Cell viability and viral replication assays

For cell viability assay, the indicated cells were infected with oHSV (MOI of 0.1) pre-incubated with ascorbate buffer ± copper (1 mg/L) ± ATN-224 (32 uM) for 30 min at RT. 48 hr post infection, the cells were fixed with 1% glutaraldehyde, and stained with 0.5% crystal violet. After washing, the crystals were dissolved in Sorenson’s buffer (0.025 M sodium citrate, 0.025 mol/l citric acid in 50% ethanol) and plates were then read on a microplate reader at 590 nm. All assays were performed in triplicate. For replication assay, the indicated cells were harvested 48 hrs post oHSV infection, and the amount of infectious virus particles in cells and media was determined by performing a standard plaque forming unit assay on Vero cells, as previously described (10).

Animal Surgery

All mice experiments were housed and handled in accordance with the Subcommittee on Research Animal Care of the Ohio State University guidelines and have been approved by the Institutional Review Board. Female athymic nu/nu mice (Charles River Laboratoties, Frederick, MD), 4–5-week-old for subcutaneous tumor model and 6–8 week-old for intracranial tumor model, were used for all studies. For subcutaneous tumor studies, glioma cells (U251T3, 1.5 × 10⁷) were implanted subcutaneously into the rear flank of female athymic nude mice. When tumors reached an average size of 100 mm³, mice were randomized and treated with PBS or ATN-224 (0.7 mg/day dissolved in 100 ul PBS) by daily gavage (n=10). Thirteen days later PBS or rQnestin34.5 was administered intratumorally (1 × 10⁵ pfu). Tumor volume was calculated using the following formula: volume = 0.5LW² as described (27).

For intracranial tumor studies, anesthetized nude mice were fixed in a stereotactic apparatus, and a burr hole was drilled 2 mm lateral to the bregma. U87ΔEGFR cells (1 × 10⁵) were then implanted at a depth of 3 mm. 3 days post tumor implantation, mice were randomized to be fed with PBS or ATN-224 (0.7 mg/day dissolved in 100 µl PBS) by daily gavage. Ten days following tumor cell implantation, the mice were anesthetized again and injected intratumorally with PBS or 5 × 10⁴ pfu of rQnestin34.5 at the same location. Animals were observed daily and were euthanized at the indicated time points when they become moribund, lethargic, anorexic, dehydrated, or distressed.

For ex vivo serum rescue assays, female nude mice were fed with PBS/ATN-224 by daily gavage for ten days. Blood was collected from sub-mandibular vein and serum was harvested as described (28). 20 µl of serum diluted with 20 µl of Hank's buffered salt solution (HBSS) was incubated with rQnestin34.5 (2 × 10³ pfu) for 1 hr at 37 °C and the amount of virus particles was measured by a standard plaque assay. For in vivo serum rescue assay, mice were fed with PBS/ATN-224 for seven days and then hrR3 (2 × 10⁷ pfu) was administered by tail vein injection. 20 minutes post virus injection serum was harvested and the number of infectious virus particles present was determined.

PCR analysis

To measure viral gene copy in vivo, tumor bearing mice were sacrificed and total DNA from the tumors was purified (Master PureTM Complete DNA & RNA Purification Kit, Epicentre Biotechnologies), as manufacturer’s instruction. Viral gene copy present in the tumors was measured by determining the total number of copies of the HSV specific ICP4 gene using quantitative real-time PCR (qPCR) analysis. ICP4 primers used were: sense, 5’-CGACACGGATCCACGACCC-3’and anti-sense, 5’-ATCCCCCTCCCGCGCTTCGTCCG-3’. Total oHSV gene copy was determined by generating a linear regression curve using a plasmid containing the ICP4 of HSV-1 viral gene (Dr. Deborah Parris at The Ohio State University). To measure changes in angiogenic gene expression, subcutaneous glioma (U251T3) bearing mice were sacrificed and RNA was purified from the ipsilateral hemisphere using RNeasy lipid tissue (Qiagen, Cat. 75842). Changes in mouse and human angiogenic gene expression was measured using commercially available primers (murine VEGF, Cat# MP218181, human VEGF, Cat# HP202779, murine IL-8, Cat# MP206804, human IL-8, Cat# HP200599, Origene, Rockville, MD).

Antibodies

The following antibodies were used for immunohistochemistry: rat anti-mouse CD31 (Pharmingen, San Jose, CA), polyclonal rabbit anti-herpes simplex virus type 1 (DAKO, Cambridge, UK), Peroxidase-conjugated donkey anti-rabbbit (Jackson ImmunoResearch Laboratories, West Grove, PA) and biotin-conjugated goat anti-rat IgG (BD Biosciences Pharmingen, San Diego, CA).

DCE-MRI imaging

Mice bearing intracranial tumors treated with rQnestin34.5 ± ATN-224 (n=4) were imaged 3 days after oHSV treatment using T2-weighted RARE imaging sequence (TR=2500 ms, TE=12 ms, Rare Factor=8, navgs=4). The imaging was performed using a Bruker Biospin 94/30 magnet (Bruker Biospin, Karlsruhe Germany) as previously described (10). The mean C(t), IAUC, and CIAUC curves were evaluated and compared for the rQnestin34.5, and rQnestin34.5+ATN-224-treated mice at day 13 from tumor implantation. The rate constant, K_trans, and the extravascular extracellular space, v_e, were determined by nonlinear curve fitting the Gd concentration, C(t), over a 20 minute time period post Gd injection. Color-coded maps for K_trans and v_e were created to visualize the spatial distribution of Gd in the tumor. Histograms of K_trans and v_e for the entire tumors were calculated and median values were compared between the treatment groups.

Combination of ATN-224 and oHSV decreases blood vessel formation and vascular hyper-permeability in tumors

To evaluate the impact of ATN-224 on glioma angiogenesis, we evaluated tumor microvessel density in subcutaneous and intracranial tumors. Fig. 2A shows photomicrographs of U251T3 subcutaneous tumor sections immunostained for CD31 to highlight endothelial cells. Quantification of CD31 staining showed a statistically significant reduction in microvessel density in tumors treated with ATN-224 (Fig. 2B). More importantly, a significant inhibition in vessel density was also observed in tumors derived from mice treated with both ATN-224 and rQnestin34.5 compared to mice treated with rQnestin34.5 alone. ATN-224 has been previously shown to reduce the expression of angiogenic cytokines (29). To evaluate if ATN-224 reduced the expression of tumoral and host angiogenic cytokines in glioma bearing mice, we measured changes in both mouse and human angiogenic factors, VEGF and IL-8. Fig. 2C shows a significant down regulation of both tumoral and host VEGF and IL-8 factors when mice are treated with ATN-224 in addition to rQnestin34.5, as compared to mice treated with rQnestin34.5 alone.

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Figure 2

Antiangiogenic effect of ATN-224 on untreated and oHSV treated tumors

A) Representative (objective: 40×) photomicrographs of tumor sections stained with anti-CD31 antibody highlighting immunopositive endothelial cells (arrow heads). B) Quantification of mean microvessel density of tumors shown in A. *: P < 0.05 compared to PBS or rQnestin34.5 treated tumors. C) Impact of ATN-224 on tumoral and host cell derived VEGF and IL-8. Real time PCR analysis for changes in gene expression of angiogenic genes in tumor bearing hemispheres of mice three days after oHSV treatment. Data presented are fold changes in gene expression ± SD relative to GAPDH. *: P < 0.05 compared to PBS or rQnestin34.5 treated tumors.

To evaluate the physiological consequence of ATN-224 on perfusion in oHSV-treated brain tumors, we performed dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of intracranial glioma-bearing mice. Fig. 3A shows representative images of coronal brain sections of mice treated with rQnestin34.5 or ATN-224 plus rQnestin34.5. The top panel shows contrast enhancement of tumor-bearing mice imaged 3 days after the oHSV treatment and immediately following administration of gadolinium diethylene-triamine penta-acetic acid (Gd-DTPA). Contrast enhancement at the site of oHSV injection was evident in all of the oHSV-treated mice. The middle and bottom panels are parametric images showing changes in K_trans (blood-to-tissue transfer constant or vessel leakiness) and v_e (volume of contrast in the extravascular and extracellular space per unit volume of tissue) between PBS- and ATN-224-fed animals treated with rQnestin34.5 respectively. Quantification of the parameters revealed a significant reduction in both K_trans (60.4%, P=0.0399) and v_e (56.3%, P=0.0316) in ATN-224 plus rQnestin34.5-treated animals compared to rQnestin34.5 alone-treated animals (Fig. 3A–B). Interestingly in mice that were not treated with rQnestin34.5, ATN-224 did not alter K_trans or v_e (Supplementary Fig. S1). The reduction in vascular perfusion and leakiness indicated by MRI analysis was further investigated histologically. Fig. 3C are representative H&E- and CD31-stained images of tumor bearing brain sections, showing increased necrosis (asterix) and reduced vessel formation in mice treated with ATN-224 in addition to rQnestin34.5 compared to mice treated with rQnestin34.5 alone. Quantification of CD31 staining revealed the reduced vessel formation to be statistically significant (Fig. 3D).

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Figure 3

Changes in vascular perfusion in oHSV treated mice ± ATN-224

A) Contrast enhanced and color coded parametric images of coronal sections of mice three days after treatment with rQnestin34.5 ± ATN-224. Top panel shows Gd enhanced image (taken immediately after Gd injection) of one representative mouse/group imaged three days post treatment with rQnestin34.5. Middle panel shows color coded parametric images of K_trans in mice treated with rQnestin34.5 or rQNestin34.5 + ATN-224. Bottom panel shows color coded parametric images of v_e in mice treated with rQnestin34.5 or rQnestin34.5 + ATN-224. A shift towards red indicates a higher value. B) Quantification of changes in K_trans and v_e in oHSV treated tumors. Data shown are mean ± SD of four different mice. C) Representative photomicrographs of tumor sections stained with Hematoxylin and Eosin (H&E) and anti-CD31 antibody highlighting endothelial cells. D) Quantification of mean microvessel density of tumors shown in C. *: P < 0.05 compared to rQnestin34.5 treated tumors.

ATN-224 increases oHSV propagation in tumors

Interestingly, even though ATN-224 was found to be antiangiogenic in intracranial U87ΔEGFR tumors, ATN-224 treatment alone did not increase survival of these mice (Fig. 1B). To evaluate if increased efficacy was also due to increased oHSV propagation in vivo, we compared viral distribution of rQnestin34.5 in mice fed with PBS to mice fed with ATN-224. Fig. 4A shows representative microphotographs of HSV-1 immuno-stained brain sections of U87ΔEGFR intracranial tumor bearing mice. While tumors from mice treated with rQnestin34.5 showed some staining for virus in tumors, the entire tumor section was highly positive for HSV-1 staining in tumors when mice were fed ATN-224 (Fig. 4A). Similarly, HSV-1 staining of U251T3 subcutaneous tumors showed enhanced virus propagation when mice were fed ATN-224 compared to mice fed PBS (Fig. 4B). No staining was observed in tumors injected with PBS instead of oHSV (Supplementary Fig. S2). Consistent with increased HSV-1 staining, we found that the total viral yield in tumors of animals treated with ATN-224 plus rQnestin34.5 was about 3.31-fold higher than in mice treated with rQnestin34.5 alone (P=0.018) (Fig. 4C).

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Figure 4

Impact of ATN-224 on oHSV propagation in vivo

A–B): Representative photomicrographs of tumor sections stained with anti-HSV-1 antibody highlighting virus propagation in mice bearing U87ΔEGFR intracranial tumors (A) and U251T3 subcutaneous tumors (B). C) Subcutaneous tumors treated with rQnestin34.5 from mice fed PBS or ATN-224 (as described in methods) were harvested three days after oHSV treatment and the total number of infectious virus was measured by a standard plaque assay (n=6). Data shown are mean pfu/ml ± SE.

ATN-224 rescues copper-mediated inhibition of oHSV replication and cytotoxicity in vitro and in vivo

Apart from increasing angiogenesis, copper (Copper) has been shown to inhibit wild-type HSV-1 replication in cultured cells in vitro (16). Thus, we tested if ATN-224’s ability to chelate copper directly increases oHSV propagation. We first tested the effect of Copper on the ability of oHSV to replicate in vitro. Stable glioma cell lines, U251T3 and GLI36ΔEGFR, and a patient-derived glioma cell, X12-V2, were infected with rQnestin34.5 or rHSVQ1 at an MOI of 0.1 ± Copper. Uninfected cells were used as negative control. As both viruses encode for GFP, fluorescent microscopy was utilized to visualize oHSV-infected cells. A significant reduction in GFP positive cells was apparent when cells were infected in the presence of copper (Fig. 5A, and Supplementary Fig. S3A).

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Figure 5

Inhibition of oHSV efficacy by Copper and its rescue with ATN-224

Glioma cells were infected with rQnestin34.5 (MOI=0.1) and incubated with buffer ± Copper (1 mg/L). A) Fluorescence microscopic images of GFP positive Gli36ΔEGFR glioma cells. B) Percent cell survival of U251T3, X12-V2, and Gli36ΔEGFR was measured 48 hrs post infection with rQnestin34.5 incubated with or without copper by standard crystal violet assay. *: P < 0.05 compared to rQnestin34.5. C–E): ATN-224 rescues copper-mediated inhibition of oHSV infection, replication and glioma cell killing. C) Fluorescent microscopy images of GFP positive Gli36ΔEGFR glioma cells infected with rQnestin34.5 incubated with Copper ± ATN-224. D) Viral titers were measured in cells infected with rQnestin34.5 pre-incubated with or without Copper ± ATN-224, 48 hrs post infection. *: P < 0.05 compared to rQnestin34.5 + Copper. E) Survival of cells infected with rQnestin34.5 pre-incubated with or without Copper ± ATN-224, 48 hrs post infection. *: P < 0.05 compared to rQnestin34.5 + Copper.

Along with reduced infection, both rQnestin 34.5 and rHSVQ1 demonstrated significantly reduced glioma cell killing when the viruses were pre-incubated with copper (Fig. 5B, and Supplementary Fig. S3B). Next, we investigated the ability of ATN-224 to rescue copper-mediated inhibition of oHSV by fluorescent microscopy and by measuring viral replication and cytolysis (Fig. 5 C–E). Fluorescent microscopy of GFP positive cells showed that ATN-224 treatment could rescue Copper-mediated inhibition of glioma cell infection (Fig. 5C). More significantly, ATN-224 treatment completely reversed the Copper-mediated inhibition of oHSV replication and oncolysis (Fig. 5D–E). ATN-224 treatment in the absence of Copper has no significant effect on the proliferation of glioma cells in vitro or the ability of rQnestin34.5 to kill glioma cells (Supplementary Fig. S4).

To examine the physiological relevance of these results, we investigated the effect of ATN-224 on oHSV replication in ex vivo and in vivo models. Serum from mice fed ATN-224 or PBS was incubated with rQnestin34.5 for 30 minutes and the ability of the virus to form plaques on vero cells was measured by a standard plaque assay. Fig. 6A, and Supplementary Fig. S5 shows an increase in GFP positive infected cells treated with virus incubated with serum obtained from ATN-224 treated mice compared to control mice. Consistent with this, quantification of oHSV showed a significant increase in survived infectious virus particles in samples treated with serum obtained from ATN-224 fed mice compared to control mice (Fig. 6B). To examine if the serum stability of oHSV was increased in vivo, mice fed with ATN-224 or PBS were injected with a single dose of oHSV (hrR3; 2 × 10⁷) via tail vein. Twenty minutes post viral injection, mouse serum was harvested to evaluate the amount of infectious virus particles. Fig. 6C shows a significant increase in the number of virus particles present in serum from ATN-224 treated mice compared to that from PBS-treated control mice (P<0.01). Collectively these results suggested that ATN-224 treatment increased the serum stability of oHSV in vivo.

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Figure 6

Increased oHSV viability (stability) in ATN-224 treated mice serum

A–B) Ex vivo serum rescue assay. Serum from mice fed PBS or ATN-224 was collected and incubated with rQnestin34.5 for 1 hour at 37 °C (n=4/group). A) Fluorescent microscopy images of GFP positive cells infected with rQnestin34.5 incubated with serum from animals fed PBS or ATN-224. B) Quantification of infectious viral plaque forming units after incubation with serum from mice fed PBS or ATN-224. Data shown are the mean viral titers obtained ± SD (n=4/group). *: P < 0.05 compared to PBS. C) Tumor bearing mice fed PBS or ATN-224 by daily gavage, were administered oHSV (hrR3) via tail vein injection. Total virus in serum was measured by a standard plaque assay. Data shown are the mean pfu/ml in serum of mice ± SD (n=6/group). *:P < 0.05 compared to PBS. D) ATN-224 treatment increased systemic delivery of oHSV. Mice with subcutaneous U251T3 tumors were fed PBS or ATN-224 by daily gavage. hrR3 was administered i.v. and tumors were analyzed for viral gene copy. Data shown are mean HSV-1 gene copy/mg of tumor DNA ± SE. *: P < 0.05 compared to PBS. E) Representative photomicrographs of tumor sections stained with anti-HSV-1 antibody highlighting virus propagation in vivo. F) Antitumor efficacy of oHSV is increased in mice bearing subcutaneous U251T3 tumors fed ATN-224 compared to PBS. *: P < 0.05 compared to oHSV.

We would like to thank Dr Qintin Pan for helpful suggestions and advice for the use of ATN-224 in animals.

Grant Support

This work was supported by funding from the National Institutes of Health grant (1R01NS064607, and 1R01CA150153 to B.K.; 5R01CA119298-5 to JCG.).