The lower compartments were filled with 20% FBS DMEM. 20 months; *p 0.05, log-rank test). We have also investigated the uPA expression in tumor tissues of 146 informative ESCC cases. Our results showed no correlation between clinicopathologic features and patients with moderate and high uPA expression in tumor tissues (Table ?(Table1).1). Kaplan-Meier analysis of survival Calcineurin Autoinhibitory Peptide curves indicated that there was no statistical difference in the overall 5-year survival rates ATN1 between patients with moderate/high uPA tumor expression and patients with negative/low uPA tumor expression (Supplementary Figure 1). Together, these data indicate a reverse correlation between uPA stroma expression and ESCC prognosis. uPA secreted by CAFs increases proliferation and migration of ESCC cells The increased uPA mRNA and protein levels in CAFs compared to NFs suggested that the uPA released from CAFs might regulate ESCC cells via a paracrine manner. To analyze the effect of uPA on ESCC tumor progression, we treated ESCC cell lines EC109 and KYSE30 with uPA, or with CAF CM containing high levels of uPA (CAF4). Cells treated with 20 ng/ml of uPA or CAF CM had significantly accelerated growth rates than cells treated with DMEM control or NF CM. After neutralizing uPA with anti-uPA antibody, the proliferation rate decreased compared to cells treated with IgG control (Figure ?(Figure4A).4A). Moreover, when EC109 and KYSE30 cells were treated with 20 ng/ml uPA or CAF CM, they exhibited increased migration and invasive potential compared to cells treated with DMEM or NF CM. Furthermore, anti-uPA antibody co-incubation with uPA or CAF CM decreased the migration and invasive potential of these cells (Figure ?(Figure4B,4B, C). Open in a separate window Figure 4 uPA secreted from CAFs functions as oncogenic protein during ESCC progressionA. Cell growth rates of EC109 and KYSE30. Cells were seeded into 96-well plate at a Calcineurin Autoinhibitory Peptide density of 3103 per well. After 6 h, cells were treated with either DMEM control or NF CM or 20 ng/ml uPA or CAF CM or 20 ng/ml uPA with 6 ug/ml IgG or CAF CM with 6 ug/ml IgG or 20 ng/ml uPA with 6 ug/ml anti-uPA antibody or CAF CM with 6 ug/ml anti-uPA antibodies. Cell growth rates were compared by WST-8 assays 48 h later. B. and C. Representative images of migratory and invasive cells per field with indicated treatment. Cells were seeded Calcineurin Autoinhibitory Peptide in the upper compartment at a density of 5104 per chamber. After 6 h, cells were treated with either DMEM control or NF CM or 20 ng/ml uPA or CAF CM or 20 ng/ml uPA with 6 ug/ml IgG or CAF CM with 6 ug/ml IgG or 20 ng/ml uPA with 6 ug/ml anti-uPA antibody or CAF CM with 6 ug/ml anti-uPA antibodies. Migrated and invaded cells were counted after 36 h. Before the experiments, EC109 and KYSE30 cells were serum-starved for 24 h, acid-washed to remove bound endogenous uPA, and then neutralized. CTL: DMEM control, u+IgG: uPA+IgG, uPA+A: uPA+Anti-uPA antibody, NFs: NF CM, CAFs: CAF CM, C+IgG: CAF CM+IgG, C+A: CAF CM+Anti-uPA antibody. Experiments in ACC were repeated at least thrice. Error bars, mean SD. Scale bar 50 um. uPA secreted by CAFs contributes to ESCC progression by activating PI3K/AKT and ERK signaling pathways To investigate the uPA-mediated signaling in Calcineurin Autoinhibitory Peptide ESCC cells, we.