Fig 2: Effects of the NT-3/TrkC axis on cells proliferation and apoptosis in the interaction between SACC cells and SCs. The proliferation of SACC-83 cells and SCs were measured by CCK8 assays (A, C). There was no significant difference among the four groups. The apoptosis rate of the SACC-83 cells and SCs were decreased when treated with NT-3 or under the co-cultured condition, while AZD7451 treatment could enhance the apoptosis rate of cells even under the co-cultured condition (B, D). *P < 0.05. The sum of the right upper quadrant (late apoptotic cell) and the right lower quadrant (early apoptotic cell) was calculated as the final apoptosis results.

Fig 3: Effects of the NT-3/TrkC axis on cells motor ability characteristics in the interaction between SACC cells and SCs. Scratch wound healing assay (A) and migration assay (B, C) demonstrated that the migration ability of the co-cultured cells and the NT-3 treatment group were significantly higher than that of the solely cultured group. On the contrary, AZD7451 inhibited the motility of SACC-83 cells and SCs significantly. *P < 0.05. 3D co-culture assay (D) showed that a stream of cells formed by SCs migrated to the SACC-83 cells (D2). Meanwhile, a stream of cells formed by SCs migrated to the region with a high concentration of NT-3 (D3), with very few SCs migrating to the control group (D1) or the SACC-83 cells with AZD7451 (D4).

Fig 4: Expression of NT-3 and TrkC in the interaction between SACC-83 cells and SCs. (A) The concentrations of NT-3 in the medium of co-cultured cells, solely cultured SACC-83 cells or SCs were detected by ELISA. NT-3 in the co-cultured conditional medium was significantly increased when compared with the medium of the solely cultured SACC-83 cells or SCs. The results of qRT-PCR (B) and western blot (C) showed that the expression of NT-3 was significantly up-regulated in the co-cultured SACC-83 cells than that in the solely cultured SACC-83 cells. The gene (D) and protein (E) expression of TrkC in the co-cultured SCs was significantly higher than that in the solely cultured SCs. *P < 0.05; NS, not significant.

Fig 5: The survival analysis of SACC patients according to the expression of NT-3 and TrkC. The high expression of NT-3 was significantly correlated with the overall survival rate and the disease-free survival rate of SACC patients (P < 0.05) (A, C). The expression of TrkC was not associated with the overall survival rate or the disease-free survival rate of SACC patients (P > 0.05) (B, D).