Liver Biology and Pathobiology
Eradication of advanced hepatocellular carcinoma in rats via repeated hepatic arterial infusions of recombinant VSV†
Article first published online: 23 DEC 2004
Copyright © 2004 American Association for the Study of Liver Diseases
Volume 41, Issue 1, pages 196–203, January 2005
How to Cite
Shinozaki, K., Ebert, O. and Woo, S. L.C. (2005), Eradication of advanced hepatocellular carcinoma in rats via repeated hepatic arterial infusions of recombinant VSV. Hepatology, 41: 196–203. doi: 10.1002/hep.20536
Conflict of interest: Nothing to report.
- Issue published online: 23 DEC 2004
- Article first published online: 23 DEC 2004
- Manuscript Accepted: 2 NOV 2004
- Manuscript Received: 26 AUG 2004
- National Institutes of Health. Grant Number: CA100830
- Deutsche Forschungsgemeinschaft. Grant Number: EB234/1-1
Viruses that replicate selectively in cancer cells hold considerable promise as novel therapeutic agents for the treatment of malignancy. Vesicular stomatitis virus (VSV) is a nonpathogenic RNA virus with intrinsic oncolytic specificity due to attenuated antiviral responses in many tumors. We report that repeated hepatic arterial infusion of recombinant syncytia-forming VSV vector in advanced multifocal hepatocellular carcinoma (HCC)-bearing rats at a 10-fold reduced vector dose resulted in sustained tumor-selective virus replication until the onset of high-titer neutralizing antibodies in blood. No significant elevations in serum transaminases and liver pathology were noted, indicating a lack of hepatotoxicity. Substantially improved tumor response was achieved with completely necrotic tumor nodules surrounded by mononuclear phagocytic cells, followed by fibrosis and calcification of the lesions, angiogenesis, and regeneration of normal hepatic parenchyma. Survival of tumor-bearing rats treated with repeated vector infusions was not only significantly improved over that of animals after a single injection at 10 times the vector dose (P = .001), but 18% of animals in the former treatment group also achieved long-term and tumor-free survival compared with 0% of animals in the latter treatment group. In conclusion, this treatment regimen will be very useful in the future development of VSV-mediated virotherapy as a novel therapeutic modality for patients with advanced HCC. (HEPATOLOGY 2005;41:196–203.)
Hepatocellular carcinoma (HCC) is the third leading cause of cancer deaths and the fifth most common cancer in the world, accounting for over 1 million cases annually.1–3 In the United States, its incidence has increased from 1.4 to 2.4 per 100,000 for the periods 1976-1980 and 1991-1995, respectively,4, 5 which may be related to an increase in HCC related to chronic hepatitis C infection.6 Survival of patients with HCC is dependent upon both extents of the malignancy and underlying liver disease. Studies in the United States have reported a median survival of 7.8 months and a 3-year survival rate of 10%.7 In view of the limited success of available treatment modalities for advanced HCC, alternative and complementary strategies need to be developed.
The use of molecularly engineered replication-competent herpes simplex virus and adenovirus to treat cancer was initially proposed by Martuza et al.8 and Bischoff et al.9 in the 1990s, which has been translated into clinical trials recently.10–12 In addition, many viruses with inherent tumor specificities due to attenuated antiviral responses are manifested by many tumor types, and these are actively being developed as oncolytic agents for cancer treatment.13 Vesicular stomatitis virus (VSV) is a nonpathogenic RNA virus that is extremely sensitive to the antiviral actions of interferon (IFN) in normal cells but not in cancer cells.14, 15 It has been postulated that this finding is due to the fact that IFN-responsive antiviral pathways are defective in many types of tumors,14, 15 including HCC.16, 17 In addition, defects in translational regulation can cooperate with impaired IFN signaling to facilitate VSV replication in tumor cells.18 VSV is particularly appealing because of its rapid replication rate of 8 to 12 hours in tumor cells,19 such that significant tumor destruction may have occurred before the initiation of potentially neutralizing antiviral immune responses in the host. Furthermore, VSV is not endemic to North America,19 implying that preexisting neutralizing antibodies in patients will not interfere with its initial infection process after intravascular administration.
For oncolytic viruses to have significant clinical benefit in advanced cancer patients with multifocal HCC, regional or systemic delivery of the therapeutic agent through the vasculature is needed. The liver has a dual blood supply, in which the portal vein supplies 75% and the hepatic artery supplies 25% of hepatic blood flow. It is also known that, in humans and animal models, malignant liver tumors have a predominantly arterial blood supply.20 Based on these considerations, regional delivery of chemotherapeutic agents via the hepatic artery has been widely performed in the clinic, thereby increasing response rates and reducing systemic toxicity.21, 22 In a correlate to hepatic arterial chemotherapy, several groups have investigated the potential of hepatic arterial infusion of viral vectors for increased transduction of liver tumors.23–28 Intrahepatic artery delivery of replication-deficient adenoviruses in transplanted or chemical-induced animal models of multifocal HCC has been shown to transduce predominantly small lesions, although significant transduction of normal hepatic parynchyma could not be avoided.23–26 More recently, replication-conditional viruses have been examined for their oncolytic potential against HCC, including adenovirus,29–31 herpes simplex virus,32, 33 and parvovirus H1.34
We have previously described the effective use of recombinant VSV vector as an oncolytic agent to treat multifocal lesions of HCC in the livers of immunocompetent rats.35 We demonstrated that VSV administered via the hepatic artery could gain access to and be selectively replicated in multifocal HCC tumors of various sizes.35 However, intratumoral virus replication peaked after only 1 day of inoculation. To improve treatment outcome, we tested the hypothesis that repeated administrations of a syncytia-inducing VSV,36 through a permanent cannula surgically implanted into the hepatic artery, would lead to sustained tumor-selective virus replication and substantially enhance its oncolytic potential in the treatment of advanced multifocal HCC in the liver of rats.
Materials and Methods
The rat HCC cell line McA-RH7777 was purchased from American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's Modified Eagle Medium (Mediatech, Herndon, VA) in a humidified atmosphere at 10% CO2 and 37°C. BHK-21 cells (American Type Culture Collection) were maintained in Dulbecco's Modified Eagle Medium (Mediatech); Vero cells (American Type Culture Collection) were grown in Minimal Essential Medium (Mediatech) in a humidified atmosphere at 5% CO2 and 37°C. All culture media were supplemented with 10% heat-inactivated fetal bovine serum (Sigma-Aldrich, St. Louis, MO) and 100 U/mL penicillin-streptomycin (Mediatech).
Recombinant VSV Vectors.
Recombinant VSV vectors expressing mutant (L289A) Newcastle disease virus fusion protein (rVSV-NDV/F(L289A)) or green fluorescent protein (rVSV-GFP) have been described.36, 37 Viral titers of working stocks were determined on BHK-21 cells by using standard plaque assays. Resulting titers for rVSV-NDV/F(L289A) and rVSV-GFP were 2.3 × 108 plaque-forming units (pfu)/mL and 6.8 × 108 pfu/mL, respectively.
Orthotopically Advanced Multifocal HCC Model in Syngeneic Rats.
Inbred male Buffalo rats (7-8 weeks old; 195 ± 18 g) were purchased from Harlan (Indianapolis, IN) and housed in a specific pathogen-free environment under standard conditions. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Mount Sinai School of Medicine and were performed according to their guidelines. To establish multifocal HCC lesions within the liver, rats were anesthetized with 100 mg/kg ketamine intraperitoneally, 1 mg/kg xylazine intraperitoneally, and isoflurane using an inhalation anesthesia system (VetEquip, Pleasanton, CA). Subsequently, rats were infused with 1 × 107 syngeneic McA-RH7777 rat HCC cells in 1 mL of Dulbecco's Modified Eagle Medium via the portal vein. Animals were anesthetized and underwent laparotomy 21 days after tumor cell implantation to assess the presence of multiple large tumor lesions visible on the liver surface (Fig. 1).
Surgical Placement of an Indwelling Intrahepatic Artery Cannula for Repeated Vector Administration.
After anesthesia, rats were placed in a supine position, and a skin incision of approximately 3 cm in length from the xiphoid process was made. The hepatic vessels (common hepatic artery, proper hepatic artery, and gastroduodenal artery) were dissected with the aid of an operating microscope. The Preclinical Mini-Port implantable access device (Deltec, St. Paul, MN) was used to administer the vector repeatedly via the hepatic artery (Fig. 1). After ligation of the gastroduodenal artery with 7-0 Prolene (Ethicon, Somerville, NJ) and temporal block of the common hepatic artery with a microvessel clip, a 2-French clear Polyurethane catheter (outer diameter 0.63 mm, inner diameter 0.30 mm) was inserted into the gastroduodenal artery. The Preclinical Mini-Port device was then implanted into a subcutaneous pocket in the umbilical area. Rats were infused via the port system either repeatedly with 1.3 × 106 pfu every other day for 4 days (3 injections total) or with a single infusion with 1.3 × 107 of rVSV-NDV/F(L289A) vector in 1 mL of phosphate-buffered saline (PBS) or an equivalent volume of buffer over 15 seconds with block of the common hepatic artery to prevent back-flow into the aorta (Fig. 1).
Recovery of Virus From Tumor and Liver Tissue Extracts.
To evaluate the kinetics of viral replication within the tumor lesions versus the normal liver, sets of animals were sacrificed at various time points after hepatic arterial infusion of VSV vector. Tissue samples were obtained using an operating microscope and were subjected to perform plaque assays to determine the viral yield. Tumor and normal liver tissues were harvested and disaggregated under sterile conditions. The suspensions were centrifuged at low speed to remove cellular debris and the supernatants were used to perform plaque assays on BHK-21 cells (sensitivity 100 pfu/mg).
In Situ Cytokine Expression Analysis.
Tumor tissues were isolated at the indicated time points after vector infusion into the hepatic artery and were homogenized in PBS (10 mL/mg tissue wet weight) containing one tablet of protease inhibitor cocktail (Roche, Indianapolis, IN). The clarified extracts were used to measure rat interleukin 12 and IFN-γ levels via enzyme-linked immunosorbent assay (Biosource, Camarillo, CA).
VSV Neutralization Assays.
Blood samples were collected from the inferior vena cava at the time of euthanization and were allowed to clot at room temperature. Samples were centrifuged and clarified sera were collected. Serum samples were heat-inactivated at 56°C for 30 minutes and two-fold serial dilutions were prepared in 96-well plates. An equal volume of rVSV-GFP (30-50 pfu) was added to each well and the plates were incubated at 37°C for 90 minutes. Subsequently, samples were transferred onto Vero cell monolayers (1.5 × 104 cells/well) in 96-well plates and incubated at 5% CO2 and 37°C for two days. Cells were fixed and stained with 1% crystal violet in 20% methanol for 15 minutes, followed by washing with tap water to remove excess color. Neutralization titer was defined as the last dilution that completely inhibited VSV-induced cytopathic effects.
Assessment of Serum Transaminase Levels.
Blood samples were collected from anesthetized animals via retro-orbital bleed. The levels of serum aspartate aminotransferase and alanine aminotransferase were determined at the Chemistry Laboratory at Mount Sinai School of Medicine.
Histology and Immunohistochemical Stainings.
At the indicated time points after vector infusion into the hepatic artery, animals were sacrificed, after which explanted livers were fixed in 4% paraformaldehyde overnight and then paraffin embedded. Five-micrometer–thin sections were subjected to either hematoxylin-eosin staining for histological analysis or immunohistochemistry using monoclonal antibodies specific for VSV-G (VSV11-M; Alpha Diagnostic, San Antonio, TX), OX-52, OX-62, NKR-P1A (10/78), and ED1 (1C7) (BD Biosciences Pharmingen, San Diego, CA). Immunohistochemistry sections were counterstained with hematoxylin.
Survival curves of animals treated with VSV vector or buffer were plotted according to the Kaplan-Meier method. Statistical significance in different treatment groups was compared using the log-rank test. Results and graphs were obtained using the GraphPad Prism 3.0 program (GraphPad Software, San Diego, CA).
Sustained Tumor-Selective Replication of VSV Prior to the Onset of High-Titer Neutralizing Antibodies in Blood.
Buffalo rats with large multifocal HCC nodules were infused with rVSV-NDV/F(L289A) via an intrahepatic artery port system on days 0, 2, and 4 at 1.3 × 106 pfu/infusion, which was only one tenth of the maximal tolerable dose used in the single vector injection experiments (Fig. 1). Groups of animals (n = 3-5 per time point) were sacrificed at 30 minutes and on days 1, 2, 3, 4, 5, 7, and 10 after the first virus infusion at day 0, and blood and tumor-bearing liver tissues were collected. To quantitatively determine the extents of viral replication in the tumors after repeated hepatic artery VSV infusions, tumor nodules were isolated and their infectious virus yields were determined via plaque assays (Fig. 2A). The results indicated that there was a greater than 1,000-fold increase in infectious virus yield in the tumor lesions from 30 minutes to day 1 after vector infusion, which was sustained for 4 days. At day 5, intratumoral virus yields significantly decreased and became undetectable after day 7. Infectious virus titers in the serum and liver tissues rapidly declined after 30 minutes and became undetectable at day 1 and 2, respectively, confirming our previous observations that VSV selectively replicated in tumor lesions without substantial virus shedding into the systemic circulation.
Neutralizing antibody titers to VSV were determined in the same animals at the same time points as above by a standard neutralization assay with serial dilutions of serum samples (Fig. 2B). Administration via the hepatic artery of VSV at the 10-fold reduced vector dose induced high titers of neutralizing anti-VSV antibodies in blood after 5 days and peaked at 7 days after inoculation, which correlated well with the kinetics of intratumoral virus clearance in the treated animals.
Lack of Hepatotoxicity.
We evaluated the potential hepatotoxicity of multiple VSV vector infusions in multifocal HCC-bearing rats after three applications of 1.3 × 106 pfu rVSV-NDV/F(L289A) through the indwelling intrahepatic artery catheter. Groups of tumor-bearing animals were infused with rVSV-NDV/F(L289A) (n = 3) or PBS control (n = 3) on days 0, 2, and 4, and blood samples were obtained daily from each rat. The kinetic profiles of serum transaminase levels (aspartate aminotransferase and alanine aminotransferase) were determined (Fig. 3A). The results indicated that repeated VSV infusions into the hepatic artery of tumor-bearing animals caused no significant elevations in serum transaminases compared with the buffer-injected control animals at all time points. Additionally, histological analyses of the livers obtained at day 3 (Fig. 3B) and day 5 (Fig. 3C) in the treated animals showed no sign of pathology, indicating that repeated virus administration at the 10-fold reduced virus dose did not result in liver injury.
Substantially Elevated Tumor Response.
To investigate the extents of tumor responses after a single versus repeated administration of VSV vector via the hepatic artery, we tested the virus in the same orthotopic multifocal HCC tumor model in Buffalo rats as described above. rVSV-NDV/F(L289A) (1.3 × 107 pfu, single administration at day 0) or rVSV-NDV/F(L289A) (1.3 × 106 pfu, repeated administration at days 0, 2, and 4) was infused through the indwelling intrahepatic artery catheter. The animals were sacrificed at the indicated time points, and the tumor-bearing livers were excised for histological analyses (Fig. 4). Tumor nodules were classified (as measured via morphometric analysis) into four groups according to the percentage of necrotic areas to the total tumor area. The spontaneous necrotic areas in all tumor nodules were less than 25% of the total tumor areas before injection (day 0). After a single virus administration at the maximal tolerable dose of 1.3 × 107 pfu, substantial tumor necrosis of up to 50% to 90% of the total tumor area was seen in 29% of all evaluated tumor nodules at day 2. Thereafter, tumors continued to grow and the percentages of necrotic areas gradually diminished over time. In contrast, repeated administration of VSV vector at one tenth the dose induced dramatically improved tumor responses with completely necrotic tumors (>90% of the total tumor area) in 29% of all evaluated tumor nodules at day 5, which was not observed in any of the animals after a single virus injection at 10 times the vector dose.
To elucidate the mechanisms that are responsible for the improved survival in the multidosing protocol, we analyzed tumor samples obtained at day 0 (before treatment), and days 1, 3, 5, and 7 after treatment from single and repeated VSV-treated animals by immunohistochemistry using the following cell-specific monoclonal antibodies: natural killer cell (NKR-P1A), pan-T cell (OX-52), dendritic cell (OX-62), and macrophage/monocyte (ED1). Intratumoral natural killer cell infiltration peaked at day 1 at similar levels in both treatment groups and subsided thereafter (data not shown). No T cells or dendritic cells could be identified in the tumor nodules until day 10. In contrast, ED1-positive cells were identified within the rim of completely necrotic tumor nodules after repeated virus administration at day 5 (Fig. 5A), although they were not seen in the single virus administration group. The number of ED1-positive cells continued to increase until day 7, which might be associated with the phagocytosis of necrotic tumor cells. Additionally, we analyzed intratumoral concentrations of interleukin 12 and IFN-γ via enzyme-linked immunosorbent assay in tumor extracts, and the results showed no remarkable difference in either group (data not shown).
To further characterize the histopathology of completely necrotic tumor nodules in detail, liver tissue sections showing the tumor border regions with normal liver were obtained and subjected to hematoxylin-eosin staining and immunohistochemistry against VSV-G protein. At day 5, completely necrotic tumor nodules were identified (Fig. 5B), and in the consecutive section, VSV-G immunostaining was weakly positive (Fig. 5C), indicating that the observed tumor necrosis was caused by VSV replication and subsequent oncolysis. Higher magnification of the tumor border area further demonstrated the presence of granulation-type tissues characterized by new blood vessel formation (Fig. 5D). At day 7, fibrotic tumor nodules with calcified necrotic areas were seen (Fig. 5E). Simultaneously, the surrounding liver parenchyma revealed binucleated hepatocytes, indicating hepatic regeneration (Fig. 5F).
Prolonged Survival and Long-Term Tumor Remission.
To assess the potential of repeated versus single administration of VSV via the hepatic artery in the treatment of multifocal HCC, rats bearing 5 to 10 visible HCC tumors in their livers with sizes ranging from 1 to 10 mm in diameter were randomly assigned to infusion via the hepatic artery port with either 1.3 × 106 pfu rVSV-NDV/F(L289A) every other day for 4 days (three injections total; n = 11), 1.3 × 107 pfu rVSV-NDV/F(L289A) (single injection; n = 11), or PBS control (single injection; n = 7), and survival was followed (Fig. 6). Buffer-treated rats began to die of tumor progression within 11 more days, and all of them expired at 17 days (median survival, 14 days). In contrast, rats treated with a single infusion of VSV vector survived for up to 25 days (median survival, 17 days), which was significantly improved over the PBS group (P = .0002). In the repeated virus administration treatment group, 2 animals survived for more than 100 days, and median survival was further extended to 27 days (P < .0001 vs. PBS). In pairwise comparison with the single virus-injected group, survival of animals treated with repeated virus infusions was significantly improved (P = .0013), with 18% of the animals showing long-term survival versus 0% in the single virus administration group.
The long-term surviving rats in the repeated virus administration group were sacrificed at 100 days after treatment and evaluated for residual malignancy. No tumor was visible macroscopically on the liver surface or elsewhere. Histologically, there was also no evidence of residual tumor cells or hepatitis. The results indicated that the large multifocal tumors (up to 10 mm in diameter at the time of first virus injection) had undergone complete remission in these animals, which enjoyed long-term and tumor-free survival.
Oncolytic viruses provide an attractive new tool for treatment of solid cancers because of their ability to replicate selectively within the tumor and kill neighboring cancer cells upon tumor lysis and secondary infection.13 We reported previously on the preferential replication of oncolytic VSV within large solitary HCC nodules in the liver after intratumoral administration.37 Although this is encouraging, advanced cancer patients often present with multifocal HCC and are not amenable to intratumoral injections to all lesions. Therefore, we have recently evaluated delivery of VSV via the vascular supply of the tumor.35 When a single dose of the vector was injected via the hepatic artery into rats bearing orthotopically implanted multifocal HCC nodules, VSV selectively replicated in the majority of tumor nodules of various sizes and caused substantial tumor destruction, which led to prolongation of animal survival.35 Importantly, no treatment toxicity—and in particular no hepatotoxicity—was observed at doses as high as 1.3 × 107 pfu, which was the maximal tolerable dose. However, although we detected large necrotic regions within multiple tumors, intratumoral virus replication peaked after only 1 day, and all treated animals succumbed to relapse.
In the present study, we envisioned that multiple infusions of VSV, through an indwelling cannula surgically implanted in the hepatic artery, would result in sustained intratumoral virus replication and much greater tumoricidal effects than a single vector injection. This procedure resembles that used in humans for regional chemotherapy or chemoembolization of malignant liver tumors. Permanent access to the hepatic artery for repeated administration of a recombinant adenovirus expressing the herpes simplex virus thymidine kinase gene has been reported.38 In our rat model of multifocal hepatic HCC lesions, we demonstrate that multiple infusions of VSV via the hepatic artery route at a 10-fold reduced vector dose than a single injection at the maximal tolerable dose dramatically increased antitumor efficacy without any toxicity, with 18% of the treated animals achieving long-term and tumor-free survival.
This much-better-than-expected treatment outcome of repeated administration at a 10-fold reduced vector dose was apparently not the result of simple arithmetic and may be attributable to a number of factors. Compared with our previously published results,35 intratumoral replication of VSV appeared to be substantially prolonged after repeated administration. We demonstrated extensive and sustained tumor-selective viral replication over a 4-day period prior to the onset of high-titer neutralizing antibodies in blood. We also detected massive accumulation of mononuclear inflammatory cells within the rim of completely necrotic tumor nodules after repeated vector delivery, which was not observed in animals after a single vector infusion at ten times the vector dose. Innate immune responses, including the recruitment and activation of mononuclear cells of the phagocytic system, could contribute to the lysis of virus-infected tumor cells.39 Whether the observed tumor-infiltrating immune cells provided a “bystander” killing effect of noninfected tumor tissue and contributed to the enhanced tumoricidal effects after repeated VSV administration will need to be explored in the future.
In summary, multifocal HCC remains a significant clinical problem with limited therapeutic options. Here, we show that repeated hepatic arterial administration of VSV at a reduced vector dose increased not only treatment efficacy, but also safety margin, for advanced multifocal HCC in the livers of immune-competent and syngeneic rats, suggesting that this virotherapy approach may have significant potential for future clinical application in patients with advanced HCC.
We thank Dr. Tian-Gui Huang for discussions, Dr. John Fallon for consultation on histological and immunohistochemical analyses of tissue samples, and Ms. Jing Xu and Ms. Sonal Harbaran for their excellent technical assistance.
- 19Rhabdoviridae: the viruses and their replication. In: KnipeDM, HowleyPM, eds. Fields Virology. 4th ed. Philadelphia: Lippincott Williams & Wilkins, 2001: 1221–1242., .
- 28Hepatic intra-arterial delivery of a retroviral vector expressing the cytosine deaminase gene, controlled by the CEA promoter and intraperitoneal treatment with 5-fluorocytosine suppresses growth of colorectal liver metastases. Gene Ther 2001; 8: 1241–1247., , , , , , et al.