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. 2013 Sep 24;21(11):2032–2042. doi: 10.1038/mt.2013.181

Antifibrotic Properties of Transarterial Oncolytic VSV Therapy for Hepatocellular Carcinoma in Rats With Thioacetamide-Induced Liver Fibrosis

Jennifer Altomonte 1, Sabrina Marozin 1, Enrico N De Toni 2, Antonia Rizzani 2, Irene Esposito 3,4, Katja Steiger 3,4, Annette Feuchtinger 3, Claus Hellerbrand 5, Roland M Schmid 1, Oliver Ebert 1,*
PMCID: PMC3831039  PMID: 23939023

Abstract

Recombinant vesicular stomatitis virus (VSV) shows promise for the treatment of hepatocellular carcinoma (HCC), but its safety and efficacy when administered in a setting of hepatic fibrosis, which occurs in the majority of clinical cases, is unknown. We hypothesized that VSV could provide a novel benefit to the underlying fibrosis, due to its ability to replicate and cause cell death specifically in activated hepatic stellate cells. In addition to the ability of VSV to produce a significant oncolytic response in HCC-bearing rats in the background of thioacetamide-induced hepatic fibrosis without signs of hepatotoxicity, we observed a significant downgrading of fibrosis stage, a decrease in collagen content in the liver, and modulation of gene expression in favor of fibrotic regression. Together, this work suggests that VSV is not only safe and effective for the treatment of HCC with underlying fibrosis, but it could potentially be developed for clinical application as a novel antifibrotic agent.

Introduction

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death and the fifth most common type of cancer in the world, accounting for over 1 million cases annually.1 In roughly 80–90% of the patients, these tumors arise from the background of liver cirrhosis,2,3 resulting from a wound-healing response to chronic liver injury, known as hepatic fibrosis. In particular, long-term alcohol abuse and chronic hepatitis C virus infection are the most prominent underlying factors responsible for liver cirrhosis in Europe and North America.4,5 The fibrotic response underlies virtually all of the complications of end-stage liver disease, including portal hypertension, ascites, encephalopathy, and metabolic dysfunction, as well as the onset of HCC.6

When HCC occurs in the setting of cirrhosis, the condition presents a great challenge for clinicians, with the degree of liver function greatly influencing the possibility for curative, or even palliative therapies. Even in patients who are diagnosed early, the course of the disease is often fatal due to the glaring deficiency of available therapies to simultaneously treat HCC and the underlying liver disease. Therefore, due to the ever-increasing incidence of cirrhosis and subsequent HCC, as well as the obvious limitations of currently available therapies, novel and effective treatments are urgently needed.

Due to recent progress in understanding the pathogenesis of liver fibrosis,7 it is now believed to be a reversible process.8,9 During fibrogenesis, hepatic stellate cells (HSCs) differentiate from the quiescent to the activated form, marked by a change to a myofibroblast phenotype coinciding with expression of α-smooth muscle actin (α-SMA). These transdifferentiated HSCs promote extracellular matrix remodeling by deregulating the secretion of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases (TIMPs), resulting in the degradation of the normal matrix and its replacement with interstitial collagen (primarily type I and III) and scar matrix. These hepatic changes cause a distortion of the normal liver architecture and lead to decompensated liver function. While it is known that the major mechanism for regression of fibrosis involves apoptosis of activated HSCs,10,11 the challenge for antifibrotic therapy is specific targeting of activated HSCs, without collateral effects on quiescent cells or myofibroblasts present in other tissues. Unfortunately, the majority of drugs under investigation have resulted in only minor antifibrotic effects, with a general lack of specificity on the HSC activation pathway.12 It has recently been reported that novel viral therapies, employing Newcastle disease virus or inactivated Orf virus, can reverse the progression of the hepatic condition.13,14

We have previously reported that recombinant vesicular stomatitis virus (VSV) vectors are effective oncolytic agents with inherent specificity for tumor cells.15,16,17 Here, we demonstrate in vitro that VSV specifically replicates in and kills activated HSCs, while sparing quiescent cells. Furthermore, we show in a thioacetamide-induced rat model of fibrosis that hepatic arterial infusion of VSV not only maintains its ability to efficiently kill tumor cells, but it also possesses antifibrotic properties which result in the unique benefit of concomitant reversal of fibrotic progression. Together, these data indicate that VSV is not only an intrinsically oncolytic virus, but its specificity can be extended to activated HSCs, resulting in fibrotic regression. Therefore, VSV has the potential to be developed into a powerful therapeutic agent for the simultaneous therapy of HCC and underlying hepatic fibrosis, which is urgently needed for the clinical management of this complex condition.

Results

VSV replicates and causes cytotoxicity in activated HSCs

To determine the relative permissiveness of HSCs to VSV replication, primary human hepatocytes, HepG2, and activated HSCs were infected with rVSV-LacZ for 24 hours. Although titers were approximately 4-logs higher in HepG2 cells as compared with primary human hepatocytes (109 versus 105, respectively), viral growth in primary HSCs was intermediate (Figure 1a). A comparison of differentially cultured LX-2 and primary HSCs revealed a striking variance in virus replication, with titers elevated by 2–3 logs in activated compared with the quiescent cells (Figure 1b), leading to a statistically significant increase in cytotoxicity at both 24 and 48 hours postinfection with VSV (Figure 1c).

Figure 1.

Figure 1

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Specificity of rVSV for activated hepatic stellate cells (HSCs). (a) Primary human hepatocytes (PHH), HepG2 cells (hepatocellular carcinoma), or activated primary human HSC were infected with rVSV-βgal at an MOI of 0.01 for 24 hours, and viral titers in the medium were measured by TCID50. (b) Quiescent (serum-starved) or TGF-β-activated LX-2 cells and quiescent-like (day 2 postplating) or activated (day 10) primary human HSCs were infected with rVSV-βgal at an MOI of 0.01, and aliquots of the conditioned medium were subjected to TCID50 analysis for determination of viral titers after 24 hours. For both cell types, statistically significant increases in titers were observed in activated cells (P < 0.05). (c) Quiescent or activated LX-2 and primary human HSCs were infected with rVSV-βgal at an MOI of 0.01, and lactate dehydrogenase in the supernatant was measured at 8, 24, and 48 hours post-transfection to determine cytotoxicity. Significant increases (P < 0.05) were observed in activated cells at 24 and 48 hours postinfection. (d) Quiescent-like (day 2 postplating) or activated (day 10) primary human HSCs treated with either PBS or rVSV at an MOI of 0.01 for 48 hours were stained with propidium iodide and subjected to FACS analysis of sub-G1 populations for determination of apoptotic cells (left panel). Additional cells were subjected to Hoechst staining for determination of nuclear changes associated with apoptotic cells (right panel). Arrows indicate chromatin condensation and nuclear fragmentation. Representative images are shown at ×20 magnification.

FACS analysis of the sub-G1 populations of propidium iodide-stained primary HSCs demonstrated a significant correlation between the number of apoptotic cells after infection with VSV and the degree of activation (10 days in culture versus 2 days) (Figure 1d, left panel). Furthermore, Hoechst staining of primary HSCs demonstrated chromatin condensation and nuclear fragmentation in the more highly activated HSCs after a 48-hour infection with VSV (Figure 1d, right panel), indicating that VSV induces apoptosis in HSCs in an activation-dependent manner.

VSV infection results in phenotypic changes of differentiated HSCs

To investigate the effects of VSV on activated HSCs at early time points after infection, immunofluorescence staining for αSMA was performed. As an internal control, cells were costained for vinculin, a protein which is expressed in both quiescent and activated HSCs.18 While cells expressed very low levels of αSMA after 2 days in culture, the expression level was substantially increased after 10 days (Figure 2a), indicating that the amount of time in culture is directly correlated with degree of activation. For simplicity, we refer to the 2-day-cultured HSCs as “quiescent” and the 10-day-cultured cells as “activated”. Activated HSCs infected with VSV for 12 hours expressed much lower levels of αSMA as compared with the PBS-treated cells at the same time point (Figure 2a), whereas vinculin expression was maintained, suggesting that the loss of αSMA was most likely due to a partial inactivation or senescence, rather than cell death. Quantitative real-time PCR using primers specific for human αSMA revealed a significant reduction in αSMA mRNA expression in activated HSCs 12 hours postinfection with VSV (Figure 2b), and western blot analysis demonstrated a similar reduction in αSMA at the protein level at the same time point (Figure 2c).

Figure 2.

Figure 2

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Loss of activation of hepatic stellate cells (HSCs) in response to vesicular stomatitis virus (VSV) infection. (a) Quiescent-like or activated primary human HSCs treated with either PBS or rVSV for 12 hours were subjected to immunofluorescent costaining for αSMA and vinculin using FITC- and Cy3-labeled secondary antibodies, respectively. Counterstaining was performed using DAPI for detection of nuclei. Representative photomicrographs are shown at ×20 magnification, and the scale bar = 100 µm. (b) Activated primary human HSCs were treated with PBS or rVSV-βgal at an MOI of 0.01 for 12 hours. As a control, quiescent-like cells were prepared. Aliquots of cDNA prepared from reverse-transcription of mRNA were subjected to real-time PCR for quantification of αSMA expression. Relative values were quantified by normalizing the expression level of αSMA to the internal housekeeping control (GAPDH) and setting the values for the quiescent cells to 1, such that the level for activated cells represents a fold increase or fold decrease. Means from each treatment group are shown, with error bars representing standard deviations. The reduction in αSMA mRNA expression in VSV-treated cells is significant (P < 0.0005) with respect to PBS-treated cells. (c) Western blots were performed from 1 µg protein from cell lysates of quiescent or activated human HSCs treated with either PBS or rVSV-βgal at an MOI of 0.01 for 12 hours. Antibodies specific for αSMA or the housekeeping protein β-tubulin were used. The image shown is representative of three independent experiments. (d) Gelatin zymography was performed from supernatants from quiescent or activated primary human HSCs (top panel) or LX-2 cells (bottom panel) after 8-hour treatment with PBS or rVSV. Negative bands indicate matrix metalloproteinases-2 activity.

Matrix metalloproteinase-2, an enzyme which degrades type IV collagen, is known to be secreted from activated HSCs, and levels are upregulated in fibrotic liver tissue.19,20 Gelatin zymography demonstrated an increase in matrix metalloproteinase-2 activity in the supernatants of activated versus quiescent primary HSCs and LX-2 cells, which was abrogated after 12-hour infection with VSV (Figure 2d). Whether these events represent a true shift toward inactivation, or they are a mere consequence of the general shutdown of host translation, remains to be determined.

Specificity of VSV replication for activated HSCs is associated with cell-cycle progression

Because interferon (IFN) signaling defects have been identified as a mechanism which allows oncolytic viruses to replicate in tumor cells,21 we investigated whether this same mechanism could explain the specificity of VSV for activated HSCs. Reporter assays for the IFN-β and IFN-stimulated response element promoters were performed to assess IFN induction and response, respectively. For these experiments, we utilized a mutant rVSV vector (M51R), which has an enhanced ability to trigger IFN responses due to an amino acid substitution in the endogenous matrix protein. Although both quiescent and activated LX-2 cells demonstrated induction of IFN-β and IFN-stimulated response element in response to polyI:C or IFN and rVSV (M51R) stimulation, there was a slight impairment of IFN signaling in the activated cells (Figure 3a). To further address this issue, multicycle growth curves of rVSV and rVSV(M51R) were compared in quiescent versus activated primary HSCs. In line with the reporter assays, we observed an attenuation of the M51R virus in comparison with VSV in both quiescent and activated cells; however, the titers of M51R at 24 and 48 hours postinfection were at least 1-log higher in the activated cells, indicating that IFN signaling in activated cells was only partially responsive or that perhaps there is an additional mechanism in place which allows the virus to replicate in activated HSCs, despite a functional IFN signaling pathway. Further investigation by IFN protection assay revealed that relatively low concentrations of IFN were sufficient to inhibit VSV replication in activated HSCs, and no significant differences in titers were detected among activated and quiescent cells regardless of the dose of IFN (Figure 3a). Taken together, we concluded that IFN signaling is perhaps not the major determinant of specificity of VSV for activated cells.

Figure 3.

Figure 3

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Mechanism of rVSV specificity for activated hepatic stellate cells (HSCs). (a) IFN-β and ISRE promoter activation in primary human HSCs was quantified using luciferase reporter plasmids. Quiescent or activated cells were cotransfected with pIFNβ-Luc or pISRE-Luc and pRL (constitutively active Renilla), and stimulated 24 hours post-transfection with pI:C or IFN, and VSV and VSV(M51R). After overnight incubation, firefly luciferase expression levels were normalized to Renilla expression and calculated as a fold-induction in comparison with mock-stimulated cells. IFN protection assay results are presented in the right panel. Quiescent or activated HSCs were pretreated overnight with increasing doses of IFN as indicated and infected with rVSV at an MOI of 0.01 for 24 hours. Aliquots of supernatant were subjected to TCID50 analysis for the determination of viral titers. Multicycle growth curves are shown for quiescent (Q) or activated (A) primary HSCs infected with VSV or VSV(M51R) at an MOI of 0.01. Viral titers were determined by TCID50, and data are presented as the mean of three experiments ± standard deviation. (b) Activated LX-2 cells were treated with a panel of cell-cycle inhibitors or DMSO for 24 hours prior to overnight infection with rVSV at an MOI of 0.01. TCID50 analysis of viral titers are shown (left panel). P value for Rosco versus DMSO <0.005. Activated cells treated with DMSO or cell cycle inhibitors for 24 hours were stained with propidium iodide (PI) for FACS analysis of percentage of cells in each phase of the cell cycle (right panel). (c) Activated LX-2 cells were transfected with siRNAs targeting cyclin B1 or topoisomerase-2α, or with scramble siRNA, and then infected with VSV at an MOI of 0.01 overnight. Cell lysates were subjected to western blot analysis to confirm knock-down (left panel), and PI-stained cells were analyzed by FACS to determine cell cycle (middle panel). Aliquots of the medium were analyzed by TCID50 to determine viral titers (P < 0.05 for siRNA versus scr). Mean values of all data are presented + SD of triplicate experiments.

To explore the role of cell proliferation on VSV replication, activated LX-2 cells were pretreated with a panel of cell cycle inhibitors prior to VSV infection. While most of the inhibitors had no affect on virus growth, pretreatment with roscovitine caused a significant attenuation (Figure 3b). FACS analysis of propidium iodide-stained cells after 24 hours of treatment with each cell cycle inhibitor revealed that roscovitine was unique in that it caused a significant cellular accumulation in G2 phase (Figure 3b). To confirm the role of G2 arrest on VSV replication, we applied siRNAs against cyclin B1 and topoisomerase-2α, which are associated with G2-S progression. After verifying that the expression of each protein was inhibited by the respective siRNA and that G2 cell cycle arrest was achieved, we measured viral titers in supernatants of LX-2 cells after overnight infection in siRNA-treated cells. Indeed, VSV was significantly attenuated in cells treated with siRNA targeting cyclin B1 and topoisomerase-2α in comparison with the scramble control (Figure 3c).

VSV replicates in HCC tumors in a fibrotic setting and also localizes to activated HSCs

To investigate the effect of liver function on VSV therapy for HCC, rats harboring HCC in the setting of a healthy versus fibrotic liver were randomized for injection of PBS or rVSV-βgal via the hepatic artery. Analysis of tissue extracted after 24 hours of treatment revealed that VSV maintains its ability to access and replicate in tumors growing in the context of hepatic fibrosis, without causing significant changes in intrahepatic titer (Figure 4a). Immunofluorescent staining of tissue sections revealed that, although the majority of VSV protein was localized within tumors, some VSV colocalized with stellate cells within fibrotic liver, as demonstrated by costaining for desmin (Figure 4b). Together, these data indicate that the replication of VSV within activated HSCs in vivo is probably limited, yet sufficient to produce a therapeutic effect. Of note, no VSV protein was detected in normal hepatocytes, and histological examination of liver tissue revealed no observable toxicity associated with rVSV therapy.

Figure 4.

Figure 4

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Specificity and safety of vesicular stomatitis virus (VSV) for hepatocellular carcinoma (HCC) with underlying hepatic fibrosis. (a) Rats harboring unifocal HCC lesions implanted into healthy livers versus livers with thioacetamide-induced hepatic fibrosis were treated by hepatic arterial infusion of PBS or rVSV-βgal (n = 5). On day 1 post-treatment, samples of liver and tumor were snap-frozen and analyzed by TCID50 to determine virus titers (left panel). Morphometric analysis of necrotic areas was performed on H/E preparations of tumor sections excised on day 3 post-treatment using ImageJ software (middle panel). Tumor areas were determined by measuring the length and width of each tumor prior to fixation (right panel). Mean values + SD are shown in each panel. (b) Additional samples obtained on day 1 post-treatment were fixed overnight and paraffin-embedded. VSV (red) and desmin (green) were visualized by immunofluorescent staining. Nuclei were detected by DAPI staining (blue). Representative images of tumor and fibrotic liver area extracted from a VSV-treated liver are shown at ×63 and ×40 magnification, respectively.White arrows indicate areas of colocalization. Scale bar = 20 µm in tumor tissue and 50 µm in liver. (c) Serum samples from control animals and animals with thioacetamide-induced hepatic fibrosis were obtained on day 1 and 3 posthepatic-arterial administration of rVSV or PBS. Mean values + SD are shown.

Morphometric analysis of tumor sections demonstrated that rVSV treatment resulted in comparable tumor responses, irrespective of liver fibrosis; however, tumors generally grew to a larger size in the fibrotic setting, resulting in a higher percentage of baseline necrosis (Figure 4a). This phenomenon, in which HCC tumors grow more aggressively in a fibrotic environment, is in line with a previous report comparing the invasiveness of HCC tumors implanted in a thioacetamide-induced fibrotic, versus a normal, liver setting.22 Serum chemistries for glutamic–pyruvic transaminase, blood urea nitrogen, and creatinine demonstrated only transient elevations, which returned to normal levels on day 3 and did not differ significantly among treatment groups, indicating no significant impairment of hepatic or renal function in response to therapy (Figure 4c).

In vivo VSV therapy results in improved liver staging and a reduction of hepatic fibrosis

Liver sections were subjected to histological examination and Desmet scoring to determine the fibrotic stages in response to PBS or VSV treatment. While PBS-treated sections were assigned a mean score of 3–4 due to the presence of portoportal and portocentral bridges, rVSV-treated sections received a mean score of 2, representing a significant improvement in fibrotic stage (Figure 5a). As a quantitative measure of fibrosis, the intrahepatic collagen contents of PBS versus rVSV-treated animals were compared by Sircol assay, which demonstrated a significant reduction in soluble collagen contents in response to rVSV therapy (P < 0.05) (Figure 5b).

Figure 5.

Figure 5

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Vesicular stomatitis virus (VSV) therapy causes improvement in liver staging and reduction in hepatic collagen content. (a) Rats harboring thioacetamide-induced hepatic fibrosis were treated by intrahepatic arterial infusion of either PBS or rVSV and sacrificed on day 1 post-treatment. Paraffin-embedded liver sections were visualized by H/E staining, and representative fields of view were photographed at ×2 or ×10 magnification. Stage 4 fibrosis (probable or definite cirrhosis) is exemplarily shown in the PBS-treated upper panel, as evidenced by fibrous bridges (arrows) connecting portal tracts (PT) and central veins (V) with formation of multiple nodules. The VSV-treated bottom panel is representative of stage 2 fibrosis, in which connective tissue links neighboring portal tracts, but the overall architecture is preserved. (b) A commercially available kit was used to determine the concentrations of acid-pepsin soluble collagen in fibrotic livers treated with PBS or VSV for 24 hours. Mean concentrations + SD are shown. P < 0.05.

Transarterial infusion of rVSV causes a reduction of αSMA expression by HSCs

Immunohistochemical staining for αSMA demonstrated a clear reduction in the intensity and number of positively stained cells on day 1 post-rVSV administration as compared with the PBS controls (Figure 6a). Semiquantitative western blot analysis similarly demonstrated a significant reduction of αSMA in fibrotic livers in response to rVSV therapy (P = 0.01) (Figure 6b). Furthermore, quantitative real-time PCR revealed that the modulation of αSMA occurs at the mRNA level, with a significant decrease (P < 0.005) in αSMA mRNA expression in fibrotic livers treated with rVSV as compared with PBS (Figure 6c). These results indicate that VSV causes either a reduction in activated HSC number or dedifferentiation into an inactivated or senescent state.

Figure 6.

Figure 6

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Vesicular stomatitis virus (VSV) therapy leads to decreased αSMA expression in vivo. (a) Rats harboring thioacetamide-induced fibrotic livers were treated with either PBS or rVSV, and paraffin sections were prepared from tissues harvested 24 hours post-treatment. Immunohistochemical staining was performed with an antibody specific for αSMA, and representative sections were imaged at ×5 (left panel) or ×20 (right panel) magnification. The black bar indicates 100 µm. (b) Homogenates of snap-frozen sections of normal liver or thioacetamide-induced fibrotic liver harvested 24 hours after intra-hepatic arterial infusion of PBS or VSV were subjected to western blot analysis for αSMA expression. Blots were stripped and reprobed with an antibody for tubulin to control for loading variations. Semiquantification of band intensities normalized to tubulin was performed using ImageJ software, and values are expressed as a relative intensity with respect to the normal liver control. P-value for VSV-treated samples versus PBS is <0.05. Three representative liver homogenates from each treatment group are shown. (c) mRNAs extracted from fibrotic liver treated with PBS or VSV for 24 hours were reverse-transcribed and used as template for quantitative real-time PCR (qPCR) to determine the relative SMA expression level after treatment. Levels were normalized to GAPDH, and VSV-treated samples are presented in terms of their relative expression level with respect to the PBS controls. Means + SD are shown. P < 0.005.

rVSV treatment is associated with the apoptosis of activated HSCs

To identify apoptotic-activated HSCs, we performed a costaining for TUNEL and αSMA in fibrotic liver tissue. Hybrid images revealed a striking colocalization of apoptotic nuclei with αSMA-positive cells in VSV-treated livers (Figure 7a), suggesting an important mechanism for the reversal of fibrosis. Of note, the number of apoptotic hepatocytes was similar among treatment groups, implying that there was no significant compromise to the normal liver paranchyma associated with VSV therapy in this model.

Figure 7.

Figure 7

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Increased TUNEL staining and NK cells in fibrotic livers following vesicular stomatitis virus (VSV) therapy. Rats harboring thioacetamide-induced hepatic fibrosis were treated with PBS or rVSV via transarterial infusion of the hepatic artery. (a) On day 3 post-treatment, animals were euthanized, and liver sections were paraffin-embedded and subjected to coimmunofluorescent staining to localize terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), appearing in yellow, and αSMA, in red. Sections were counterstained with DAPI for localization of nuclei. Representative sections are shown at ×40 magnification with oil (white bar indicates 100 µm). (b) Liver sections obtained on day 3 after treatment with PBS (left panel) or VSV (right panel) were subjected to immunohistochemical staining for natural killer cell marker (ANK61). Representative sections are shown at ×20 magnification (scale bar = 100 µm). The number of positively stained cells per ×10 field of view was quantified and is shown as mean + standard deviation. P value <0.01.

Natural killer (NK) cell accumulation corresponds with rVSV treatment in fibrotic livers

Increasing evidence demonstrates that NK cells inhibit hepatic fibrosis via direct killing of activated HSCs and by induction of IFNγ, justifying the clinical development of NK cell therapy for hepatic fibrosis.23,24 Since we have previously observed a striking intratumoral infiltration of NK cells to sites of VSV replication,25,26 we performed immunohistochemical staining of fibrotic liver tissue to quantify the hepatic accumulation of NK cells in response VSV therapy. Of note, we observed a significant increase in the number of NK cells in VSV- versus PBS-treated livers (Figure 7b), which accumulated along the connective tissue. We speculate that virus-induced activation of NK cells could potentiate bystander cell killing, causing apoptosis even in those HSCs not directly infected by VSV. The precise role of NK cells in VSV-mediated antifibrotic therapy is an important topic of ongoing investigations. In addition, to further characterize the immune cell infiltrate in response to antifibrotic VSV therapy, we performed immunohistochemical staining for myeloperoxidase-positive granulocytes, CD68+ macrophages, and CD3+ T-lymphocytes; however, no significant differences in cell numbers were detected within fibrotic liver tissue treated with VSV versus PBS (data not shown).

VSV treatment results in therapeutic modulation of key genes associated with fibrotic progression

To investigate the effects of rVSV therapy on relevant mRNA expression, we performed quantitative real-time PCR. Consistent with a repression of the fibrotic condition, we observed significant decreases in TGF-β, collagen 1, and TIMP-1 mRNA, as well as an increase in IFN-α mRNA in response to VSV therapy after 24 and 72 hours; representative data from 24 hours are shown (Figure 8). Of particular importance, TGF-β has been identified as a potential anti-inflammatory and antifibrotic target, and therapies aimed at inhibition of TGF-β signaling are under development.27 Therefore, VSV-mediated inhibition of TGF-β expression could provide a significant indirect mechanism of antifibrotic activity. In addition, the upregulation of IFNα mRNA is noteworthy, as it was recently demonstrated that the administration of IFNα ameliorates fibrosis via a reduction in TGFβ and TIMP-1 expression.28

Figure 8.

Figure 8

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Modulation of mRNA expression following rVSV administration. Thioacetamide-induced fibrotic livers in rats were treated by transarterial infusion of PBS or rVSV via the hepatic artery. Liver samples snap frozen 24 hours post-treatment were subjected to mRNA purification and subsequent cDNA preparation using commercially available kits. Aliquots of cDNA were used for the quantification of relevant genes associated with fibrotic progression by real-time PCR, as indicated. PBS values were set to 1, and vesicular stomatitis virus values were calculated as a fold-expression with respect to PBS. Means from each treatment group are shown, with error bars representing standard deviations. P values <0.05 were considered significant.

Discussion

The rising incidence of cirrhosis and subsequent onset of HCC, coupled with the obvious deficiency of currently available therapies to manage this complex clinical scenario, highlight the urgent need for alternative and effective treatment modalities. A major weakness of many preclinical investigations into novel HCC therapies is that they rely on inappropriate animal models, which poorly reflect the clinical setting. We have previously reported in an immune-competent rat model of orthotopic HCC that hepatic arterial infusion of recombinant VSV results in significant tumor responses and subsequent prolongation of survival.16,29,30 Here, we investigate the use of VSV in a more challenging and clinically relevant model of HCC with underlying hepatic fibrosis. We demonstrate that hepatic arterial infusion of VSV in this setting is not only safe and effective as an HCC therapy, but it also possesses inherent antifibrotic properties. In particular, VSV administration resulted in a reduction of activated HSCs, decreased fibrotic content in the liver, and a qualitative improvement in liver staging as determined by Desmet score.

The data presented here demonstrate that the antifibrotic effects of VSV are potentially the result of three distinct, although potentially interconnected, mechanisms: (i) direct cell killing of infected HSCs, (ii) recruitment of activated NK cells, and (iii) modulation of gene expression in favor of fibrotic resolution. Whether the modulatory effects on NK cells and gene expression truly represent a direct response to VSV infection, or they are merely a consequence of the reduction of activated HSCs, remains to be seen and is a topic of ongoing investigations. However, regardless of the mechanism, we speculate that these indirect aspects of VSV therapy are crucial in providing a favorable therapeutic outcome, as it is improbable that the virus can homogenously infect HSCs throughout the liver, especially in the fibrotic context, where large amounts of extracellular matrix provide a physical barrier to efficient virus spread. Furthermore, since these studies focused on the therapy of HCC in a cirrhotic context, we did not investigate the efficacy of VSV therapy for fibrosis in the absence of hepatic tumors. Therefore, we can only presume that the antifibrotic properties of VSV are not dependent on the presence of HCC; however, since the treatment of the fibrotic condition prior to the onset of HCC would be a highly desirable application for future VSV therapy, these investigations are warranted and are ongoing.

As a critical endpoint, we closely examined the safety of VSV therapy for HCC in the fibrotic setting. Because of the correlation between VSV replication and cell cycle progression in activated HSCs, it was an obvious concern that the virus could inadvertently replicate in other proliferating cells in the setting of chronic liver injury; however, we saw no indication that this was the case in our model. Intrahepatic viral titers were comparable in fibrotic versus healthy livers, and careful examination of histology and immunofluorescent staining failed to reveal any signs of toxicity or viral replication in hepatocytes in the fibrotic setting. This could be in part due to the fact that hepatocyte proliferation is thought to occur only as a first-line defense, after which time if the injury becomes chronic, hepatocytes lose their ability to replicate, and the majority of liver regeneration occurs via differentiation of hepatic progenitor cells (also known as oval cells).31 Furthermore, we have previously shown that in HCC cells, proliferation does not play a role in determining the ability of VSV to replicate.32 Therefore, we speculate that cell cycle progression is merely a component of HSC permissiveness to VSV, and that other aspects of the activation process surely contribute to this phenomenon. Further investigations into the mechanism of VSV specificity for activated HSCs are ongoing.

Due to significant improvements over the last decade in the treatment of hepatitis C virus, it is now possible to fully resolve the infection;33 however, in the absence of an effective antifibrotic therapy, those patients with established cirrhosis nevertheless face a high risk of developing HCC. It is therefore crucial that novel therapies are developed to safely and effectively treat hepatic fibrosis. Here, we have shown that VSV replicates selectively both in tumor cells and activated HSCs, providing an ideal agent for the simultaneous treatment of both HCC and underlying fibrosis. Furthermore, if VSV is administered at the early stages of fibrosis development, it is possible that the progression of the disease can be interrupted, and the onset of HCC and other complications of end-stage liver disease can be delayed or potentially avoided. However, we acknowledge that further modifications of the VSV platform will surely be necessary, as the long-term survival benefit provided by the wild-type vector may be minor, if any. Therefore, the current studies are meant to report our novel findings as proof-of-principle for future long-term survival studies, which will employ optimized vectors and combination therapies, as well as additional animal models of hepatic fibrosis, to comprehensively assess the potential clinical benefit of VSV as an antifibrotic and oncolytic agent.

In conclusion, the data presented here support the development of VSV vectors as safe and effective tools for the clearance of activated HSCs and subsequent regression of fibrosis. This work suggests that the use of VSV, and possibly other negative-strand RNA viruses, is not limited to cancer therapy, but can potentially be applied to additional therapeutic targets. The simultaneous therapy of HCC and underlying hepatic fibrosis with a single agent provides a significant therapeutic advantage over the current state of the art, and therefore, VSV has the potential to be developed into a powerful oncolytic and antifibrotic agent for future clinical application.

Materials and Methods

Virus and cell lines. Production of rVSV-LacZ and rVSV (M51R) and propagation of BHK-21 cells, the rat HCC cell line, McA-RH7777 (ATCC, Manassas, VA), and HepG2 cells was performed as described.30,32 Unless otherwise stated, infections were performed at a multiplicity of infection (MOI) of 0.01 for 24 hours, and viral titers were quantified by TCID50 analysis on BHK-21 cells. Multicycle growth curves were performed in quiescent and activated human HSCs by infection with viruses at an MOI of 0.01 for 1 hour, followed by washing and replacement with complete medium. Aliquots of supernatant were harvested at various time points postinfection (0, 16, 24, and 48 hours) and analyzed by TCID50. LX-2 cells, a kind gift from Scott Friedman (Mount Sinai School of Medicine, NY), were either serum-starved or supplemented with 10% FBS and 2 ng/ml TGFβ (R&D Systems, Minneapolis, MN) for 48 hours for differentiation into quiescent or activated phenotypes. Cryopreserved activated primary human HSCs were cultured in DMEM (ATCC) supplemented with 10% FBS and plated for either 2 or 10 days, to achieve either a quiescent-like or activated status, respectively.

Lactate dehydrogenase assays (Roche Applied Science, Indianapolois, IN) were performed as recommended, or cells were incubated with 1 µg/ml Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) and visualized by fluorescence microscopy for the detection of nuclear blebbing. Propidium iodide (Sigma-Aldrich)-stained cells were analyzed for cell cycle phases or sub-G1 events using flow cytometry (FACS calibur with Cell Quest Software; Becton Dickinson, San Jose, CA) as previously described.34

Gelatin zymography. LX-2 and primary HSCs were mock-treated or infected with rVSV for 12 hours prior to loading aliquots of conditioned medium onto polyacrylamide gels containing 0.1% gelatin (Sigma-Aldrich). Gels were developed and stained with 5% coomassie, according to established protocols35 and scanned using an Odyssey infrared imager (LI-COR Biosciences, Lincoln, NE).

IFN assays. Luciferase reporter assays were performed as previously described.36 Cells were either mock-treated, or stimulated with poly I:C (2.5 µg/ml), universal type I IFN (500 IU), or rVSV-LacZ, or rVSV(M51R)-GFP at a MOI of 1 overnight. For IFN protection assays, cell monolayers were mock-treated or treated overnight with Universal type I IFN (PBL Interferon Source, Piscataway, NJ), followed by infection at a MOI of 1. Viral titers were quantified after 24 hours.

Cell cycle inhibitors and siRNA. Activated HSCs were treated overnight with DMSO or the following chemical cell cycle inhibitors prior to infection with rVSV-LacZ: MNK1 (40 µmol/l), Roscovitine (50 µmol/l), Rapamycin (100 nmol/l), CDK4 (250 nmol/l), LY294002 (40 µmol/l), or Aphidicolin (2.5 µg/ml) (Calbiochem-Merck, Gibbstown, NJ). Inhibitor doses were chosen based on that shown to produce the strongest inhibition in the absence of toxicity in preliminary dose–response experiments in activated HSCs. In addition, LX-2 cells were transfected with siRNAs (Dharmacon, Waltham, MA), using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA). After 48 hours, the transfected cells were infected overnight with rVSV-LacZ.

Animal studies. All procedures involving animals were performed according to the guidelines of the institution's animal care and use committee, and the local government. Six-week-old male Buffalo rats (Harlan Winkelmann; Borchen, Germany) received either normal drinking water or a 0.01% solution of thioacetamide continuously over the course of the experiment. After 19 weeks, 106 McA-RH7777 cells suspended in 20 µl of DMEM were implanted orthotopically in the liver. After 10 days, when HCC nodules reached 0.5–1 cm in diameter, PBS or 107 pfu of rVSV-LacZ (n = 10) was infused via the hepatic artery, and animals were euthanized on day 1 or 3 after treatment (n = 5). Serum chemistry measurements were performed at the Institute for Clinical Chemistry and Pathobiochemistry at the Klinikum rechts der Isar. Tumor and liver sections were flash frozen or fixed overnight in 4% paraformaldehyde.

Histology, immunohistochemistry, and immunofluorescence. 3 µm thin paraffin sections were stained with hematoxylin–eosin or immunohistochemically using antibodies for αSMA (Sigma-Aldrich) or the NK cell marker ANK61 (Santa Cruz Biotechnology, Santa Cruz, CA). Double-immunofluorescent stainings were performed using the following combinations of antibodies: desmin (Thermo Scientific, Waltham, MA) and VSV-G (Alpha Diagnostic, San Antonio, TX) in conjunction with FITC-conjugated antirabbit and Cy3-conjugated antimouse antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA); αSMA (Sigma-Aldrich) and Alexa Fluor 568–conjugated goat anti-mouse IgG (Invitrogen) together with the Apoptosis Detection Kit (Millipore, Billerica, MA). ImageJ software (National Institutes of Health, Bethesda, MD) was used for morphometric analysis of tumor necrosis. Fibrotic stages were assessed on a 0–4 scale according to the criteria of Desmet37 by a pathologist who was blind to the study groups.

Collagen assay. Snap-frozen liver sections obtained on day 1 post-treatment were subjected to analysis of acid–pepsin soluble collagen content using the Sircol Collagen Assay (Biocolor, Carrickfergus, UK) according to the manufacturer's instructions.

Western blot and real-time PCR. Nitrocellulose membranes (Amersham, Buckinghamshire, UK) were probed with the following antibodies: αSMA (Sigma-Aldrich), cyclin B1 (Cell Signaling, Boston, MA), topoisomerase 2α (Cell Signaling), and tubulin (Sigma-Aldrich) and antimouse or rabbit secondary antibodies conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories). The ECL chemiluminescence kit (Amersham) was used for detection. ImageJ software (NIH) was used for semiquantification.

RNA extracted from snap-frozen tissue using the RNeasy Mini Kit (QIAGEN, Valencia, CA) was reverse transcribed with Quantitect Reverse Transcriptase (QIAGEN) and amplified by quantitative real-time PCR (Roche, Indianapolois, IN) using the KAPA SYBR Fast LightCycler 480 kit (PEQLAB, Biotechnology GmbH, Germany) and the primers listed in Table 1. To take into account any variation due to heterogeneity of RNA expression in different areas of the liver, we sampled tissue from two different lobes of each liver, such that in total 10 events were analyzed for each treatment group. Expression levels of the gene of interest were normalized to that of the internal housekeeping control (GAPDH).

Table 1. Real-time polymerase chain reaction primer sequences.

graphic file with name mt2013181t1.jpg

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Statistical analysis. Data were analyzed for statistical significance using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Individual data points were compared by applying a two-sided Student t-test, and P values of less than 0.05 were considered statistically significant.

Acknowledgments

We would like to thank Scott Friedman (Mount Sinai School of Medicine, NY) for sharing LX-2 cells and protocols. In addition, we are thankful to Johannes Schwarz and Daniela Dietel (Comprehensive Pneumology Center, Munich, Germany) for performing apoptosis staining and to Barbara Lindner and Sabrina Goldmann for excellent technical assistance. This work was supported in part by the German Research Aid (Max-Eder Research Program), the Federal Ministry of Education and Research (Grant 01GU0505) and the SFB 824 (DFG Sonderforschungsbereich 824), German Research Foundation, Bonn, Germany. With regards to disclosure of financial conflicts of interest, this work forms the scientific basis of an application for a patent, with JA and OE being the co-inventors.

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