Abstract
Patients with oral squamous cell carcinoma (OSCC) present significant alterations in their saliva proteome. We have used the shotgun Phage Display (PD) technology to identify candidate proteins that were upregulated in saliva of OSCC by selecting ligands to salivary proteins from a single-chain variable fragment (scFv) PD combinatorial library. After two selection cycles, the highly reactive clone scFv-D09 was able to distinguish saliva of OSCC patients from healthy subjects by enzyme-linked immunosorbent assay (ELISA) with sensitivity and specificity of 96.67%. Additionally, the scFv-D09 clone presented a positive immunostaining for invasive malignant epithelial cells in the connective tissue, keratin pearls in the OSCC, and ducts of salivary glands. We have further identified the target protein as the tropomyosin alpha-4 chain (TPM4) by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, and its binding to the scFV-D09 was demonstrated by bioinformatics. Briefly, we have identified TPM4 as upregulated salivary protein in patients with OSCC, which plays a central role in stabilizing cytoskeleton actin filaments, probably linked with tumor tissue remodeling. Long-term longitudinal studies are needed to validate TPM4 as a potential marker of a malignant process.
Highlights
Patients with oral squamous cell carcinoma (OSCC) present significant alterations in their saliva proteome
The predicted amino-acid sequence for both heavy and light chains of the scFv-D09 clone is shown in Fig. 1A, including all three complementarity determining regions (CDRs) and conserved framework regions (FWRs)
This study describes the identification of Tropomyosin alpha-4 (TPM4) chain as an antigen in saliva of OSCC patients
Summary
Patients with oral squamous cell carcinoma (OSCC) present significant alterations in their saliva proteome. Several salivary protein markers have been detected at high concentrations in saliva of OSCC, for example, interleukin 610 and 811, epidermal growth factor receptor (EGFR)[12], Cyfra 21.114, catalase[15], tumor necrosis factor (TNF-α)[10], the peptide defensin- 116 and S100A8 acid protein[17], which may be considered putative biomarkers for OSCC diagnosis, but none of them were validated Combinatorial technologies, such as Phage Display, have led to the detection of specific proteins differentially expressed between cancer and non-cancer samples[18,19,20], and this technology is highly desirable since it allows selection of interesting combinatorial antibodies targeting specific antigens from different samples[21]
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