real-time PCR analyses and western blotting analysis of ACTA2 and COL1A1 expression in mono-cultured, indirect co-cultured (ID), and direct co-cultured (D) fibroblasts (PSC-GFP) with tumor cells (BxPC-3). FACS was performed to collect GFP⁺ fibroblasts after 24 h and 48 h for mRNA and protein samples, respectively. For qPCR analyses, values were presented as mean ± SD (n = 3). q Western blotting analysis of proteins harvested from mono-cultured and direct co-cultured (D) fibroblasts (PSC-GFP) treated with IgG (2 μg/ml) or anti-activin A-neutralizing antibody (1 and 2 μg/ml). FACS was performed to collect GFP⁺ fibroblasts after 48 h of mono- or co-cultured conditions. r, s BxPC-3 cells and mixed PSCs/BxPC-3 cells treated with IgG (2 μg/ml) or anti-activin A-neutralizing antibody (2 μg/ml) every 3 days and collected tumor samples at day 14 after inoculation. Representative IHC images r and H Score s of αSMA expression. Scale bar, 0.5 mm. Values were presented as mean ± SD (n = 6). t, u BxPC-3 cells and mixed PSCs/BxPC-3 cells treated with follistatin (20 ng/ml) every 3 days and collected tumor samples at day 14 after inoculation. Representative IHC images t and H score u of αSMA expression. Scale bar, 0.5 mm. Values were presented as mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-tailed t-test).