Abstract
A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.
Highlights
A promising avenue toward the development of more selective, better anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers [1,2,3,4]
A subsequent proteolytic digestion followed by nanocapillary HPLC peptide separation and MALDI-TOF/TOF mass spectrometric analysis permitted the identification of 637 proteins cumulated in all specimens
A number of antibody-based fusion proteins are currently in clinical development, including the human antibody L19, which is specific to the extra domain B of fibronectin (18 –20)
Summary
Patients—This study was started upon approval by the ethical committee of the University Hospital of Liege (Belgium). Afterward, a second perfusion step with 450 ml of PBS containing a 50 mM solution of the primary amine Tris was performed for 8 –9 min to quench unreacted biotinylation reagent. All perfusion solutions contained 10% dextran 40 as a plasma expander and were prewarmed to 40 °C Both perfusion steps were performed with a pressure of 100 –150 mm Hg. Successful perfusion was indicated by the wash out of blood during the first minutes of perfusion and subsequent flow of clear perfusate out of the renal vein. Antibodies were used in a dilution of 1:500 to stain sections from paraformaldehyde-fixed, paraffin-embedded tissue specimens and were detected by the immunoperoxidase technique (Vectastain Elite ABC kit, Vector Laboratories) according to standard procedures. The products of the PCR were analyzed by 2% agarose gel electrophoresis, stained by ethidium bromide, and imaged using the BioDoc-It imaging system (UVP, Upland, CA)
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