ABSTRACT
Biomarkers are needed to elucidate the biological background and to improve the detection of cancer. Therefore, we have analyzed laser-microdissected cryostat sections from head and neck tumors and adjacent mucosa on ProteinChip arrays. Two differentially expressed proteins (P = 3.34 x 10(-5) and 4.6 x 10(-5)) were isolated by two-dimensional gel electrophoresis and identified as S100A8 (calgranulin A) and S100A9 (calgranulin B) by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immunodepletion assay. The relevance of these single marker proteins was evaluated by immunohistochemistry. Positive tissue areas were reanalyzed on ProteinChip arrays to confirm the identity of these proteins. As a control, a peak with low P was identified as calgizzarin (S100A11) and characterized in the same way. This technical triade of tissue microdissection, ProteinChip technology, and immunohistochemistry opens up the possibility to find, identify, and characterize tumor relevant biomarkers, which will allow the movement toward the clonal heterogeneity of malignant tumors. Taking this approach, proteins were identified that might be responsible for invasion and metastasis.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/metabolism , Head and Neck Neoplasms/metabolism , Proteomics/methods , Calgranulin A/metabolism , Calgranulin B/metabolism , Cryoultramicrotomy , Epithelium/metabolism , Humans , Immunohistochemistry , Pharynx/metabolism , Protein Array Analysis/methods , S100 Proteins/metabolismABSTRACT
Head and neck cancer is a frequent malignancy with a complex, and up to now not clear etiology. Therefore, despite of improvements in diagnosis and therapy, the survival rate with head and neck squamous-cell carcinomas is poor. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, we have analyzed changes of protein expression between microdissected normal pharyngeal epithelium and tumor tissue by ProteinChip technology. For this, cryostat sections from head and neck tumors (n = 57) and adjacent mucosa (n = 44) were laser-microdissected and analyzed on ProteinChip arrays. The derived mass spectrometry profiles exhibited numerous statistical differences. One peak significantly higher expressed in the tumor (p = 0.000029) was isolated by two-dimensional gel electrophoresis and identified as annexin V by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immuno-deplete assay. The relevance of this single marker protein was further evaluated by immunohistochemistry. Annexin-positive tissue areas were re-analyzed on ProteinChip arrays to confirm the identity of this protein. In this study, we could show that biomarker in head and neck cancer can be found, identified, and assessed by combination of ProteinChip technology, two-dimensional gel electrophoresis, and immunohistochemistry. In our experience, however, such studies only make sense if a relatively pure microdissected tumor tissue is used. Only then minute changes in protein expression between normal pharyngeal epithelium and tumor tissue can be detected, and it will become possible to educe a tumor-associated protein pattern that might be used as a marker for tumorigenesis and progression.