Biomarkers are needed to evaluate the stage of progression and potential for invasion and metastasis by oral squamous cell carcinomas. Objectives: To develop methods to detect tumor biomarkers in small samples of serum, saliva, or other fluids using SELDI-TOF (surface-enhanced laser desorption and ionization - time of flight) mass spectrometry (MS). Aims of this study were to detect and measure proteins in serum-free conditioned medium from three tumor cell clones with differing metastatic potentials. Methods: Conditioned media were prepared from the non-metastatic primary rat mammary carcinoma MTC clone and two lung metastases clones MTLn2 and MTLn3 of low and high metastatic potential, respectively. Proteins were concentrated by acid precipitation, washed, dissolved in sample buffer with urea, thiourea, and CHAPS detergent, reduced and alkylated, precipitated, and washed again. Samples of 5 µL containing 1 µg protein in binding buffers were applied to hydrophobic (H50), anion exchange (Q10), and cationic exchange (CM10) Ciphergen ProteinChip arrays. Unbound proteins and salts were removed by washing with binding buffer and then with water. Proteins were separated and measured in a Ciphergen PBS IIc spectrometer. Results: Media from the three clones had differing mass spectra on each of the three arrays. A total of 38, 56, and 52 protein peaks in the 5,000 to 100,000 M/z range were detected in average spectra from the samples on H50, Q10, and CM10 arrays. Peaks of interest were selected as those with greater intensity in media from the high metastatic potential clone than the low potential clone and with at least 4 times greater intensity than the primary non-metastatic clone. Eight peaks with M/z values from 5,665 to 68,152 met these criteria. Conclusions: SELDI-TOF MS is a rapid and sensitive method to detect and measure biomarkers that may be associated with tumor invasion and metastasis. Supported by USPHS DE07258

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