Abstract C40: Proteome profiling and detecting method of lung cancer cell-derived exosomes as tumor biomarkers.
Authors: Kamada Haruhiko , Yamashita Takuya , Inoue Masaki , Nagano Kazuya , Katayama Sumie , Mukai Yohei , Yoshioka Yasuo , Higashisaka Kazuma , Tsutsumi Yasuo , Tsunoda Shin-ichi 
Published: August 2014

Abstract
Introduction: Exosomes are small membrane vesicles that can mediate intercellular and cell surface transfer of RNAs and proteins to regulate the growth and metastasis of tumors. Recently, the finding of some tumor cell-derived exosomes suggests that blood-based exosomes may provide important diagnostic information and aid in therapeutic decisions for cancer patients. In this study, we performed a proteomic analysis of exosomes derived from cultured lung cancer cell lines to detect membrane proteins as cancer biomarkers and evaluated their usefulness for tumor diagnosis.
Material and Method: Exosome preparation: The culture medium derived from human lung cancer cell lineswere sequentially centrifuged to eliminate cells and debris, followed by filtration through a nitrocellulose membrane. Then, exosomes were precipitated by ultracentrifugation.
Proteomic analysis: Exosome samples were subjected to reduction, alkylation with iodoacetamide, and tryptic digestion. Extracted tryptic peptides from each sample were concentrated by centrifugal lyophilization and analyzed by LC-MS/MS fitted with a nanoflow RP-HPLC. RP-HPLC of peptide mixtures was performed on an C18 column developed using a linear 60-min gradient from 0% to 100% B (acetonitrile).
ELISA protocol: Microtiter plates were coated with mouse anti human CD81 monoclonal antibody, which is one of the molecular biomarkers detecting exosome and incubated overnight. After blocking, several concentrations of purified exosomes were added and incubated. After 3 washes with PBS, antibody recognized a target molecule was added. After the final 3 washes with PBS, the reaction was developed with TNBZ reagents.
Results and Conclusion: The isolated vesicles were classified as exosomes based on structural analysis using transmission electron microscopy, and the exosomes were detected as small extracellular membrane vesicles 40-100 nm in diameter. The proteome analysis identified more than 50 proteins that were expressed in the exosomes derived from three lung cancer cell lines, but not in normal cells. The amount of identified proteins expressed in plasma exosomes can be quantified using different antibodies against exosome markers and tumor-expressing proteins. The ELISA results for the detected proteins suggested that the ELISA platform using anti-CD81 and anti-target protein antibodies could detect tumor-derived exosomes as a type of tumor biomarker. The results of this study will provide a new diagnostic method for detecting exosomes secreted from cancer into blood, which will be particularly useful for developing tests for minimally invasive personalized cancer therapy.
Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C40.
Citation Format: Haruhiko Kamada, Takuya Yamashita, Masaki Inoue, Kazuya Nagano, Sumie Katayama, Yohei Mukai, Yasuo Yoshioka, Kazuma Higashisaka, Yasuo Tsutsumi, Shin-ichi Tsunoda. Proteome profiling and detecting method of lung cancer cell-derived exosomes as tumor biomarkers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C40.