Fig 2: In vitro blockage of SC-secreted proteins results in decreased invasion of PC cells. The effects of SC-CM on PC cells (PANC-1 and MIA PaCa-2) invasiveness were studied in vitro using Transwell invasion assay. For both treatment and control, cells (~60,000) were plated to the insert of a Transwell plate coated with collagen and allowed to invade for 24 h through a collagen-coated matrix toward SC-CM or control media as applicable. Serum-free media was used as negative control and serum-supplemented media was used as positive control. The non-invaded cells were removed from the top of the insert, and invaded cells were quantified using the QCM ECMatrix Cell Invasion Assay Kit described in section “Materials and Methods.” A significantly greater number of invaded PC cells in presence of SC-CM was observed compared to negative control (p < 0.05). To determine if the proteins of interest were involved in the stimulation of PC cells by SCs, the invasion assay was performed with blocking antibodies. Among the tested blocking antibodies, neutralization of MMP-2, cathepsin D, PAI-1, and Gal-1 decreased the invasiveness of both types of PC cell line compared to SC-CM alone (p < 0.05). Blocking antibodies were used at 6 µg/mL concentration. Blocking Gal-3BP, biglycan, and TIMP-2 in SC-CM had no significant inhibitory effect on PC invasiveness (p > 0.05). Representative data are shown from three independent experiments, performed in at least three replicates each. Statistical significance was obtained by one-way ANOVA. The error bars represent the SD of the mean. p-values are displayed as *p < 0.05, **p < 0.01, and ***p < 0.001. SC-CM, Schwann cell–conditioned media; PC, pancreatic cancer; Gal-3BP, galectin-3 binding protein; MMP-2, matrix metalloproteinase-2; PAI-1, plasminogen activator inhibitor-1; TIMP-2, tissue inhibitor of metalloproteinases-2; Gal-1, galectin-1; RFU, relative fluorescence unit; ns, non-significant.

Fig 3: Schematic representation of SC-stimulation of PC cells. Gal-3BP, MMP-2, cathepsin D, PAI-1, biglycan, TIMP-2, and Gal-1 are the SC-secreted proteins that have been identified as potential promoters of PC cell proliferation and invasion in this study. SC, Schwann cell; PC, pancreatic cancer; Gal-3BP, galectin-3 binding protein; MMP-2, matrix metalloproteinase-2; PAI-1, plasminogen activator inhibitor-1; TIMP-2, tissue inhibitor of metalloproteinases-2; Gal-1, galectin-1.

Fig 4: WB validation of candidate molecules. WB analysis confirmed the presence of secreted candidate molecules in SC-CM. WB, western blot; SC-CM, Schwann cell–conditioned media; Gal-3BP, galectin-3 binding protein; MMP-2, matrix metalloproteinase-2; PAI-1, plasminogen activator inhibitor-1; TIMP-2, tissue inhibitor of metalloproteinases-2; Gal-1, galectin-1.

Fig 5: Prognostic value of mRNA expression corresponding to the identified candidate proteins. Using cBioportal, Kaplan–Meier estimates of survival for PC patients with alterations in different gene combination were performed using The Cancer Genome Atlas (TCGA) on pancreatic adenocarcinoma. (A) Survival analysis showed that patients with alterations in the seven-gene proliferation panel (Gal-3BP, MMP-2, cathepsin D, PAI-1, biglycan, TIMP-2, and Gal-1) had worse overall survival than those without alterations (log-rank test p = 0.0215). (B) In the cases of invasion panel proteins (MMP-2, cathepsin D, PAI-1, and Gal-1), worse prognosis was also observed for patients with alterations in the four genes than those without alterations (log-rank test p = 0.0058). PC, pancreatic cancer; Gal-3BP, galectin-3 binding protein; MMP-2, matrix metalloproteinase-2; PAI-1, plasminogen activator inhibitor-1; TIMP-2, tissue inhibitor of metalloproteinases-2; Gal-1, galectin-1.