Two tumor cell lines were used in the experiments: hepatocellular carcinoma cell line H22 and retroperitoneal sarcoma cell line MethA. Both cell lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). They were maintained in RPMI-1640 medium or Dulbecco's modified Eagle's medium (DMEM) tissue culture medium. Media were supplemented using 10% fetal bovine serum (FBS) (Gemini), 1% glutamine and 1% antibiotic mixture (Cellgro) unless indicated otherwise. Cells were grown in a humidified atmosphere containing 5% CO₂ at 37°C. These two cell lines have the capacity to grow intraperitoneally (i.p.) in BALB/c female mice.

∆51M recombinant IFN-inducing mutants of wild-type VSV were used in the animal study and they showed no toxic and durable cures in a previous study (21). VSV-∆51 expressing GFP was a recombinant derivative of VSV-∆51, and kindly provided by Yan-Jun Wen (Sichuan University). Plaque-forming units (PFU) were used for calculating infectious titers. Inactivated virus was produced by exposure to UV light for 45 min. Viruses were propagated in A549 cells (ATCC) and purified as previously described (22).

For viral infection, 24 h after seeding, the medium containing FBS was removed and replaced by serum-free medium 24 h before infection at a multiplicity of infection (MOI) of 1.0. One hour later, the infection medium was removed and replaced by complete serum-free medium with or without 600 mg/l L-glutamine, 2.5% glucose or 110 mg/l sodium pyruvate as indicated. Culture media were supernatants and cells were harvested at 0, 12, 24 and 48 h post-infection and processed for metabolites and protein quantifications. At each time point the cellular proteins were extracted, and cell supernatants were analyzed.

The virus was purified from the supernatants by passing through a 0.2-µm filter and centrifuging at 30,000 × g, and then re-suspension in PBS. After A549 tumor cells were challenged with VSV at infection of 1.0 PFU/cell for 24 h, the supernatant was harvested for a progeny virus assay. PFU were used for calculating infectious titers as previously described (23).

β-actin antibodies were purchased from Sigma (Milano, Italy). VSV whole-virus antisera was provided by Yan-Jun Wen (Sichuan University). Sodium oxamate, 2-deoxy-D-glucose (2-DG), pyruvate, bis-2-(5-phen-ylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and dimethyl-α-ketoglutarate were purchased from Sigma. 2-DG and oxamate were directly solubilized in cell culture medium and BPTES was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 10 mM, used at specified concentrations. In this research, all of the antibodies were used at the manufacturer's recommended dilution.

After 3 weeks of incubation, ascites cells were collected, washed with phosphate-buffered saline (PBS) three times, and used fresh for assays as described previous (24,25). After washing, the H22 and MethA cells from the ascites were used for further study immediately or maintained in RPMI-1640 medium. They were grown in a sealed modular chamber flushed with contained 5% CO₂ at 37°C. For in vitro experiments, cells obtained from ascites were counted to 1×10⁵ cells per milliliter medium and cultured in 6-well plates, 24-well plates or 10-cm dishes. When the H22 and MethA cells were incubated for 12 h, they were treated with VSV as previously described (26). In brief, the virus was added to the culture at an MOI of 1.0 PFU per cell. After VSV incubation for 1 h at 37°C, the cells were washed once with PBS and 5 ml of RPMI-1640 or DMEM. Following incubation, the cells were maintained for an additional period of 0–48 h and harvested, washed 3 times with PBS, scraped into RIPA buffer, centrifuged at 8,000 rpm at 4°C for 10 min and stored at −80°C for further analysis, and the media were collected to conduct further investigation. At the end of the incubation period, viable cells in each well were counted using the trypan blue method and the final results were normalized as described.

Total RNA was isolated by an Isogen RNA extraction kit (Nippon Gene, Inc., Tokyo, Japan) and reverse-transcribed by murine leukemia virus reverse transcriptase using a High Pure RNA Isolation kit (Invitrogen, Sydney, Australia) according to the manufacturer's protocol. cDNA amplicons were amplified with specific primers (data not shown). We used the β-actin gene as an internal control (202 bp; β-actin sense, 5V-CTTCCTGGGCATGGAGTCCT-3V; antisense, 5V-GGAGCAATGATCTTGATCTT-3V). The amplification was carried out in a Palm Cycler. The initial cDNA synthesis was performed at 54°C for 30 and 5 min at 94°C for denaturation. This was followed by 30 rounds of amplification of PCR consisting of 1 min at 94°C, 1 min at 55°C, 2 min at 72°C with a Gene Amp PCR System 9600 (Perkin Elmer) and Taq DNA polymerase (Toyobo, Osaka, Japan). The amplified DNA fragments were visualized by electrophoresis at 120 V on 2% agarosegel in 1X TAE buffer containing ethidium bromide. DNA molecular weight marker was also electrophoresed for comparison.

Metabolites were quantified from cell supernatants using a Glucose (GO) Assay kit, a Glutamine Determination kit (GLN1) (Sigma-Aldrich, Saint-Quentin Fallavier, France) and a pyruvate assay kit (BioVision, Nanterre, France). Assays were performed according to the manufacturer's instructions. At each time point the cellular proteins were extracted, and cell supernatants were analyzed. For each assay, concentrations in the cell culture medium were normalized with total protein amounts in the well and are expressed as nmol consumed (compared to time zero) per µg of protein (nmol/µg).

For western blot analysis, protein samples were subjected to 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in TBS [20 mM Tris and 137 mM NaCl (pH 7.3)] containing 0.1% Tween-20, and then the membrane was incubated with the specific antibodies. After being washed, the membrane was incubated with peroxidase-conjugated goat anti-mouse immunoglobulin G or anti-rabbit immunoglobulin G. Kodak Molecular Imaging software was used to quantification the intensity of the bands (Eastman Kodak Co., Rochester, NY, USA).

After obtaining the cell-free supernatants of ascites and plasma of mice, VEGF levels were measured with ELISA kits (Quantikine; R&D Systems, Minneapolis, MN, USA) as described by the manufacturer's protocol.

Malignant ascites H22 and MethA or cells obtained from ascites or cultured in a dish were infected with GFP-VSV as previously described. To evaluate infectivity, GFP fluorescence intensity was measured by flow cytometry 48 h after incubation; data were analyzed using FLOWJO software. For analysis of apoptosis, flow cytometric analysis (FCM) was performed to detect sub-G1/apoptotic cells in hypotonic buffer as previously described (24).

Fluorescent in situ terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to analyze the apoptotic cells within malignant ascites or cultured cancer cells after VSV infection using a commercially available apoptotic cell detection kit (Promega, Madison, WI, USA). After the cells were incubated with the VSV for 48 h, the cells were rinsed with PBS 3 times. The samples were counted to 1×10⁶ cells and subsequently spread on a slide to perform TUNEL assay following the manufacturer's protocol.

BALB/c female mice were used to establish H22 and MethA cell models, respectively. Before injection, the mice were housed in autoclaved microisolator cages in specific pathogen-free conditions and fed autoclaved pellets and sterile water ad libitum. All animal research was approved by the Animal Care and Use Committee of Sichuan University and was in compliance with all regulatory guidelines. The health status of each animal was monitored daily. Following a week of acclimatization, the mice were injected i.p. with 10×10⁶ H22 and MethA cells. Malignant ascites was collected three weeks later, red blood cells were schizolysed, and ascites fluid free of red blood cells was washed with PBS three times and centrifuged at 300 × g at 4°C for 5 min. Each type of tumor cell (1×10⁶) suspended in 100 µl of PBS was then injected i.p. into the mice with a 14-gauge needle.

After approximately 7 days of cancer cell incubation, ascites formation was observed. On day 10, 12 and 14, the mice were treated with 1×10⁸ VSV-∆51 or 1×10⁸ PFU equivalent of UV-inactivated VSV-∆51 by intra-peritoneal injection. Five mice from each group were left to develop ascites, and the weight of each mouse was recorded every other day until day 24 after implantion when the mice were euthanized. After euthanasia, the ascites fluid was collected, and the numbers of tumor cells and red blood cells were counted per milliliter per mouse. Before sacrifice, a blood sample was collected through the ophthalmic artery; ascites fluid was completely aspirated as previously described (24). The other five mice of each group were monitored on a daily basis for tumor burden, cachexia and abdominal distension. Mice were euthanized when their abdominal circumference reached 9.5 cm or they were becoming moribund. The survival time of each animal was calculated from the day of the beginning of the treatment to euthanasia. The ascites volume of each mouse was calculated as previously described (24). All of the specimens including cells, cell-free ascites fluid, tumors dissected from the peritoneal cavity and the plasma were stored in liquid nitrogen for further analysis. Ascites fluids of the control group were centrifuged and supernatants were liquidated into 1 ml samples and stored at −80°C until needed.

The relevant indices concerning side effect, such as diarrhea, anorexia, skin ulceration, or cachexia were observed every day. The heart, liver, lung, spleen and kidney were harvested and fixed in 4% paraformaldehyde in PBS, then sectioned and stained with H&E. All of the sections were observed by two different pathologists.

All data are reported as the means ± SE. Comparison of the different group samples was carried out by analysis of variance (ANOVA) and an unpaired Student's t-test. The level of significance was set as P<0.05. We used standard statistical software SPSS, version 13, for all statistical analyses.

To evaluate the effect of VSV on malignant ascite formation, mice bearing i.p. H22 and MethA tumor cells were treated with VSV 3 times on day 10, 12 and 14 after incubation. Using a well-established animal model of malignant ascites formation, here we showed an inhibitory effect of VSV on intraperitoneal tumor growth, malignant ascites formation. Fig. 1A shows the survival curves of the mice bearing H22 or MethA tumor cells from the three groups. In several mice, VSV treatment abolished the malignant ascites completely; nearly 80% survived the full intended length of study (40 days). In contrast, all of the mice from the NS and UV-VSV-treated group died by day 40, before the treatment period was over. At the end of the treatment period, the mean volume of ascites in the control groups was 7.44±1.45 and 4.76±0.63 ml per mouse in the H22 and MethA model, respectively. The mean volume of ascites in the VSV-treated groups were 1.09±0.45 and 1.06±0.66 ml per mouse (P<0.05); ascites in several mice were abolished completely (Fig. 1B). The health condition of the control mice was poorer than that of the treated group and exhibited anemia, asthenia or asitia. VSV treatment significantly reduced the number of floating tumor cells and the red blood cells (P<0.05) in the peritoneal cavity (Fig. 1C). In the H22 and MethA models only 41.6±4.21×10⁶ and 23.43±3.37×10⁶ tumor cells and 21.6±4.21×10⁶ and 35.43±3.37×10⁶ red blood cells per milliliter were found in the mice treated with VSV, while in the controls and UV-VSV group there were nearly 2-fold more tumor cells and 8-fold more red blood cells than that in the VSV-treated group. Therefore, treatment of VSV in the malignant models reduced the tumor burden and improved the health condition of the mice. Differences between the VSV and UV-VSV group achieved statistical significance (P<0.05). These results suggest that administration of VSV within the peritoneal cavity was sufficient to suppress malignant ascite formation and prolong survival time. This higher level of inhibition suggests that VSV is efficient for the treatment of ascites particularly in the late phase.

VEGF is the most widely implicated growth factor involved in the initiation and progression of malignant ascite formation. We therefore measured VEGF (Fig. 1D) concentrations in plasma and ascites and found a significant decrease in the concentration levels in the treated mice. Compared to the values in the control group, plasma and ascite VEGF levels were significantly reduced. In the VSV-treated group total ascite VEGF levels decreased from 637.33±118.72 to 133.40±46.45 pg/ml (P<0.05) in the H22 group and 372.12±6.78l to 104.25±39.44 pg/ml (P<0.05) in the MethA group, respectively. Furthermore, the plasma VEGF level was significantly reduced from 257.72±43.26 in the control mice to 34.53±15.68 pg/ml (P<0.05) in the H22 group and from 175.45±26.76 in the control mice to 23.22±13.12 pg/ml (P<0.05) in the MethA group. Together, these results showed that VSV effectively blocks cancer and hypoxia-induced elevation of VEGF levels in these mice. This level of VEGF suppression is correlation with significant regression of disease within the peritoneal cavity.

To investigate the apoptotic effect of VSV on the malignant ascites cells, we treated ascites cells and cultured cells with VSV. We used flow cytometry to assess the percentage of sub-G1 phase cells in order to estimate the number of apoptotic cells. Cells from the ascites exhibited increased apoptosis compared to the cells cultured in a dish treated with VSV (Fig. 2A). In malignant ascites of late phase, we showed that VSV induced cell apoptosis by 64.2% in the cultured H22 cells whereas in ascites H22 cells the rate of apoptosis was 81.3% after 48 h of infection. Regarding the MethA cells, the rate of apoptosis was 60.2% in the cultured cells whereas the rate was 89.3% in the ascites cells. Furthermore, TUNEL assay was also carried out to detect early DNA fragmentation associated with apoptosis (Fig. 2B). Although the apoptotic effects of VSV were variable among the different cells, cells from ascites illustrated increased apoptosis. Taken together, these results suggest that VSV induces a high rate of apoptosis in cells from malignant ascites fluid.

To quantitatively analyze the expression of viral and cellular proteins, we performed viral infection at an MOI of 1.0 in the following experiments. We assessed the effect of VSV replication in the H22 and MethA cancer cells from the malignant ascites. H22 and MethA cells were infected with VSV-∆ 51-GFP as previously described. The expression of VSV-∆ 51-GFP in the H22 and MethA cells was examined using flow cytometry (Fig. 3A). We observed that VSV replication in malignant ascites H22 and MethA cancer cells was enhanced. Immunoblot analysis using anti-VSV serum also revealed higher levels of expression of VSV proteins in the cells from the ascites than that in cultured cells at 24 h after VSV infection (Fig. 3B). Twenty-four hours following infection, the number of VSV-infected cells increased 2- to 4-fold in the H22 cells from the malignant ascites and 2- to 4-fold in the MethA cells from the malignant ascites (Fig. 3C) compared to the cultured cells. These results indicate that the production of viral proteins and the viral replication were enhanced in cancer cells from the malignant ascites.

Hypoxia promotes the shift from OXP HOS to a glycolytic mode, leading to increased glucose capture and lactic acid production by tumor cells (anaerobic 'glycolysis'). Here, we aimed to gain further insight into the glycolytic activity of malignant ascites H22 cells and compare this activity to that of normoxic cultured cells. We first investigated the expression of genes involved in glycolytic metabolism such as hexokinase 2 (Hk2), PKM2 and lactate dehydrogenasea (LDHA) as well as glucose transporters such as glucose transporter 1 (Glut1). We also determined the expression of glutaminase GLS2, the key enzyme which catalyzes glutamate production from glutamine in a hypoxic condition. All glycolytic activity was standardized to that noted in the nomoxia dish-cultured cells. The ascites H22 cells exhibited a relatively higher level of expression of all these genes than levels noted in the control cells cultured under normoxia, resulting in high glycolytic activity and glutamine metabolism. Statistical significance was found for GLUT1, PKM2, Hk2, GLS2 and LDHA (P<0.05). Of note, GLUT1 and LDHA are known to be responsible for increased glucose uptake and consumption via anaerobic glycolysis but not oxidative phosphorylation. The result indicates that cancer cells in malignant ascites are highly glycolytic and the glucose and glutamine consumption are elevated (Fig. 4A).

It is known that viruses make use of cell nutrition for replication. We next assessed the three main carbon sources of hypoxia cells: glucose, glutamine and pyruvate. We then measured key metabolite consumption within the supernatants of the ascites or cultured H22 cells infected with VSV (MOI 1.0) or UVI-VSV at different times post-infection in the medium with indicated concentrations of glucose pyruvate lactate or glutamine (i.e., 0, 12, 24 and 48 h). Following infection with VSV, the consumption of glucose, glutamine and pyruvate was elevated because of virus replication (Fig. 4B). Interestingly, the consumption of glucose, pyruvate and glutamine in the VSV-infected ascites cancer cells was more rapid than that noted in the VSV-infected cultured cells. Moreover, the gap in consumption between the VSV infection and UVI-VSV infection in ascites cancer cells was much more obvious than that in the cultured cancer cells (data not shown).

Since pyruvate, glucose and glutamine are consumed for virus production, we hypothesized that glycolysis and glutamine metabolism are necessary for efficient viral replication. We investigated the impact of both glucose and glutamine deprivation on VSV replication. As expected, the replication of the virus was restrained in medium without glutamine or glucose, as shown in Fig. 5A, and replication of the virus was restored when glutamine or glucose was added. Thus, we next examined VSV replication following treatment with oxamate and 2-DG, a glycolytic inhibitor. Oxamate is a pyruvate analog that can block the conversion of pyruvate to lactate or acetyl-CoA (27). 2-DG, a glucose analog that inhibits hexokinase, is the first enzyme in the glycolytic pathway. We first investigated the effect of 2-DG on glycolysis in the ascites H22 cancer cells. Glucose uptake, ATP production and pyruvate production were increased (data not shown) in the ascites cancer cells. VSV-infected ascites H22 cells treated with oxamate or 2DG exhibited a decrease in the release of extracellular infectious virus (Fig. 5B). Notably, the replication of the VSV was restored, when the medium was supplemented with 4 mM pyruvate. These data indicate that glycolysis is a critical metabolic pathway for optimal VSV replication.

It has been shown that hypoxic cancer cells also use glutamine as a carbon fuel source for survival (28–30). Viruses require exogenous glutamine for efficient replication, and inhibition of glutamine metabolism blocks certain virus protein synthesis (14). We examined infectious virus replication following pharmacological inhibition of glutamine metabolism. VSV-infected cells were treated with BPTES to inhibit glutaminase, the key enzyme that catalyzes the first step of glutaminolysis, which converts glutamine to glutamate. Fig. 5C shows that blockage of glutaminolysis reduced viral replication. Moreover, virus production in the BPTES-treated cells was significantly recovered by α-ketoglutarate supplementation.