A Cell Proteomic Approach for the Detection of Secretable Biomarkers of Invasiveness in Oral Squamous Cell Carcinoma

Abstract

To identify potential biomarkers of invasiveness in oral squamous cell carcinoma. Design: A pilot proteomic study for the identification of secreted and cleaved proteins that can serve as potential biomarkers for head and neck carcinoma invasiveness. Subjects: Two primary cell lines and their variants were established from 2 oral squamous cell carcinoma human tissue samples with distinct invasive phenotypes. The cell lines were confirmed to maintain the invasive capacity of the original cancer when implanted into the tongues of immunocompromised RAG-2/␥(c) mice. Interventions: Invasiveness was assessed by the capacity of cells to invade through a matrigel matrix using the Boyden chamber assay and correlated with the invasiveness seen clinically and histologically in patients. In parallel, cell lines were grown in serum-free conditioned medium, which was then used to identify secreted and/or cleaved proteins that emanate from cancer cells, using 2-dimensional gel electrophoresis and matrix-assisted laser desorption-ionization combined with tandem mass spectrometry. Results: The invasion assays revealed a correlation between cell migration capacity through matrigel matrix and the aggressive phenotype seen in the clinical and histopathological assessments. More than 50 proteins were identified as being differentially secreted in media between the least and the more aggressive cell lines (PϽ.05). These include proteins that regulate cell metabolism, cell structure, cell adhesion, and cell motility, as well as proteins with undefined function. Conclusions: We report a sensitive and clinically relevant approach to screen for secreted biomarkers of oral squamous cell carcinoma invasiveness using proteomic technology. Both high-and low-abundant secreted proteins were identified and can represent potential biomarkers for oral cancer.

TWIST1 is a molecular marker for a poor prognosis in oral cancer and represents a potential therapeutic target

“…We addressed the impact of Twist1 manipulation on the EMT phenotype using an NOE cell line, which was established from normal human tongue tissue, and the OSCC1.2 cell line, which was established from a poorly differentiated, metastatic, human oral cancer (stage T4N2b) that had vascular, lymphatic, and perineural invasion. 23,24 Exposure of NOE cells to TGFb induced a clear EMT phenotype in almost 100% of the population (Fig. 5A).…”

“…22 The OSCC1.2 cell line was established by our group from a metastatic OSCC and was maintained in culture as previously described. 23,24 Small Interfering RNA Expression…”

“…Exponentially growing cells were suspended in PBS (10 6 cells per 0.1 mL) and injected orthotopically into the tongues of male severe combinedimmunodeficiency (SCID) mice; or, alternatively, cells were implanted into the mice subcutaneously to address distinct tumor microenvironment, as previously described. 23,24 Tumor length (L) and width (W) were monitored every second day using a caliper that was accurate to 0.5 mm. Tumor volume (V) was calculated using the following formula: V 5 L 3 W 3 1=2 W. Once the tumor reached the maximal size allowed by institutional guidelines, the mice were killed, and the tumors were dissected and weighted using a precision balance.…”

Identification of Mac-2-binding Protein as a Putative Marker of Neuroendocrine Tumors from the Analysis of Cell Line Secretomes

“…In the present study, the established NET serum markers, chromogranins A, B, and C, were identified in the CM of all three lines, consistent with the neuroendocrine phenotype of these cells. These proteins have not been reported in the secretomes of several non-NET cell lines (11,13,(22)(23)(24) and were found in normal neuroendocrine secretory vesicles (21). However, these proteins possess limitations as NET biomarkers (see below).…”

“…This approach is based upon the assumption that the proteins secreted by the cell line will be representative of tumor cells in vivo. Studies have been performed using cell line models of prostate cancer, breast cancer (11), and head and neck cancer (13). Some putative markers were subsequently validated using immunological methods on limited numbers of clinical samples.…”