Extra-cellular release and blood diffusion of BART viral micro-RNAs produced by EBV-infected nasopharyngeal carcinoma cells

Expression of a panel of 5 BART miRNAs was investigated in total RNA extracted from the C15, C17 and C666-1 NPC xenografts by quantitative PCR following multiplexed reverse-transcription (RT). Reverse transcription was performed on a multiplex mode using a set of primers specific for all 5 BART miRNAs, followed by single-mode PCR using one universal primer and one primer specific for each BART miRNA. The small non-coding RNA RNU44 was used as an endogenous reference. Our panel of BART miRNAs included members of cluster 1 (miR-BART 1-5p and 5) and cluster 2 (miR-BART 7-3p, 12 and 13). On the basis of previous publications, these microRNAs were expected to be among the most abundant BART miRNAs produced by NPC cells [4, 14, 15, 17, 18]. As anticipated, they were readily amplified from the RNA of NPC xenografts. The RNA extracted from the CAPI tumor, an EBV-negative non-NPC epithelial xenografted tumor was used as a negative control (Figure 1). In order to allow comparative analysis of BART miRNAs in NPC and EBV-infected lymphoid cells, total RNAs from 2 lymphoid cell lines were processed using the same primers and experimental conditions. Daudi was derived from a Burkitt lymphoma and carries its own EBV isolate. NAD+C15 is an LCL (lymphoblastoid cell line) derived from normal B-cells in vitro transformed by artificial infection using the C15 EBV isolate [4, 19]. As previously reported, no BART miRNA was detected in Daudi [4]. In contrast, all 5 BART miRNAs of our panel were detected in the NAD+C15 LCL with a profile somehow similar to the C15 NPC xenograft profile (Figure 1). It is noteworthy that in NPC tumors as well as in the NAD+C15 LCL, miR-BART 7-3p was expressed at a higher level than the 4 other BART miRNAs.

Several reports have shown that at least a fraction of extra-cellular microRNAs are secreted in association with exosomes [7, 12, 20]. Therefore, we undertook to investigate the distribution of BART miRNAs in exosomes released by malignant NPC cells. Epithelial cells from the C15 and C17 NPC xenografts were dispersed by collagenase treatment and incubated in vitro for 48 h in order to produce conditioned culture media. Exosomes were prepared from these conditioned media as explained in Figure 2A. Simultaneously, exosomes were prepared from permanently propagated cell lines: the NAD+C15 LCL, Daudi and Hela cells. Quality of these exosome preparations was assessed using ordinary morphological and biochemical criteria. Round vesicles of 50 to 100 nm in diameter often with a plate-like shape were observed under electron microscopy. High concentrations of the CD63 tetraspanin were obtained in the exosome extracts constrasting with the absence of the gp96 cytoplasmic protein (Figure 2B)[21]. The distribution of miR-BART 1-5p, 5, 7-3p, 12 and 13 was investigated in total RNA extracted from these exosomes using multiplexed RT combined to real-time PCR. The cellular miR-21 which is abundant in most types of human malignant cells was used as an endogenous control [22]. The highest relative concentrations of BART miRNAs were detected in exosomes released by the C15 NPC cells, followed by exosomes from the NAD+C15 LCL (Figure 3). Lower but still significant amounts of BART miRNAs were detected in exosomes from C17 NPC cells. In contrast, no BART miRNA were detected in exosomes from Daudi and Hela cells. Like for tumor RNAs, miR-BART 7-3p was more abundant than other BART miRNAs in all exosome RNA preparations.

Our NPC xenografts are propagated in nude mice by sub-cutaneous inoculation of small tumor fragments which grow subcutaneously without invasion of underlying organs and tissues and therefore are well tolerated. We could collect plasma samples from mice carrying relatively large NPC xenografts (C15, C666-1 and C17) and also from mice carrying a xenografted EBV-negative human epithelial tumor (CAPI) used as a negative control [21]. The average ratio of tumor to mouse body mass was about 6 to 8%. Samples from 3 or 4 mice carrying the same xenografted tumor line were pooled and assessed for EBV DNA load. High DNA copy numbers were obtained for C15, C666-1 and C17 but not CAPI mice (Table 1). Total RNA was extracted from 100 μl of each plasma pool and subjected to multiplexed RT for the panel of miR-BART 1-5p, 5, 7-3p, 12 and 13 followed by single mode real-time PCR. The cellular microRNA miR-146a which is known to be abundant in blood plasma was used as an endogenous reference [23]. As shown in Figure 4 and Table 1, the most abundant BART miRNAs were found in plasma samples from mice carrying the C15 or C666-1 NPC tumors, consistent with the relative abundance of these microRNAs in the corresponding xenografted tumors. In contrast, low amounts of BART miRNAs were found in plasmas from mice carrying the C17 NPC. The miR-BART 7-3p was the most abundant in all cases. In contrast, the 2^-ΔCT was very low for miR-BART1-5p. There was no miR-BART detection in the pool of plasma samples from CAPI mice.

To demonstrate that the data obtained in our murine NPC model were relevant to human pathology we investigated the dissemination of the miR-BART 7-3p in plasma samples obtained from five consecutive NPC patients prior to any treatment. We used single-mode RT combined to real-time PCR. Plasma from three healthy EBV-carriers, a healthy donor not infected by EBV and two patients bearing non-NPC tumors were used as controls (Table 2). For each plasma sample, RNA was extracted from a total volume of 100 μl. The cellular miR-146a was used as an endogenous reference [23]. As shown in Figure 5, miR-BART7-3p was detected in the plasma samples from NPC patients at much higher levels than in samples from control donors, except for one of them (HEP 1). Overall the difference was statistically significant (p = 0.026 using the Mann-Whitney test).

C15 and C17 are xenografted EBV-positive NPC tumor lines permanently propagated by subcutaneous passage into nude mice [25]. Suspensions of NPC cells were obtained by dispersion of xenografted tumors using type II collagenase, sometimes combined with trypsin pre-treatment, as previously described [28]. C666-1 is an EBV-positive NPC tumor line which has been adapted to in vitro culture [29]. It was grown in RPMI supplemented with 25 mM Hepes and 7.5% FCS. Alternatively C666-1 cells were injected sub-cutaneously into nude mice for obtention of xenografted tumors (3 million cells mixed with 100 μl culture medium and 100 μl Matrigel, BD Biosciences, Le Pont-de-Claix, France). All experiments on xenografted NPC tumors were conducted in the animal facility of the Institut de Cancérologie Gustave Roussy, according to institutional guidelines. Daudi is an EBV-positive Burkitt lymphoma cell line [4]. NAD+C15 is a lymphoblastoid cell line (LCL) resulting from transformation of B lymphocytes from a normal adult donor by the C15 EBV-strain [19]. Daudi and NAD+ C15 were grown in RPMI supplemented with 10% FCS. The HeLa cervix carcinoma cell line was cultured in DMEM with 5% FCS.

Cells of various types were incubated at appropriate concentrations in culture medium supplemented with 1.5% fetal calf serum (FCS) for 48 h, at 37°C under 5% CO2. C15 and C17 NPC cells were obtained by dispersion of xenografted tumors and incubated in 24 well plates at 1.2 million cells/well in 1.5 ml RPMI medium. HeLa cells were grown to 70% confluency in 175 cm² flasks and then incubated in 20 ml culture medium (DMEM). Daudi and NAD+C15 cells were incubated at 1 million cells/ml in RPMI (100 million cells/100 ml culture medium/175 cm² flasks). Following collection, conditioned media were clarified by centrifugation at 300 g for 10 min and at 1890 g for 15 minutes at 4°C to remove biggest cell remnants and debris and frozen at - 80°C.

This procedure was adapted from the method described by Lamparski et al. [30]. All steps were performed at 4°C. Thawed conditioned culture supernatants (at least 400 ml) were first clarified by a centrifugation at 12 000 g for 35 min and then subjected to ultracentrifugation at 66 000 g for 2 h using a Ti45 Beckman rotor, resulting in a pellet designated as "nano-material pellet". Exosomes contained in this pellet were further purified by flotation on a cushion made of a sucrose solution in deuterium oxide (D₂O). Practically, the nano-material pellet was redissolved in filtrated PBS (2 × 9 ml for an initial volume of 400 ml supernatant). One ml of sucrose/D₂O solution (20 mM Tris/30% sucrose/D₂O pH 7.4) was layed down carefully under 9 ml of nano-material solution at the bottom of a SW41 Ti polycarbonate tube. This two phase discontinuous gradient was subjected to ultracentrifugation at 76 000 g for 75 min on a SW41 Ti Beckman rotor. The faint band containing the exosomes at the surface of the cushion was then collected without disturbing the pellet. The exosomes were diluted 1:5 in PBS and pelleted by ultracentrifugation at 110 000 g in a SW41 Ti rotor for 90 min. Two additional washing steps were performed in a smaller volume (ultracentrifugation at 110 000 g using a TLA100.3 Beckman rotor). Washed exosomes were then processed for protein or RNA extraction. Exosome proteins were extracted in 20 μl of RIPA buffer (150 mM NaCl 5M, 50 mM Tris HCl pH:7,4, 5 mM EDTA, 0,1% SDS, 0,5% NaDOC, 0,5% NP40) supplemented with Complete anti-proteases (Roche, Basel, Switzerland). RNA extraction was started by solubilization in 800 μl of TRI REAGENT (Molecular Research Center, Cincinnati, OH).

For negative staining, exosome fractions were observed after dilution in salt buffer (Tris 10 mM, pH 7.5, NaCl 150). Five microliters of solution was adsorbed onto a 300 mesh copper grid coated with a collodion film covered by a thin carbon film, activated by glow-discharge. After 1 min, grids were washed with aqueous 2% (w/vol) uranyl acetate (Merck, France) and then dried with ashless filter paper (VWR, France). TEM observations were carried out on a Zeiss 912AB transmission electron microscope in filtered low loss mode. Electron micrographs were obtained using a ProScan 1024 HSC digital camera and Soft Imaging Software system.

Exosome lysates were clarified by centrifugation at 16 000 g for 15 minutes at 4°C. Protein concentrations were determined using the Bradford protein Assay (Biorad Laboratories, Gif-sur-Yvette, France). The protein extracts (12.3 μg) were loaded on a Nupage Bis Tris MiniGel (Invitrogen, Carlsbad, New-Mexico) and migration was performed in non-reducing conditions. Monoclonal antibody against CD63 (TS63) was previously described (Charrin, Rubinstein at al, 2001). The gp96 cytoplasmic protein was detected with a rat monoclonal antibody (Stressgen, Ann Harbor, MI) and β-actin was visualized using a monoclonal antibody (AC-74) from Sigma Aldrich (St. Louis, MO).

Blood samples were collected from mice carrying xenografted NPC tumors under anesthesia by intra-cardiac puncture in EDTA tubes. Eight human plasma samples were collected after signature of informed consent from patients of the Institut de Cancérologie Gustave Roussy or Paris hospitals working in collaboration with this institute (Table 2). Five of these samples were collected from NPC patients prior to any treatment whereas two control samples were obtained from patients bearing non-NPC tumors (one adenocarcinoma of unknown primary and one larynx squamous cell carcinoma). Tumor staging was done according to ESMO (European Society of Medical Oncology) guidelines [31]. Additional control plasma samples were obtained from four healthy donors including three EBV-carriers and one EBV-sero-negative adult. Plasma was separated from blood cells by centrifugation at 1700 g at 20°C for 15 min and frozen at - 80°C.

EBERs (Epstein-Barr encoded RNAs) which are small untranslated RNAs from EBV - totally distinct from the viral microRNAs and generally very abundant in NPC cells - were detected on tissue sections from the tumor biopsies by in situ hybridization using commercial kits, mainly from Ventana Medical System (Illkirch, France) [2]. Circulating antibodies directed to VCA (viral capsid antigen) and EBNA (Epstein-Barr nuclear antigen) were assessed in human plasma samples using the Vidas(r) EBV kit from Biomerieux (Lyon, France). EBV viral load in plasma samples was quantified as previously described [32]. Briefly: total DNA was extracted from 200 μl plasma aliquots using the QIAmp blood kit (Qiagen Inc., Courtaboeuf, France). Viral load was then determined by real-time quantitative PCR with primers designed to amplify the thymidine kinase gene of EBV (BXLF1). The copy number was determined by reference to a standard curve based on a tenfold serial dilution of a plasmid containing a unique copy of the BXLF1 genomic segment.

Detection of BART miRNAs was performed using reagents and protocols of the TaqMan MicroRNA Reverse Transcription and TaqMan MicroRNA Assay kits (Applied Biosystems, Foster City, CA). In this experimental system, reverse transcription (RT) is primed using a stem-loop primer specific of each microRNA. Each stem-loop primer has a specific linear portion complementary of the 3' end of the target microRNA and a loop portion containing a universal invariant target sequence. This RT results in a c-DNA joining the microRNA complementary sequence to the invariant sequence. This c-DNA is amplified by TaqMan PCR using a specific forward primer and a universal reverse primer in the presence of a specific hydrolysis probe. Due to spatial constraint of the stem-loop structure, this system is about 100 times more efficient at amplification of mature microRNAs than their precursors [33]. Reverse transcription was done in 15 μl reaction mix including 90 ng total RNA for cells and exosomes or 9.16 μl of the eluted RNA for plasma samples, 3 μl of the RT primer solution (final concentration: 50 nM), 0.15 μl dNTP (1 mM), 1 μl Multiscribe Reverse transcriptase (3.33 U/μl), 1.50 μl of 10× Buffer, 0.19 μl RNase inhibitor (0.25 U/μl) and nuclease free water. The reaction mix was incubated at 16°C for 30 min, 42°c for 30 min, 85°C for 5 min then frozen at -20°C. Single-mode real-time PCR was performed in a 20 μl reaction volume, containing 1.33 μl RT reaction mix providing the cDNA template, 1 μl of the primer mix including - for a given microRNA - the universal reverse primer (0.7 μM), the specific primer (1.5 μM) and the hydrolysis probe (0.2 μM) (TaqMan MicroRNA Assays, Applied Biosystems, foster City, CA), 10 μl of Fast Start Universal Probe Master mix (Roche, Basel, Switzerland) and RNase-free water. The first cycle included one step of 2 min at 50°C and one step of 10 minutes at 95°C. It was followed by 45 cycles including one step of 15 sec at 95°C and one step of 60 sec at 60°C. The following sets of primers and probes were purchased from Applied Biosystems (TaqMan MicroRNA assays): ebv-miR-BART 1-5p (197199_mat), ebv-miR-BART5 (197237_mat), ebv-miR-BART7-3p (197206), ebv-miR-BART12 (005725), ebv-miR-BART13 (005446), RNU44 (001094), hsa-miR-146a (000468), hsa-miR-21(000397). Amplification reactions were performed in an Applied Biosystems Step One Detection System. Data from RT-Q-PCR were analysed using the comparative C_T method with RNU44, hsa-miR-21, hsa-miR-146a as endogenous references for tumor samples, exosomes and plasma samples, respectively. The 2 ^-ΔCT parameter was used as the index of target microRNA relative concentrations.

Detection of EBV-miR-BART 1-5p, 5, 7-3p, 12 and 13 was also performed in a multiplex mode, combining a multiplex Reverse Transcription (RT) stage and a stage of single PCR as recommended by the manufacturer. For this aim, a pool of RT stem-loop primers was made by mixing 6.25 pmoles of each primer. Practically, 25 μl of each primer solution were loaded in a 1.5 ml microtube and dried in a speed vacuum for 1 hour at 50°C. All RT dried primers were then solubilised in 100 μl of RNase free water. The same Taqman MicroRNA reverse Transcription kit used for single RT was used for multiplex with a few modifications: 90 ng input RNA was mixed with 4 μl of the RT primer mix (final concentration: 12.5 nM), 0.4 μl dNTPs (2 mM), 2 μl Multiscribe Reverse Transcriptase (5U/μl), 2 μl 10× RT Buffer, 0.25 μl RNase Inhibitor (0.25U/μl) and nuclease free water to reach a volume of 20 μl. Reaction parameters were identical to those used for single reverse transcription. The resulting cDNA was diluted by adding 180 μl of RNase-free water to the 20 μl reaction mix and stored at -80°C. Subsequent real time PCR was performed in the same conditions as when it was combined to single-mode RT, except that 9 μl of final RT reaction mix was mixed with other PCR reagents instead of 1.33 μl.