Citation
- Authors: Zheng Y. et al.
- Year: 2023
- Journal: J Cancer 14 114-128
- Applications: in vivo / DNA / in vivo-jetPEI
Method
Generation and purification of anti-PAI-1 monoclonal antibodies
BALB/C mouse were used for immunization. PAI-1 plasmid mixed with In vivo-jetPEI (Polyplus) transfection reagent was used as antigen. Ten days after the last boost the sera of the immunized mice were tested for the PAI-1 specific antibody by ELISA, and the spleen cells were isolated and fused with myeloma cells to obtain hybridomas. Limited dilution was performed as standard step to selected hybridoma cell clones. We used GST-PAI-1 prokaryotic protein and GST protein (2 μg/mL) as coating antigen of primary screening, and His-PAI-1 (400 ng/mL) recombinant protein as coating antigen in the next few rounds of screening. Antibodies were prepared by intra-peritoneal injection of hybridomas into 8-week-old BALB/C female mice. One week before the hybridoma injection, 500 μL of incomplete Freund's adjuvant was injected. After 10-15 days, the ascites was collected, and protein G agarose beads were used for antibody purification. After being concentrated by PEG20000, antibodies were dialyzed by PBS and identified with SDS-PAGE.
Abstract
Plasminogen activator inhibitor (PAI-1) is highly expressed in esophageal squamous cell carcinoma (ESCC) and strongly contributes to metastasis, making it a potential target for ESCC therapy. However, the antibodies and inhibitors targeting PAI-1 have not shown good therapeutic effect in the in vivo experiments yet. Here, we generated a panel of novel monoclonal antibodies (mAbs) against PAI-1. Analysis of PAI-1 expression in 90 tissue specimens and 128 serum specimens from ESCC patients with these mAbs confirmed that PAI-1 levels was significantly correlated with metastasis and poor survival. In addition, we found that high PAI-1 expression contributed to the enhanced motility and invasiveness of two ESCC cell lines. Next, mAb-1E2 and mAb-2E3, which have highest affinity with PAI-1, were shown to possess strong inhibitory effects on ESCC migration and invasion. Anti-tumor and anti-metastatic effects of mAb-2E3 were further demonstrated in the experimental animal models. Finally, LRP1 was identified as key factor mediating the pro-invasive function of PAI-1 and the anti-invasive capacity of mAb-2E3 in ESCC cells. The mAb-2E3 markedly decreased STAT1 phosphorylation levels and blocked the binding between PAI-1 and LRP1-ClusterII domain. Collectively, mAb-2E3 developed by our lab may be an effective antibody drug which can be used for anti-metastatic therapy in ESCC.