¹Department of Neurosurgery and Clinical Neuroscience, Kyoto University Graduate School of Medicine, Kyoto, Japan

² Department of Neurosurgery, Georgetown University Medical Center, Washington DC, USA

³Department of Ophthalmology and Visual Sciences, Kyoto University Hospital, Kyoto, Japan

⁴ Department of Microbiology and Immunology, Georgetown University Medical Center, Washington DC, USA

^aCorrespondence: SD Rabkin, Department of Neurosurgery, Georgetown University Medical Center, 3970 Reservoir Road NW, Washington, DC 20007, USA

Targeting viral vectors to appropriate cell types so that normal cells are not adversely affected is an important goal for gene therapy. Previously, we described a novel approach to viral gene therapy using a conditional, replication-competent herpes simplex virus (HSV), where replication and associated cytotoxicity are limited to a specific cell-type by the regulated expression of an essential immediate-early viral gene product. In this report we analyze the hepatoma-specific replication, cytotoxicity and anti-tumor effect of recombinant HSV G92A, regulated by the albumin enhancer/promoter. G92A efficiently replicated in vitro in two human hepatoma cell lines expressing albumin, but not in four human non-hepatoma, albumin-non- expressing tumor cell lines, while all cell lines were equally susceptible to a tissue nonspecific HSV recombinant, hrR3. In vivo, G92A replicated well in subcutaneous xenografts of human hepatoma cells (Hep3B) in athymic mice, but not in non-hepatoma subcutaneous tumors (PC3 and HeLa), whereas, hrR3 replicated well in both tumor types. Intratumoral inoculation of G92A inhibited the growth of established subcutaneous hepatoma tumors in nude mice, but not prostate tumors. Replication-competent viral vectors controlled by cell-specific transcriptional regulatory sequences provide a new therapeutic strategy for tumor therapy.

HSV; hepatocellular carcinoma; transcriptional regulation; virus replication; albumin enhancer

Introduction

A number of approaches are being developed that may be effective for cancer treatment using gene therapy. Most of them involve the transfer of genes, such as suicide genes,^1,2,3 cytokine genes,^4,5 or tumor suppressor genes,^6,7 to tumor cells using replication-defective vectors such as recombinant viruses, naked DNA, liposomes, etc. Replication-competent vectors have advantages over replication-defective vectors for cancer gene therapy, if replication can be safely controlled and confined to the target tumor cells. Examples of such viral vectors include: an attenuated, multi-mutated herpes simplex virus (HSV) that replicates in brain tumor cells but not normal brain,^8,9 and an adenovirus mutant (E1B^-) that selectively replicates in p53-deficient human tumor cells.¹⁰ Other types of tissue- or cell-specific targeting of vectors will be important for the success of cancer gene therapy. Tissue-specific targeting of retrovirus vectors has been demonstrated using chimeric envelope proteins to generate new viral ligand-receptor interactions.^11,12,13 In addition, tissue-specific regulatory sequences have been used to drive expression of therapeutic genes in replication-defective viral vectors.^13,14,15,16 In a similar strategy as described here, a prostate-specific replication-competent adenovirus has been constructed using a prostate-specific antigen (PSA) enhancer/promoter to drive expression of the adenovirus E1A gene and consequent viral replication.¹⁷

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, especially in Asia and Africa.^18,19 Surgical resection is the therapy of choice with a 5-year survival of up to 45%.²⁰ However, curative surgery is not practical in the majority of cases because of the association with liver cirrhosis, usually due to chronic viral hepatitis B and C and recurrent disease. Other therapeutic options, including liver transplantation, chemotherapy, transarterial chemoembolization and percutaneous ethanol injection, are mostly palliative with overall survival rates remaining low.^20,21,22

HSV exhibits a broad host range and can efficiently destroy infected target cells leading to a spreading infection. One approach to the use of HSV vectors for cancer gene therapy is to attenuate the virus, reducing its toxicity to normal tissue. A number of HSV mutants, where viral genes contributing to neurovirulence have been deleted or inactivated, have been used for tumor therapy in animal models.^{8,9,23,24,25,26,27,28,29} We describe an alternative approach to virus-mediated gene therapy, where HSV replication and associated cytotoxicity are limited to a specific cell-type by the regulated expression of the HSV essential immediate-early gene product, ICP4.³⁰ A recombinant HSV vector was constructed that was regulated by the albumin enhancer/promoter (e/p) and termed G92A.³⁰ G92A contains a deletion in both copies of the ICP4 gene and an insertion of a chimeric transgene (albumin e/p-ICP4) in the thymidine kinase (tk) gene. To identify cells containing replicating virus easily, a second transgene (TK promoter-E. coli lacZ) was also inserted in the tk gene. G92A is derived from d120 which, in addition to the ICP4 deletions,³¹ contains a second mutation in the US3 gene.³² The US3 gene encodes a protein kinase that blocks virally induced apoptosis³² and viral mutants lacking US3 have greatly reduced neuropathogenicity.³³

The plaquing ability of G92A is over 1000 times higher on albumin-expressing human hepatoma cells than on nonexpressing human cells, however growth of G92A was much slower than that of wild-type HSV.³⁰ In this study we examined whether this in vitro cell specificity would be maintained in vivo in a subcutaneous tumor model and whether G92A would be therapeutically efficacious and safe.

Results

G92A replication in human hepatoma and non-hepatoma cells in vitro

The ability of G92A to replicate specifically in albumin-expressing hepatoma cells was examined using a viral single-step growth experiment in two human hepatoma and four non-hepatoma tumor cell lines. Human Hep3B and HuH7 hepatoma cells express albumin.¹⁴ Infection of either hepatoma or non-hepatoma cells with wild-type, parental HSV-1 strain KOS resulted in similar viral replication kinetics and virus yield, whereas no significant viral burst was detected in the population of G92A-infected non-hepatoma cells (Figure 1 HeLa, Caki-2, PC3, T24). The viral yield was about 100-fold less than the input virus and this may represent a very small number of cells in the population that are somewhat permissive to G92A replication. G92A-infected Hep3B and HuH7 cells produced a significant viral burst, with lower virus yield and somewhat delayed replication compared with KOS-infected cells (Figure 1). These data, in combination with the results obtained previously with non-albumin expressing human SW480, MCF7 and Det551 cells,³⁰ demonstrates the distinct target cell specificity of G92A to albumin-producing hepatoma cells in vitro.

Target cell specificity in vitro

We assessed the ability of G92A to spread and destroy target cells in a growing monolayer. In contrast to plaque assays, virus spread was not limited to direct cell-cell contact and cells were kept growing with 10% IFCS. HrR3 was used as a tissue nonspecific HSV. Both G92A and hrR3 express -galactosidase, with the lacZ gene under control of the HSVtk promoter and HSV ICP6 promoter, respectively. We found that the expression of -galactosidase was somewhat greater in hrR3-infected cells than in G92A-infected cells when assayed by X-gal intensity, likely due to differences in promoter regulation and ICP4 expression. Both hepatoma cell types were efficiently destroyed by G92A and hrR3 (Figure 2 A-F), while the three non-hepatoma cells were destroyed only by hrR3 (Figure 2G-O). No X-gal-positive cells were observed after G92A infection of Caki-2, and a few X-gal-positive cells were observed after G92A infection of PC3 and HeLa cells. However, there was no cell destruction in non-hepatoma cells infected with G92A even on day 5, whereas all target cell types were almost completely destroyed by hrR3 on day 5 (data not shown).

Virus spread in the subcutaneous tumor nodule in nude mice

Virus spread in vivo was detected by X-gal staining of infected s.c. tumor nodules, because lacZ expression in G92A-infected cells is dependent upon ICP4 expression. HrR3 showed extensive virus spread in Hep3B, as well as PC3 and HeLa tumor nodules (Figures 3 and 4c, f and i). G92A spread well in vivo only in Hep3B tumor nodules and not at all macroscopically in PC3 or HeLa tumor nodules even with a three times higher dose of virus (Figure 3b and c). Microscopic examination of sections from tumor nodules inoculated with 3 ´ 10⁷ p.f.u. of G92A revealed only two blue cells in a PC3 tumor nodule (Figure 4e) and several blue cells along the needle track of a HeLa tumor nodule (Figure 4h). No blue cells were seen in PC3 tumor nodules inoculated with 1 ´ 10⁷ p.f.u. of G92A.

G92A inhibition of tumor growth in vivo

Athymic BALB/c (nu/nu) mice harboring s.c. Hep3B tumor nodules of similar size (5-8 mm in maximal diameter) were inoculated intraneoplastically with 3 x 10⁷ pfu of G92A in 0.06 ml virus buffer or with virus buffer alone for mock. Some tumor nodules were re-inoculated on day 6. Mean tumor volume, as measured by external caliper, was significantly reduced in tumors treated with G92A (both single and double injections) compared with the mock infection (P < 0.0001, ANOVA followed by Scheffe's analysis at 4 weeks after treatment) (Figure 5 left, Hep3B). Two out of six tumor nodules in the G92A-single injection group and five out of five tumor nodules in the G92A-double injection group had smaller tumor nodules at 4 weeks after treatment than at day 0, before treatment. Two of the five tumor nodules in the G92A-double injection group were 'cures' with no tumor growth 3 months after treatment. The G92A treated Hep3B tumors had marked necrotic areas. G92A did not show any antitumor activity to PC3 tumor nodules in nude mice (no statistical significance between tumor growth ratio of mock and G92A-treated nodules, P = 0.96) (Figure 5 right, PC3), whereas PC3 tumors were susceptible to hrR3 tumor growth inhibition (data not shown).

Lack of G92A virulence in vivo

Albumin is highly expressed in normal hepatocytes.³⁴ It was therefore important to determine whether G92A would be virulent when injected directly intrahepatically in young mice. None of the G92A treated s.c. tumor-bearing animals exhibited any signs of HSV disease. Injection of the parental HSV-1 strain KOS (10⁵ p.f.u.) into the livers of susceptible BALB/c mice resulted in numerous symptoms of HSV infection within 2 days of injection, including lethargy, decreased hind limb movement and food intake. In non-survivors, mortality occurred within 6 days of KOS infection (Table 1). Infectious virus could be isolated from the livers of deceased animals (data not shown). In comparison, animals injected intrahepatically with G92A (1 or 7 ´ 10⁶ p.f.u.) exhibited no symptoms of disease and were healthy and alive 75 days after infection (Table 1).

Discussion

In this report we demonstrate the cell type-specific replication, viral spread and associated oncolytic activity of G92A to albumin-expressing human hepatoma tumors in nude mice. G92A efficiently suppressed the s.c. tumor growth only of hepatoma cells in vivo and not non-hepatoma tumor nodules. In a previous report, we demonstrated that G92A replicated well in vitro in three human albumin-expressing hepatoma cell lines but not in human albumin non-expressing cells MCF7 (breast adeno- carcinoma), SW480 (colon adenocarcinoma) and Detroit 551 (diploid fibroblast cells).³⁰ We have been unable to generate s.c. tumors reproducibly in nude mice using HepG2, MCF7 or SW480 cells. To generate a human albumin non-expressing s.c. tumor model, we examined human non-hepatoma tumor cell lines, PC3, HeLa and Caki-2, which are known to be tumorigenic in nude mice.^35,36,37 These three non-hepatoma tumor cell lines are susceptible to HSV infection, including wild-type KOS and mutant hrR3, while G92A is unable to replicate or spread in these cells. These studies were done at low multiplicity of infection (MOI) in human tumor cells, as opposed to other studies that demonstrated cytotoxicity of d120 (the parental virus of G92A) in vitro at high MOI in normal human cells.^38,39 It is possible that G92A infected non-hepatoma cells were killed but not detected in the assays we used. However, for this tumor therapy paradigm, the spread of virus rather than individual cell death is a critical feature. These studies extend the number of human non-hepatoma cell lines that G92A is unable to replicate in to seven, while it efficiently replicated in all three human hepatoma cell lines.

We and others have used hrR3, which contains a lacZ insertion in the ICP6 gene,⁴⁰ as an attenuated, replication-competent HSV vector for tumor therapy.^25,41,42 Potential safety concerns about hrR3 are likely to limit its clinical application. In a disseminated brain tumor model, intrathecal inoculation of hrR3 was found to cause marked neurologic morbidity and vector-related mortality.⁴³ HSV ribonucleotide reductase mutants (ICP6^-) have greatly reduced pathogenicity in adult mice,^44,45 however, they do form productive lesions in guinea pigs and significant virulence in newborn mice.^45,46

In previous studies, promoter elements inserted into the HSV-1 genome were affected by the regulatory properties of surrounding HSV-1 sequences.^47,48,49 There are several possible reasons why G92A demonstrated cell type-specific replication and therefore hepatoma-specific regulation of the ICP4 gene. As we described previously,³⁰ the tissue-specific regulatory element (albumin e/p) drives expression of ICP4, an essential immediate-early gene that is required for transactivation of HSV early and late genes. Thus, other HSV promoters are theoretically silent in non-permissive (albumin non-expressing) conditions. In addition, the 5' region of the albumin e/p was flanked by the SV40 polyadenylation sequence, which may suppress nonspecific expression. It has been reported that addition of the SV40 polyadenylation signal 5' to the tissue-specific -fetoprotein (AFP) promoter in recombinant adenovirus, suppressed the expression of a marker gene in non AFP-expressing cells, and suppressed the low level of expression in a promoterless construct.⁵⁰

This strategy of using tumor cell-specific promoters and/or enhancers may be applicable to many types of neoplasm. There are several criteria important for the successful application of a tissue-specific regulatory sequence: (1) The sequence should have a restricted cell specificity that is tightly regulated; (2) regulation is at the level of transcriptional initiation; (3) transcription should be limited to tumor cells, with as few other sites of expression as possible. In this respect, the AFP promoter may be a better choice than the albumin e/p for the treatment of hepatocellular carcinoma. AFP expression is often highly elevated in hepatocellular carcinoma and to a lesser degree in liver cirrhosis,^51,52 but is not expressed in normal adult liver,⁵³ as is the case with albumin; and (4) the promoter should be strong enough to drive sufficient ICP4 expression. The data of Huber et al¹⁴ indicated that the albumin e/p was more potent than the AFP promoter, which was one reason why we used the albumin e/p instead of the AFP promoter to construct G92A. Recently Rodriguez et al¹⁷ applied a similar strategy to recombinant adenovirus. They replaced the adenovirus E1A promoter with a minimal human prostate-specific antigen (PSA) e/p to generate an attenuated replication-competent adenovirus which grew significantly better in human PSA expressing than non-expressing prostate cancer cells.

The chimeric ICP4 transgene was inserted in the tk locus in d120, which makes G92A tk^-. The reasons for inserting the transgene into the tk locus include: easy selection of recombinant tk^- mutants with the aid of ganciclovir, HSVtk^- mutants grow poorly in nondividing cells^24,54,55 and have reduced neurovirulence.^33,56 Replication of HSVtk^- mutants in mouse liver was reported to be much less than wild-type HSV, except after partial hepatectomy when liver cells are replicating.⁵⁷ From a safety viewpoint, deletion of tk is a disadvantage because of the loss of sensitivity to commonly used antiviral drugs such as acyclovir.

G92A contains the E. coli lacZ gene regulated by an E HSV-1 promoter (tk). LacZ expression was for the most part only detected in those infected cells where the tk promoter would be active, which is during G92A replication. Therefore X-gal staining, to detect lacZ expression, was used to identify cells undergoing active G92A replication both in vitro and in vivo. Other genes, such as those encoding immune modulatory proteins, could be inserted in place of or in addition to lacZ to enhance antitumor efficacy, as recently demonstrated by Andreansky et al.⁵⁸ G92A could also be used as a helper virus in combination with defective HSV vectors expressing immune modulatory or 'suicide' genes which might have advantages over recombinant vectors alone.^59,60

These studies were performed in immune-deficient athymic mice, however in the clinic, patients are immunocompetent even if immunosuppressed. An active immune system could be beneficial to G92A therapy if it resulted in a local inflammatory response and an antitumor immune response, or it could be detrimental if the anti-HSV immune response were to limit the replication and spread of G92A in the tumor. Intratumoral injection of an attentuated, replication-competent HSV vector, G207, induces a systemic tumor cell-specific antitumor immune response⁶¹ and such HSV vector-mediated responses can be augmented by the expression of various cytokines.⁶⁰ We have found that prior immunization of mice with HSV does not affect the efficacy of G207- mediated antitumor therapy (Chahlavi et al, unpublished data). This suggests that treatment of hepatomas with G92A in immunocompetent animals should be similar or more effective than was seen in the human xenografts described here. It will be of interest to determine whether the immune system limits the replication and spread of G92A in tumors, and what contribution the immune response versus the cell specificity of viral replication plays in limiting the pathogenesis of G92A.

The parental virus d120, and presumably G92A, also contains a mutation in the US3 gene.³² A US3^- HSV-2 mutant was reported to replicate normally in vitro but had greatly reduced virulence (>10⁴-fold) after intraperitoneal inoculation which was associated with restricted replication in the liver.⁶² Direct inoculation of G92A into the livers of BALB/c mice had no effect on the overall health and survival of these animals, whereas the same or lower doses of wild-type HSV-1 KOS was rapidly and uniformly lethal. This suggests that the mutations in tk and/or US3 attenuate G92A replication and pathogenicity in the liver, but not in hepatoma tumors. As with any potentially pathogenic, replication-competent viral vector, safety is an important concern. Although HSV hepatitis is a rare clinical manifestation of HSV infection, usually associated with impaired immunity such as during organ transplant, it has a high mortality.^63,64 This strategy of targeting viral growth and spread to specific tumor cell types, in a non-virulent virus backbone provides new opportunities for tumor therapy.

Materials and methods

Cells and viruses

Human hepatoma cell lines Hep3B and HuH7 were grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% heat-inactivated FCS (IFCS) (Hyclone, Logan, UT, USA). Human non-albumin-expressing tumor cell lines, HeLa (cervical epitheloid carcinoma), Caki-2 (renal clear cell carcinoma), and T24 (bladder carcinoma) were cultured in the same medium and PC3 (prostate cancer) was grown in RPMI 1640 supplemented with 10% IFCS. African green monkey kidney cells, Vero and E5, were grown in DME supplemented with 10% newborn calf serum (Hyclone). Hep3B, HeLa and Vero cells were obtained from American Type Culture Collection (Rockville, MD, USA), HuH7 cells were kindly supplied by J Gerin (Georgetown University Medical Center, Washington, DC, USA) and other urological tumor cell lines were kindly supplied by Dr Y Mizutani (Department of Urology, Kyoto University, Kyoto, Japan). E5 cells are ICP4 complementing cells derived from Vero⁶⁵ and were kindly supplied by N DeLuca (University of Pittsburgh School of Medicine, Pittsburgh, PA, USA). Viral stocks of wild-type HSV-1 strain KOS, obtained from D Knipe (Harvard Medical School, Boston, MA, USA), and ICP6^- recombinant hrR3 (parental strain KOS), obtained from S Weller (University of Connecticut Health Center, Farmington, CT, USA), were generated from low multiplicity infections of Vero cells. G92A (parental strain KOS) was generated from low multiplicity infection of Hep3B cells as described previously.³⁰ The structures of G92A and hrR3 have been described in detail.^30,40 Virus was prepared from infected cells by freeze-thaw/sonication, low-speed centrifugation, ultracentrifugation of supernatant and resuspension of virus pellet in virus buffer (150 mm NaCl/20 mm Tris pH 7.5).⁸

Cell destruction assay in vitro

Hep3B, HuH7, PC3, HeLa and Caki-2 cells (10⁵) were plated in six-well dishes (Falcon, Becton Dickinson, Franklin Lakes, NJ, USA), 24 h before virus infection. The cells were infected with G92A or hrR3 at an MOI of 0.07 in 0.7 ml PBS supplemented with 1% IFCS. Virus inoculum was removed after 60 min and the cells incubated in DME supplemented with 10% IFCS at 37°C in humidified 5% CO₂. At the times indicated, the cells were fixed (0.5% glutaraldehyde/2% formaldehyde) and histochemically stained in X-gal solution (1 mg/ml 5-bromo-4-chloro-3-indolyl--d-galactopyranoside (X-gal), 5 mm potassium ferricyanide, 5 mm potassium ferrocyanide, 2 mm magnesium chloride) at 37°C for several hours.

Single-step viral growth

Monolayer cultures of 2 ´ 10⁵ cells in 12-well dishes (Falcon) were infected with HSV-1 strain KOS or G92A at a MOI of 1.0 in 0.5 ml PBS supplemented with 1% IFCS. Virus inoculum was removed after 60 min and the cells incubated in DME supplemented with 1% IFCS at 37°C in humidified 5% CO₂. At the times indicated, virus was harvested from the wells and titrated on E5 (ICP4⁺) cells. Plaques were counted and expressed as p.f.u./ml.

Virus spread in vivo

Ten-week-old BALB/c-nu/nu athymic mice were kept in groups of five or less and had free access to food and water. All animal procedures using tumor implants were approved by the Institute of Laboratory Animals, Faculty of Medicine, Kyoto University. For subcutaneous (s.c.) tumor nodule formation, mice were injected with: 5 ´ 10⁶ Hep3B cells in the flank subcutaneously and approximately 2 to 3 weeks later, tumor nodules of 5 to 8 mm in maximal diameter were obtained; or 2 ´ 10⁷ PC3 cells in the flank subcutaneously and approximately 4 to 6 weeks later, tumor nodules of 5 to 8 mm in maximal diameter were obtained in about 70% of the animals; or 1 ´ 10⁷ HeLa cells in the flank subcutaneously and approximately 4 to 6 weeks later, tumor nodules of 5 to 10 mm in maximal diameter were obtained in about 80% of the animals. Mice were anesthetized with an i.p. injection of a 0.25-0.3 ml solution consisting of 84% bacteriostatic saline, 10% pentobarbital (1 mg/kg; Abbott Laboratories, Chicago, IL, USA) and 6% ethyl alcohol for all surgical procedures. To measure virus spread in vivo, s.c. tumors (approximately 7-10 mm in maximal diameter) were inoculated with 1 or 3 ´ 10⁷ p.f.u. of G92A or hrR3 in 60 l virus buffer or virus buffer alone for mock. Six days later, the tumor nodules were removed, fixed with 2% paraformaldehyde, 5 mm EGTA, 2 mm MgCl₂ in 0.1 m Pipes buffer (pH 7.3), histochemically stained in X-gal solution with 0.02% NP-40 and 0.01% sodium deoxycholate, and 20 m sections cut by cryostat.

Subcutaneous tumor therapy

For the s.c. tumor growth studies, mice harboring s.c. tumors (approximately 5 to 8 mm in maximal diameter) were randomly divided and intraneoplastically inoculated with 3 ´ 10⁷ p.f.u. of G92A in 60 l virus buffer or virus buffer alone for mock. Where indicated, tumors were re-inoculated a second time on day 6. Tumor nodules were injected with multiple trajectories. Tumor size was measured by external caliper and the volume calculated as (a ´ b²)/2, where a is the maximum length of the tumor nodule and b is the length perpendicular to a.^10,66 Tumor growth ratio was determined by volume_dayX/ volume_day0. Statistical differences in growth ratio were assessed by analysis of variance.

In vivo virulence

Four to 5-week-old female BALB/c mice were obtained from the National Cancer Institute (Rockville, MD, USA). These animal procedures were approved by the Georgetown University Animal Care and Use Committee. Mice were anesthetized as above and a 1.5 cm subcostal incision made to divide the peritoneum and expose the liver. Injections (30 or 100 l) were made into the right hepatic lobe with a Hamilton syringe (Reno, NV, USA) and 30-gauge needle. Evidence of intrahepatic inoculation was seen as a brief blanching of the hepatic tissue upon inoculation. The incision was closed and animals followed.

Acknowledgements

We thank N Deluca, J Gerin, D Knipe, S Weller and Y Mizutani for providing reagents, I Saito and E Hatano for helpful discussions, and T Toda for assistance with the liver injections. This work was supported in part by a Grant-in-Aid for Scientific Research (07671513) from the Japanese Ministry of Education, Science and Culture and grant from the Takeda Science Foundation (to S-IM), and National Institutes of Health grant NS32677 (to RLM).

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Figure 1 Single-step growth curve for G92A and KOS in hepatoma and non-hepatoma tumor cells. Monolayers of cells in 12-well dishes, were infected with KOS () or G92A () at MOI of 1.0 and at the times after infection indicated, virus was harvested from the wells and titrated on E5 (ICP4⁺) cells. The virus yield (p.f.u.) per well of infected cells was determined. The target cell type is indicated at the top of each graph. The initial decrease in viral titer is due to the eclipse phase of infection. KOS showed marked replication irrespective of cell type, but G92A showed definitive replication only in hepatoma cells, with the virus yield on non-albumin-expressing cells about two logs less than input.

Figure 2 In vitro cell destruction assay. Target cells (10⁵) were plated in six-well dishes 24 h before virus infection. The cells were infected with G92A (B, E, H, K, N) or hrR3 (C, F, I, L, O) at MOI of 0.07 or with virus buffer (A, D, G, J, M). Infected cells were fixed on day 3 after infection and histochemically stained with X-gal (black precipitate). The cells were Hep3B (A, B, C), HuH7 (D, E, F), PC3 (G, H, I), HeLa (J, K, L), and Caki-2 (M, N, O). HrR3 demonstrated marked cell destruction irrespective of cell type, whereas G92A demonstrated replication and cell destruction only in hepatoma cells.

Figure 3 Macroscopic virus spread in the subcutaneous tumor nodule in nude mice. Palpably growing (approximately 7-10 mm in maximal diameter) s.c. tumor nodules (Hep3B, PC3 and HeLa) were inoculated with 1 or 3 ´ 10⁷ p.f.u. of G92A or hrR3. For mock infection, the same volume of virus buffer was used. Six days later, the nodules were removed and histochemically stained with X-gal. G92A could spread well only in the Hep3B tumor nodule and not at all macroscopically in PC3 and HeLa even with a three times higher dose of the virus. HrR3 showed effective virus spread in Hep3B, PC3 and HeLa tumor nodules. Bar, 5 mm. (a) Hep3B. Left is a nodule infected with mock, middle with G92A (1 ´ 10⁷ p.f.u.) and right with hrR3 (1 ´ 10⁷ p.f.u.). In the tumor nodule infected with G92A, there are two distinct blue areas (X-gal stain) at the top and middle. (b) PC3. Upper left is a tumor nodule infected with G92A (1 ´ 10⁷ p.f.u.), upper right with G92A (3 ´ 10⁷ p.f.u.), lower left with mock and lower right with hrR3 (1 ´ 10⁷ p.f.u.). (c) HeLa. Left is a tumor nodule infected with G92A (3 ´ 10⁷⁷ p.f.u.), middle with mock and right with hrR3 (1 ´ 10⁷ p.f.u.).

Figure 4 Microscopic virus spread in the subcutaneous tumor nodule in nude mice. Inoculated Hep3B (a, b, c), PC3 (d, e, f) and HeLa (g, h, i) tumor nodules were sectioned after X-gal staining and counterstained with nuclear fast red. Tumors were inoculated with virus buffer (a, d, g), 1 ´ 10⁷ p.f.u. of G92A (b), 3 ´ 10⁷ p.f.u. of G92A (e, h) or 1 ´ 10⁷ p.f.u. of hrR3 (c, f, i). HrR3 showed effective virus spread in Hep3B, PC3 and HeLa tumor nodules. G92A could spread well only in the Hep3B tumor nodule, with only several X-gal-stained cells (blue) in the PC3 and HeLa tumor nodules.

Figure 5 Tumor growth inhibition of G92A to Hep3B and PC3 in nude mice. Athymic BALB/c (nu/nu) mice harboring s.c. Hep3B or PC3 tumor nodules (5-8 mm in maximal diameter) were treated intraneoplastically with either 3 ´ 10⁷ p.f.u. of G92A in 0.06 ml virus buffer or with virus buffer alone for mock (day 0). Some mice were re-inoculated with 3 ´ 10⁷ p.f.u. of G92A on day 6. Bars indicate standard error. Analysis of variance followed by Schaffe's analysis was performed on the tumor growth ratio at 4 weeks after treatment. (a) Hep3B. The number of inoculated tumor nodules was: seven for mock, six for G92A (single inoculation) and five for G92A (double inoculation). Mean tumor volume, measured by external caliper, was significantly reduced in tumors treated with G92A (both single and double injection) compared with the mock infection (P = 0.0001). (b) PC3. The number of inoculated tumor nodules was: six for mock and seven for G92A. G92A did not show any antitumor activity against PC3 tumor nodules (P = 0.96, between mock and G92A-treated tumor growth ratio).