Glioblastoma (GBM) is known as malignant brain tumor, and GBM biomarkers are required to detect cancer at earlier stage for improving therapeutic outcomes. However, GBM biomarkers possessing the potential for clinical applications have not been identified yet. In the previous studies, GBM biomarkers were screened from body-fluid. However, abundant proteins in the body-fluid interfere identification of proteins secreted from tumor tissues. Therefore, the purpose of the present study was to identify GBM-selective secreted proteins from the conditioned-mediums (CMs) of GBM cells and validated their expression in the cerebrospinal fluid (CSF) of GBM or non-brain tumor patients.
GBM (U87, U251, T98G) and non-GBM (MDA-MB-231, MCF-7, Caco-2) cells were cultured with FBS-free medium. After incubation, CMs were collected and the proteins were concentrated by acetone precipitation. The peptides digested by Lys-C and trypsin were identified by data-dependent acquisition and quantitated by data-independent acquisition (SWATH-MS) using TripleTOF5600 (SCIEX, USA). The identified proteins were quantified by targeted proteomics with spiking internal-standard peptides by MRM of QTRAP6500 (SCIEX).
We identified 2,371 proteins from the CMs, and 1,338 proteins were identified by more than 3 peptides. Among 1,338 proteins, 19 proteins were detected only in the CMs of GBM cells. To validate the GBM-selective secretion, the expression of 19 proteins were quantified in the CMs and CSF by targeted proteomics. As a result, 19 proteins exhibited higher expression levels in CMs of GBM cells than those of non-GBM cells. The expression of laminin subunit alpha-4 (LAMA4) and osteopontin (OPN) were significantly greater in CSF of 22 GBM patients than in 11 non-brain tumor patients and showed AUC greater than 0.86. Therefore, LAMA4 and OPN were suggested to be secreted from GBM tissues. In conclusion, LAMA4 and OPN were identified GBM-selective secreted proteins, and are candidates as biomarkers and therapeutic targets for GBM.