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Blood, Vol. 113, Issue 9, 1957-1966, February 26, 2009
Blood diffusion and Th1-suppressive effects of galectin-9–containing exosomes released by Epstein-Barr virus–infected nasopharyngeal carcinoma cells
Blood Klibi et al.
113: 1957
Supplemental materials for: Klibi et al
Files in this Data Supplement:
- Table S1. Clinical and pathological data on NPC plasma donors from Tunisia (Salah Azaiz Institute, Tunis)* (PDF, 33.7 KB)
- Table S2. Clinical and pathological data on NPC plasma donors from France (Gustave Roussy Institute of Oncology and Paris hospitals) (PDF, 29.8 KB)
- Table S3. Data about non-NPC plasma donors from Tunisia (Salah Azaiz Institute, Tunis)* (PDF, 14 KB)
- Table S4. Clinical and pathological data about non-NPC plasma donors from France (Gustave Roussy Institute of Oncology and Paris hospitals) (PDF, 26.3 KB)
- Figure S1. In situ hybridization of EBER viral RNAs on tumorsections from 5 NPC plasma donors (JPG, 79.5 KB)
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For all 5 NPC patients recruited in the Gustave Roussy Institute and Paris hospitals (NPC N to R), EBER RNAs were detected on primary tumor sections by in situ hybridizations (ISH). Intense staining is visible in most malignant epithelial cells providing confirmation of EBV involvement. EBER ISH was performed using kits from Dako (Glostrup, Denmark) (NPC N), Ventana Medical Systems (Illkirch, France) (NPC O, P and R) and Enzo Diagnostics (Farmingdale, NY) (NPC Q).
- Figure S2. Specific detection of HLA class II/galectin-9–positive exosomes in the plasma of NPC patients (JPG, 48.9 KB)
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Low density vesicles (110 Kg pellets)derived from human plasma samples were subjected to capture with anti-HLA class II magnetic beads in order to isolate HLA class II-positive exosomes (in the same way as for Figures 1 and 2). Proteins eluted from the beads were analyzed by western blotting for detection of galectin-9. In panel A, a protein extract from the C17 xenograft provides a positive control. Plasma samples named NPC G to L were from Tunisian patients (clinical and pathological data are summarized in Table S1). Sample NPC E is the same as NPC E displayed in Fig. 2. Plasma samples named NPC M to Q were from patients treated at the Gustave Roussy Institute or Paris hospitals (Table S2). In panels A and B, the healthy donor plasma sample was the same as in Fig. 2. Five other controls named TD to TG were from patients suffering from malignancies distinct from NPC (Table S4). Galectin-9 bound to HLA-class II exosomes is found in all but one NPC plasma samples (NPC I is the exception). In many cases, the m-isoform (low molecular weight) is more abundant than the l-isoform (see references 20 and 38). No galectin-9 is detected in control plasma samples except a possible low amount in the sample from patient TF, who suffered from an epithelioid sarcoma.
- Figure S3. Production of HLA class II/galectin-9–positive exosomes and microparticlesis not dependent on NPC cell apoptosis (JPG, 41.5 KB)
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Methods: NPCcells derived from the C17 xenograft were dispersed and used to prepare conditioned medium as described in the Materials and Methods section with the following modifications. Following a 24h incubation period, half the cells were treated with the 7C11 antibody, an agonist of the CD95 receptor, which is known to be a potent inducer of apoptosis in C17 cells (reference 28). 7C11 is an IgM monoclonal antibody from Beckman Coulter Immunotech (Villepinte, France) which was used at a final concentration of 75 ng/ml. The rest of the cells were used for a control condition and incubated with a non-specific IgM. After 16h of incubation with 7C11 or the control IgM, cell samples (6 × 106)were collected for measurement of caspase 3/7 activation using a luminescent luciferase-bound substrate (Promega, Madison, WI). Finally, culture supernatants were collected 24h after addition of the antibodies. Light density vesicles (110 Kg pellets) were prepared according to the procedure described in the Materials and Methods section, from the same volumes of conditioned media derived for control cells and apoptotic cells, respectively. Pellets were analyzed by western blotting for galectin-9 and DR-α detection (same antibodies as in Figs 1 to 4). Results: On the left panel, measurement of caspase3/7 activity confirms apoptosis induction by the 7C11 antibody. On the right panel, western blot analysis shows that galectin-9 and HLA class II DR-α molecules bound to exosomes and other light-density vesicules (110 Kg pellet) are less abundant when apoptosis is induced in C17 cells. This is one of 2 similar experiments.
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