Specific detection of galectin-9–containing exosomes in the plasma of mice xenografted with NPC tumor lines. (A) A significant amount of galectin-9 is detected by ELISA in crude plasma samples from mice xenografted with NPC tumor lines (C15, C17, and C666-1), but not control mice either without xenografts (tumor-free) or xenografted with a non-NPC carcinoma tumor line (non-NPC). Assays were performed in triplicate on 3 plasma samples from 3 different mice. (B) A series of plasma samples were collected from 6 mice carrying C15 tumors of various sizes. Galectin-9 concentration in these samples is proportional to the tumor volume (y = 3.2× + 1.7, R² = 0.994). (C) Captures with anti–HLA class II beads were performed on low-density vesicles (110-kg pellets) derived from pools of murine plasmas. Samples collected with anti–class II beads are designated nos. 3 and 6 (class II). Control samples collected with beads carrying nonspecific Ig are nos. 2 and 5 (NS Ig). In addition to plasma material, protein extracts from the C17 and the non-NPC xenografted tumors were used as controls for Western blot analyses (nos. 1 and 4). (D) Numerous vesicles approximately 70 nm in diameter are captured by HLA class II beads and visualized by electron microscopy when these procedures are applied to plasma vesicles from C17-xenografted but not control mice. Scale bar represents 500 nm. In the inset, 2 vesicles at original magnification (× 4). (E) Western blot analysis detects galectin-9, CD9, CD63, and the DR-α chain in C17 plasma vesicles. In contrast, none of these molecules are detected when the capture is performed on control mouse plasma. Note that CD63 is much more concentrated in C17 exosomes than in the tumor extract. Gp96, which is a cytoplasmic membrane protein, is detected in the C17 and non-NPC tumor extracts (lanes 1 and 4) but not in C17 exosomes (lane 3).