Cell Surface Vimentin Detection in Cancer Cells by Peptide-Based Monoclonal Antibody

Abstract

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.

Extracellular Vimentin is a Damage-Associated Molecular Pattern Protein Serving as an Agonist of TLR4 in Human Neutrophils

“…The presence of Vim on the cell surface results from its release from Vim-expressing cells (11, 21, 34). Our study uncovers a novel binding interaction between Vim and TLR4, adding a crucial dimension to our understanding of Vim’s functional role.…”

“…Vimentin (Vim), an intermediate filament protein, is predominantly expressed in mesenchymal cells and forms part of the cytoskeleton, maintaining cellular integrity and facilitating migration (9,10). New work is highlighting however that these intracellular vimentin filaments can be disassembled into subunits that are released into the extracellular environment, coined as extracellular vimentin (eVim), which can then attach to the cell surface or the ECM (11)(12)(13). Increased expression of eVim and its post-translationally modified (PTM) form, citrullinated vimentin (CitVim), has been observed in various acute inflammatory conditions such as sepsis and chronic ones like pulmonary fibrosis or rheumatoid arthritis (RA) (14)(15)(16).…”

Extracellular vimentin as a modulator of the immune response and an important player during infectious diseases

“…4,[28][29][30] During inflammation, endothelial cells and immune cells, such as neutrophils and macrophages, may release vimentin as part of their immune response. 12,31,32 In addition, when cells undergo injury or necrosis, their contents, including vimentin, can be passively released into the extracellular space as a result of physical trauma, chemical exposure or ischemic damage. 32,33 Notably, eVim may play a significant role in tumor progression, invasion and metastasis.…”

“…Elevated blood levels of eVim have been observed in patients with pulmonary lung fibrosis, sepsis, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and coagulopathy 4,28–30 . During inflammation, endothelial cells and immune cells, such as neutrophils and macrophages, may release vimentin as part of their immune response 12,31,32 . In addition, when cells undergo injury or necrosis, their contents, including vimentin, can be passively released into the extracellular space as a result of physical trauma, chemical exposure or ischemic damage 32,33 …”