Background: Vimentin is a prominent Intermediate Filaments (IFs) protein expressed in different mesenchymal origin cell types. Besides a wide range of cellular function roles associated with vimentin expression, its dysregulation and cell surface expression in the induction of malignancy properties have been reported extensively, making it a promising cancer-specific target. Therefore, this study aimed to generate and characterize anti-vimentin monoclonal antibodies. Methods: A 14-mer synthetic peptide from vimentin was conjugated to Keyhole Limpet Hemocyanin (KLH) and used for immunization of Blab/C mice and monoclonal production by conventional hybridoma technology. The monoclonal antibody was purified using affinity chromatography of supernatants from the selected hybridoma cells. ELISA, Immunoprecipitation-Western blotting (IP-WB), Immunocytochemistry (ICC), and flow cytometry were employed to characterize the produced monoclonal antibody in terms of interaction with vimentin immunizing peptide as well as vimentin protein. Results: Amid the several obtained producing anti-vimentin antibody hybridomas, the 7C11-D9 clone (IgG1 isotype with kappa light chain) showed higher reactivity with the immunizing peptide, and led to its selection for purification and characterization. The purified antibody could detect vimentin protein in IP-WB, ICC and flow cytometry of the normal and cancerous cells with different origin. No vimentin expression was found in normal healthy Peripheral Blood Mononuclear Cell (PBMC). Conclusion: Taken together, 7C11-D9 anti-vimentin monoclonal antibody might be used as immune diagnostic or immune therapeutic tool where detection or targeting of vimentin in a wide range of organisms is required.

Pancreatic carcinoma is the fourth-leading cause of cancer death in the United States and due to its late presentation, only few patients would be candidates for the curative treatment of pancreactomy. Monoclonal antibodies have brought hope to targeted therapy.Objectives: To identify new biomarkers, a panel of monoclonal antibodies was generated against newly established cell line, Faraz-ICR from a patient with pancreatic acinar cell carcinoma.Material and Methods: Balb/c female mice were immunized with Faraz-ICR cell line and their spleenocytes fused with SP2/0 myeloma cell line. Highly reactive hybridoma producing antibodies against Faraz-ICR was detected using ELISA, immunofluorescence staining and flow cytometry. Western blot and 2D immunoblot were utilized for further characterization of the target antibodies.Results: Among highly reactive clones, the reactivity of 7C11 clone was assessed in comparison to other epithelial tumors. The antibody isotype was IgM that reacted with a 55 kDa protein in western blot analysis. To further characterize the target antigen, immunoproteome of the Faraz-ICR cell line was performed. By LC-MS analysis, the target of 7C11 clone was identified to be vimentin.Conclusions: Pancreatic cancer is a highly lethal malignancy with no reliable biomarker for early detection and diagnosis. In this study, by establishing a pancreatic acinar carcinoma cell line, a panel of monoclonal antibodies was generated to identify specific or associated cancer targets. Furthermore, 7C11 mAb was introduced that can specifically recognizes vimentin as a tumor marker. This antibody may serve as a new tool for prognostic and therapeutic strategies.

Vimentin is a cellular marker that has recently been considered in the prognosis of colorectal cancer (CRC), and its expression appears to indicate increased malignancy. The aim of this study was to investigate the expression of vimentin in CRC patients and its association with prognostic factors. This retrospective study was performed on CRC patients who had undergone colectomy at Imam Khomeini Hospital, Ahvaz, Iran, in 2019 and 2020. Data around the microscopic degree of the tumor differentiation and the status of lymph node involvement were extracted from the patients' pathology reports. Immunohistochemistry staining for vimentin was performed on biopsy specimens, and its expression was assessed and compared in both CRC specimens and normal colon tissues. Appropriate statistics were used with P<0. 05 considered as statistically significant. Out of 31 CRC patients, vimentin expression was moderate-positive in 20 (64. 5%) and strong-positive in 11 patients (35. 5%). Mean percentage of stained cells, the intensity of staining, and vimentin expression in the immunohistochemistry evaluations had no significant relationship with tumor grade and tumor invasion rate (P>0. 05), but they showed a significant relationship with lymph node involvement (P<0. 05) and mean percentage of stained cells, the intensity of staining, and expression of vimentin marker increased with increasing lymph node involvement. In the normal tissue samples, 5 out of 30 samples showed weak-tomoderate vimentin expression. Vimentin expression was significantly associated with lymph node involvement,however, further studies with larger sample sizes are required to determine its probable association with other prognostic variables.

Vimentin, an intermediate filament of mesenchymal cells, is upregulated in epithelial-mesenchymal transition (EMT) and has a main role in cancer metastasis. As a new strategy to control metastatic outgrowth, EMT markers are generally inhibited using some drugs or specific siRNA. In this study, AGS gastric cancer cells were treated with metformin and vimentin-specific siRNA (vim-siRNA) for 48 h. The impact of metformin and vimsiRNA on vimentin downregulation in AGS cells were analyzed by quantitative PCR and Western blot. Following treatment with metformin and vim-siRNA, cell motility, migration and invasion abilities of AGS cells were also analyzed. The results showed that inhibition of vimentin due to metformin was comparable with the vim-siRNA. Furthermore, wound-healing and invasion assays showed a significant decrease in migration and invasion of AGS cells following metformin and vim-siRNA treatment. Our finding for the first time indicated that metformin can be an alternative to specific siRNA for inhibition of vimentin expression and migration of AGS cell line. Taken together, our data indicates that the use of metformin might have a priority to siRNA for inhibition of gastric cancer cell behaviors siRNA is more unstable and expensive than metformin, and needs special vehicles and delivery strategies for efficient transfection of cells. Further in vivo studies can reveal metformin's potential in inhibition of EMT and metastasis of cancer cells.