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The murine fibroblastic cell line L929 was obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% (v/v) glutamine (Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). The prostate cancer cell line PC-3, colorectal carcinoma cell line DLD-1, and large cell lung carcinoma cell line NCI-H460 were obtained from China Center for Type Culture Collection (CCTCC), and cultured in RPMI-1640 at 37°C, 5% CO2 with 95% humidity. Reovirus type 3 Dearing strain was obtained from ATCC (VR-824) and stored in -80°C until use. Reovirus was propagated in L929 cells, titrated by a standard plaque assay. For generation of UV-inactivated reovirus, reovirus in PBS were exposed to UV light (shortwave 254nm) for 30 minutes. The UV-induced loss of reoviral replicability was confirmed with L929 cell viability assay.
To assess junctional adhesion molecule-A (JAM-A) expression, cells were stained with FITC-conjugated anti-JAM-A mAb (clone 1H2A9, Santa Cruz Biotechnology). In experiments evaluating reovirus to CIK cell attachment, reovirus treated CIK cells were incubated with anti-reovirus–σ3 primary antibody (1:100, 4F2; DSHB, University of Iowa, Department of Biology, Iowa City, IA, USA) at 4°C overnight. This was followed by incubation with FITC-goat anti-mouse IgG (1:100, Jackson ImmunoResearch, Inc.) secondary antibody for 30 min at 4°C. The cells were subsequently washed and stained with APC-conjugated mouse-anti-human CD3(SK7), PE-conjugated mouse-anti-human CD8(RPA-T8) or PE-conjugated mouse-anti-human CD56(MY31) antibodies, respectively. Appropriate FITC, PE, or APC isotype control antibodies were used as negative controls; all antibodies were obtained from BD Biosciences, and used according to the manufacturer’s instructions. Stained cells were analyzed on a FC500 flow cytometer (Beckman Coulter), with data analyzed with FlowJo software (Tree Star, Ashland, OR, USA).
Generation of CIK cells and loading with reovirus: The study protocol was approved by the Ethics Committee of Guizhou Medical University, and all participants provided written informed consent. PBMCs were isolated from peripheral blood from healthy donors, by density gradient centrifugation with Ficoll-Hypaque (GE Healthcare Life Sciences; Milan, Italy). CIK cells were generated from PBMCs as previously described [19]. Briefly, PBMCs were cultured in GT-T551 medium (Takara Bio Inc.) containing 1000 U/ml human interferon γ (PeproTech) for 24 hours. PBMCs were then stimulated with 100 ng/ml anti-CD3 antibody (R&D) and 500 U/ml rHuIL-2 (PeproTech). Fresh medium containing 500 U/ml rHuIL-2 was added every 3 days. To assess CIK cell quality, aliquots of cells were harvested after 14–16 days of incubation, and characterized by phenotypic analysis and cytotoxicity assay. The major effector cells were NKT (CD3+CD56+NKT ≥ 20%) and CTL cells (CD3+CD8+CTL ≥ 60%). To establish the optimal reovirus loading condition, CIK cells were infected with reovirus of 1 plaque forming units (pfu)/cell at both 4°C or 37°C for 2 or 4 hours, respectively. CIK cells were then washed with PBS to remove unbound viruses, and reovirus binding on CIK cells was assessed by flow cytometry. Optimal reovirus loading occurred at an MOI of 1 at 4°C for 2 hours, and this condition was used in subsequent experiments.
To determine reovirus-mediated cell lysis, tumor cell lines (DLD-1, PC-3, and NCI-H460) and CIK cells were seeded in 96-well plates at a density of 5×103 cells per well in 100μl medium and incubated overnight. After 12 hours, cells were treated with reovirus in the amounts of 0, 0.001, 0.01, 0.1 and 1 pfu/cell. After 2 hours of incubation, cells were washed with PBS to remove unbound viruses, and fresh growth medium was added. Cell viability was measured with Cell Counting Kit-8 (CCK-8, Dojindo Lab, Tokyo, Japan) following the manufacturer's instructions, and calculated as percentage of viable cells in the reovirus-treated group versus untreated controls.
Total RNA was extracted from DLD-1, PC-3, NCI-H460, and CIK cells at 12, 24, 48, and 72 hours after infection with 1pfu/cell reovirus, respectively, with TRIZOL reagent (Invitrogen). First strand cDNA synthesis was performed using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) with 0.5μg total RNA per 10μl reaction. Gene expression was quantified by real-time quantitative RT-PCR with iQSYBR Green supermix (Bio-Rad Laboratories, Hercules, CA); detection was performed by measuring the binding of the fluorescence dye SYBR Green I to double-stranded DNA. The primer sets were provided by Generay Biotechnology (see Table 1). The comparative Ct method was used for data analysis. Relative quantity was defined as 2−ΔΔCt, with β-actin used as the reference gene.
Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0184816.t001 Primers used in qRT-PCR.Gene short names, Genbank accession numbers, PCR primer sequences and amplicon length are shown. Tumor or CIK cells were seeded at a density of 2×104 cells/well into glass bottom dishes, and incubated overnight at 37°C. Then, the medium was removed, and 1 pfu/cell of reovirus was incubated with tumor or CIK cells at 4°C for 2 hours. Virus inocula were then aspirated, and cells were cultured in fresh media for 12 hours. To prepare the cells for microscopy, reovirus-infected cells were washed twice with PBS, and fixed with 4% paraformaldehyde. Then, mouse anti-reovirus–σ3 primary antibody (4F2, 1:100) was applied at 4°C and incubated overnight. Afterwards, the cells were washed three times with PBS, and FITC-conjugated goat anti-mouse IgG secondary antibodies (1:50, Jackson ImmunoResearch, Inc.) were applied in 3% BSA for 30 min at 4°C. Counterstaining was performed with 4′, 6-diamidino-2-phenylindole (DAPI). Confocal images were acquired on a confocal microscope (Olympus, FV1000, Olympus Optical, Japan) in sequential scanning mode.
DLD-1, PC-3, NCI-H460, and CIK cells were incubated with 1 pfu/cell of reovirus at 4°C for 2 hours. Then, the cells were washed with PBS twice and incubated at 37°C for 12 hours. Following incubation, the cells were harvested and fixed in 2.5% glutaraldehyde in 0.1M sodium phosphate buffer (pH 7.4) at room temperature for 2 hours. The cells were then washed once in sodium phosphate buffer and dehydrated using a graded series of ethanol. After dehydration, the samples were subjected to two changes of propylene oxide and embedded in epoxy resin. After ultra-microtome sectioning, sections were analyzed on a FEI Tecnai G2 20 TWIN electron microscope operating at 200 kV.
Reovirus hand-off in the presence of human AB serum: Human AB serum was collected from 4 healthy donors. The neutralizing effect of human AB serum was confirmed with the L929 cell line. DLD-1, PC-3, and NCI-H460 tumor cells were used as target cells; they were kept in the log phase and passaged the day before the cytotoxicity assay. For the CIK cell transfer reovirus-mediated cytotoxicity assay, cell viability was assessed by CCK-8 assay. Briefly, DLD-1, PC-3 and NCI-H460 cells were seeded into 96-well plates at 8 ×105/ml in 100μl per well for 12 h. Then, CIK cells were re-suspended at 2×106 cells/ml and incubated with reovirus or UV-inactivated reovirus at 1 pfu per cell at 4°C for 2 hours and washed 3 times with PBS to remove unbound viruses. CIK cells loaded with reovirus were co-cultured with tumor cells at an E/T ratio of 10:1 in the presence of 7.5% human AB serum for 2 hours. Afterwards, CIK cells were washed, and adherent tumor cells were cultured for an additional 24, 48, and 72 hours, respectively. Cell viability was measured by CCK-8 assay according to manufacturer’s instructions. Calculate the percentage of cell death as follows: (1-Atest /Acontrol) ×100, where Atest is the absorbance of experimental wells and Acontrol is the absorbance of control wells. All experiments were performed in biological triplicate.
For the reovirus-loaded CIK cell mediated cytotoxicity assay, the CytoTox 96® Non-Radioactive Cytotoxicity Assay kit (Promega; Madison, WI) was used to quantify LDH release following the manufacturer’s protocol. Briefly, CIK cells were resuspended at 2 × 106 cells/ml, and incubated with reovirus at 1 pfu per cell at 4°C for 2 hours. Then, the cells were washed twice to remove the unbound reovirus. Using an E/T ratio of 20:1, reovirus-loaded CIK cells (1.6 ×106) were plated into 96-well plates, and mixed with tumor cells (8 ×104) in the presence of 7.5% human AB serum (v/v). The experiment was performed in triplicate. After incubation at 37°C in 5% CO2 for 6 hours, culture plates were centrifuged at 250×g for 5 minutes, and 10μl of supernatant from each well was collected and analyzed by measuring the optical density (OD) value to estimate cell death. OD values were converted into percent specific cytotoxicity (%) as: (ODExperimental−ODEffector spontaneous−ODTarget spontaneous)/(ODTarget maximum–ODTarget spontaneous) ×100.All assays were performed in triplicate, with CIK cells isolated from 3 unique donors.
GraphPad Prism version 6 (GraphPad, San Diego, CA) was used for statistical analysis. For normally distributed variables, comparisons between two given groups were performed by the parametric Student's t test; multiple group comparisons were carried out by one-way or two-way analysis of variance (ANOVA).
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