Fig 2: Homophilic ATP1A1 interactions promote activin A secretion.a ELISA analysis of activin A in fibroblasts generated from two distinct individuals (PSC and PSC-2) after direct contact with purified tumor membrane proteins (from BxPC-3, SU.86.86, MIA PaCa-2, or PANC-1 cells). Values were presented as mean ± SD (n = 3). b Western blotting analysis of no cross-linking control and cross-linked membrane protein complexes with streptavidin-HRP under reducing condition. Since 2-mercaptoethanol broke disulfide bond on the cross-linker, a band (*) of biotin-labeled tumor membrane proteins from BxPC-3 and SU.86.86 cells were identified. c Five plasma membrane proteins were identified by mass spectrometry analysis. d ELISA analysis of activin A in mono-cultured or direct co-cultured PSCs with BxPC-3 cells stably expressing lentiviral-based LacZshRNA, ATP1A1shRNA Clone#1, ATP1A2shRNA, ATP1A3shRNA, EMC1shRNA Clone#1, ITGB1shRNA. Values were presented as mean ± SD (n = 3). e ELISA analysis of activin A in fibroblasts co-cultured with 2% paraformaldehyde fixed BxPC-3 cells and treated with IgG (4 µg/ml), anti-ATP1A1-neutralizing antibody (2 and 4 µg/ml, 14418-1-AP, Proteintech), or anti-EMC1-neutralizing antibody (2 and 4 µg/ml, GTX119884, Genetex). Values were presented as mean ± SD (n = 3). f Left panel, western blotting analysis of biotinylated fibroblast membrane proteins with streptavidin-HRP under reducing condition. A band (*) of biotin-labeled fibroblast membrane proteins which bound to ATP1A1 was identified. Right panel, re-probing the western blot membrane from left panel with anti-Flag antibody to confirm that the identified band (*) was not ATP1A1-Flag. g ATP1A1 was identified by mass spectrometry analysis. h ELISA analysis of activin A in mono-cultured and direct co-cultured BxPC-3 cells with PSCs stably expressing lentiviral-based LacZshRNA and ATP1A1shRNA Clone#1. Values were presented as mean ± SD (n = 3). i Immunofluorescence staining of ATP1A1 in fibroblasts (PSC-GFP) and cancer cells (BxPC-3-RFP and SU.86.86-RFP) measured by confocal microscope. Scale bar, 2 µm. j Live-cell confocal imaging of BxPC-3 cells stably expressing ATP1A1-GFP and PSCs stably expressing ATP1A1-RFP. Co-localization of ATP1A1 was compared between initiation and establishment of tumor-fibroblast contact within 15 mins. Scale bar, 5 µm. k, l Correlation of ATP1A1 and INHBA expression levels in PDAC patient specimen. k Representative images of ATP1A1 expression (brown color, IHC) and INHBA expression (activin A, pink, in situ hybridization, RNAscope). Scale bar, 25 µm. l Pearson’s correlation coefficient analysis of ATP1A1 and INHBA according to H score and tumor-adjacent INHBA+ cells (%) (n = 100, r = 0.52, p < 0.0001). **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-tailed t-test).

Fig 3: Activin A was mainly secreted from fibroblasts upon direct contact with tumor cells.a, b Immunofluorescence staining of activin A in direct co-cultured PSCs and BxPC-3-cherry cells with or without contact with the other cell type measured by confocal microscope. A protein transport inhibitor (containing Monensin) was used for activin A accumulation in the Golgi complex before staining. Representative images a and quantification results of activin A intensity analyzed by Image J b. Scale bar, 5 µm. Values were presented as mean ± SD (n = 5 for BxPC-3 cells without direct contact with PSCs (due to low cell number); n = 50 for the other three groups). c ELISA analysis of activin A (R&D Systems) in supernatant of co-cultured fibroblasts with 2% paraformaldehyde fixed tumor cells (BxPC-3, SU.86.86, HPAF-II, AsPC-1, MIA PaCa-2, or PANC-1 cells), or reciprocally in supernatant of co-cultured tumor cells with 2% paraformaldehyde fixed fibroblasts. Values were presented as mean ± SD (n = 3). (d) Representative images of in situ hybridization (RNAscope, ACDBio) using probes for co-staining of activin A (INHBA, pink) and aSMA (ACTA2, blue) in PDAC patient specimen. Pink (activin A) and blue (aSMA) signal dots were co-localized in peri-tumoral cells. The nucleus was stained with hematoxylin. Scale bar, 50 µm. e Percentage of INHBA+ cells in regions with 0-10 µm and 10-20 µm distance from tumor–stromal interface in human PDAC specimens (n = 50). f Representative images of VIM and CK19 expression (brown color, IHC) and ACTA2/INHBA co-expression (ACTA2, aSMA, blue; INHBA, activin A, pink; RNAscope) in serial sections of human PDAC specimens. The nucleus was stained with hematoxylin. Scale bar, 50 µm. g H score of VIM expression levels in tumor cells with tumor-adjacent INHBA- or INHBA+ CAFs. Values were presented as mean ± SD (n = 30). h Representative images of in situ hybridization (RNAscope) using a probe for the single stain of INHBA in PDAC patient specimen. The nucleus was stained with hematoxylin. Scale bar, 100 µm. The expression levels of INHBA in tumor-adjacent stromal cells were quantified as 0 (no expression), 1 + (less than 10% of tumor-adjacent cells), 2 + (10–50% of tumor-adjacent cells), and 3 + (>50% of tumor-adjacent cells). i Kaplan–Meier analysis of overall survival according to INHBA expression levels in regions within 10 µm distance from tumor (p = 0.01, Log-rank test). For statistical analyses, the score of 0 and 1+ were combined as the low expression group, and the score of 2+ and 3+ were combined as the high expression group. Blue line, INHBAlow in 0–10 µm, n = 55; Red line, INHBAhigh in 0–10 µm, n = 11. *p < 0.05, ***p < 0.001, ****p < 0.0001 (two-tailed t-test).