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

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1 originl rticle The Americn Society of Gene & Cell Therpy Antifirotic Properties of Trnsrteril Oncolytic Therpy for Heptocellulr Crcinom in Rts With Thiocetmide-Induced Liver Firosis Jennifer Altomonte 1, Srin Mrozin 1, Enrico N De Toni 2, Antoni Rizzni 2, Irene Esposito 3,4, Ktj Steiger 3,4, Annette Feuchtinger 3, Clus Hellerrnd 5, Rolnd M Schmid 1 nd Oliver Eert 1 1 II. Medizinische Klinik und Poliklinik, Klinikum rechts der Isr, TU, München, Germny; 2 Medizinische Klinik und Poliklinik II, Klinikum G roßhdern, LMU, München, Germny; 3 Institut für Pthologie, Helmholtz Zentrum München, München, Germny; 4 Institute of Pthology, Klinikum rechts der Isr, TU, München, Germny; 5 Klinik und Poliklinik für Innere Medizin I, Universitätsklinikum Regensurg, Regensurg, Germny Recominnt vesiculr stomtitis virus () shows promise for the tretment of heptocellulr crcinom (HCC), ut its sfety nd efficcy when dministered in setting of heptic firosis, which occurs in the mjority of clinicl cses, is unknown. We hypothesized tht could provide novel enefit to the underlying firosis, due to its ility to replicte nd cuse cell deth specificlly in ctivted heptic stellte cells. In ddition to the ility of to produce significnt oncolytic response in HCC-ering rts in the ckground of thiocetmideinduced heptic firosis without signs of heptotoxicity, we oserved significnt downgrding of firosis stge, decrese in collgen content in the liver, nd modultion of gene expression in fvor of firotic regression. Together, this work suggests tht is not only sfe nd effective for the tretment of HCC with underlying firosis, ut it could potentilly e developed for clinicl ppliction s novel ntifirotic gent. Received 17 Decemer 212; ccepted 25 July 213; dvnce online puliction 24 Septemer 213. doi:1.138/mt INTRODUCTION Heptocellulr crcinom (HCC) is the third leding cuse of cncer-relted deth nd the fifth most common type of cncer in the world, ccounting for over 1 million cses nnully. 1 In roughly 8 9% of the ptients, these tumors rise from the ckground of liver cirrhosis, 2,3 resulting from wound-heling response to chronic liver injury, known s heptic firosis. In prticulr, longterm lcohol use nd chronic heptitis C virus infection re the most prominent underlying fctors responsile for liver cirrhosis in Europe nd North Americ. 4,5 The firotic response underlies virtully ll of the complictions of end-stge liver disese, including portl hypertension, scites, encephlopthy, nd metolic dysfunction, s well s the onset of HCC. 6 When HCC occurs in the setting of cirrhosis, the condition presents gret chllenge for clinicins, with the degree of liver function gretly influencing the possiility for curtive, or even pllitive therpies. Even in ptients who re dignosed erly, the course of the disese is often ftl due to the glring deficiency of ville therpies to simultneously tret HCC nd the underlying liver disese. Therefore, due to the ever-incresing incidence of cirrhosis nd susequent HCC, s well s the ovious limittions of currently ville therpies, novel nd effective tretments re urgently needed. Due to recent progress in understnding the pthogenesis of liver firosis, 7 it is now elieved to e reversile process. 8,9 During firogenesis, heptic stellte cells (HSCs) differentite from the quiescent to the ctivted form, mrked y chnge to myofirolst phenotype coinciding with expression of α-smooth muscle ctin (α-sma). These trnsdifferentited HSCs promote extrcellulr mtrix remodeling y deregulting the secretion of mtrix metlloproteinses nd tissue inhiitors of mtrix metlloproteinses (TIMPs), resulting in the degrdtion of the norml mtrix nd its replcement with interstitil collgen (primrily type I nd III) nd scr mtrix. These heptic chnges cuse distortion of the norml liver rchitecture nd led to decompensted liver function. While it is known tht the mjor mechnism for regression of firosis involves poptosis of ctivted HSCs, 1,11 the chllenge for ntifirotic therpy is specific trgeting of ctivted HSCs, without collterl effects on quiescent cells or myofirolsts present in other tissues. Unfortuntely, the mjority of drugs under investigtion hve resulted in only minor ntifirotic effects, with generl lck of specificity on the HSC ctivtion pthwy. 12 It hs recently een reported tht novel virl therpies, employing Newcstle disese virus or inctivted Orf virus, cn reverse the progression of the heptic condition. 13,14 We hve previously reported tht recominnt vesiculr stomtitis virus () vectors re effective oncolytic gents with inherent specificity for tumor cells Here, we demonstrte in vitro tht specificlly replictes in nd kills ctivted HSCs, while spring quiescent cells. Furthermore, we show in thiocetmide-induced rt model of firosis tht heptic rteril infusion of not only mintins its ility to efficiently kill tumor cells, ut it lso possesses ntifirotic properties which result in the unique enefit of concomitnt reversl of firotic progression. Together, these dt indicte tht is not only n intrinsiclly oncolytic virus, ut its specificity cn e extended to ctivted Correspondence: Oliver Eert, II. Medizinische Klinik und Poliklinik, Klinikum rechts der Isr, TU München, Ismningerstr. 22, Munich, Germny. E-mil: Oliver.eert@lrz.tum.de vol. 21 no. 11, nov. 213

2 The Americn Society of Gene & Cell Therpy Antifirotic Properties of for HCC nd Firosis HSCs, resulting in firotic regression. Therefore, hs the potentil to e developed into powerful therpeutic gent for the simultneous therpy of HCC nd underlying heptic firosis, which is urgently needed for the clinicl mngement of this complex condition. RESULTS replictes nd cuses cytotoxicity in ctivted HSCs To determine the reltive permissiveness of HSCs to repliction, primry humn heptocytes, HepG2, nd ctivted HSCs were infected with r-lcz for 24 hours. Although titers were pproximtely 4-logs higher in HepG2 cells s compred with primry humn heptocytes (1 9 versus 1 5, respectively), virl growth in primry HSCs ws intermedite (Figure 1). A comprison of differentilly cultured LX-2 nd primry HSCs reveled striking vrince in virus repliction, with titers elevted y 2 3 logs in ctivted compred with the quiescent cells (Figure 1), leding to sttisticlly significnt increse in cytotoxicity t oth 24 nd 48 hours postinfection with (Figure 1c). FACS nlysis of the su-g1 popultions of propidium iodidestined primry HSCs demonstrted significnt correltion etween the numer of poptotic cells fter infection with nd the degree of ctivtion (1 dys in culture versus 2 dys) (Figure 1d, left pnel). Furthermore, Hoechst stining of primry HSCs demonstrted chromtin condenstion nd nucler TCID5/ml c % Cytotoxicity LX-2 Primry humn HSC PHH HCC HSC Dy 2 Dy LX-2 TCID5/ml Log 1 % Cytotoxicity Primry humn HSC Dy 2 Dy 1 TCID5/ml Log 1 d Apoptosis (%) Time postinfection (hours) Time postinfection (hours) 48 hour 48 hours Figure 1 Specificity of r for ctivted heptic stellte cells (HSCs). () Primry humn heptocytes (PHH), HepG2 cells (heptocellulr crcinom), or ctivted primry humn HSC were infected with r-βgl t n MOI of.1 for 24 hours, nd virl titers in the medium were mesured y TCID5. () (serum-strved) or TGF-β-ctivted LX-2 cells nd quiescent-like (dy 2 postplting) or ctivted (dy 1) primry humn HSCs were infected with r-βgl t n MOI of.1, nd liquots of the conditioned medium were sujected to TCID5 nlysis for determintion of virl titers fter 24 hours. For oth cell types, sttisticlly significnt increses in titers were oserved in ctivted cells (P <.5). (c) or ctivted LX-2 nd primry humn HSCs were infected with r-βgl t n MOI of.1, nd lctte dehydrogense in the superntnt ws mesured t 8, 24, nd 48 hours post-trnsfection to determine cytotoxicity. Significnt increses (P <.5) were oserved in ctivted cells t 24 nd 48 hours postinfection. (d) -like (dy 2 postplting) or ctivted (dy 1) primry humn HSCs treted with either or r t n MOI of.1 for 48 hours were stined with propidium iodide nd sujected to FACS nlysis of su-g1 popultions for determintion of poptotic cells (left pnel). Additionl cells were sujected to Hoechst stining for determintion of nucler chnges ssocited with poptotic cells (right pnel). Arrows indicte chromtin condenstion nd nucler frgmenttion. Representtive imges re shown t 2 mgnifiction. Moleculr Therpy vol. 21 no. 11 nov

3 Antifirotic Properties of for HCC nd Firosis The Americn Society of Gene & Cell Therpy frgmenttion in the more highly ctivted HSCs fter 48-hour infection with (Figure 1d, right pnel), indicting tht induces poptosis in HSCs in n ctivtion-dependent mnner. infection results in phenotypic chnges of differentited HSCs To investigte the effects of on ctivted HSCs t erly time points fter infection, immunofluorescence stining for αsma ws performed. As n internl control, cells were costined for vinculin, protein which is expressed in oth quiescent nd ctivted HSCs. 18 While cells expressed very low levels of αsma fter 2 dys in culture, the expression level ws sustntilly incresed fter 1 dys (Figure 2), indicting tht the mount of time in culture is directly correlted with degree of ctivtion. For simplicity, we refer to the 2-dy-cultured HSCs s quiescent nd the 1-dy-cultured cells s ctivted. HSCs infected with for 12 hours expressed much lower levels of αsma s compred with the -treted cells t the sme time point (Figure 2), wheres vinculin expression ws mintined, suggesting tht the loss of αsma ws most likely due to prtil Vinculin αsma Dpi Merge + c d αsma Fold mrna expression 1..5 αsma β-tuulin Primry HSC LX2. Figure 2 Loss of ctivtion of heptic stellte cells (HSCs) in response to vesiculr stomtitis virus () infection. () -like or ctivted primry humn HSCs treted with either or r for 12 hours were sujected to immunofluorescent costining for αsma nd vinculin using FITC- nd Cy3-leled secondry ntiodies, respectively. Counterstining ws performed using DAPI for detection of nuclei. Representtive photomicrogrphs re shown t 2 mgnifiction, nd the scle r = 1 μm. () primry humn HSCs were treted with or rβgl t n MOI of.1 for 12 hours. As control, quiescent-like cells were prepred. Aliquots of cdna prepred from reverse-trnscription of mrna were sujected to rel-time PCR for quntifiction of αsma expression. Reltive vlues were quntified y normlizing the expression level of αsma to the internl housekeeping control (GAPDH) nd setting the vlues for the quiescent cells to 1, such tht the level for ctivted cells represents fold increse or fold decrese. Mens from ech tretment group re shown, with error rs representing stndrd devitions. The reduction in αsma mrna expression in -treted cells is significnt (P <.5) with respect to -treted cells. (c) Western lots were performed from 1 μg protein from cell lystes of quiescent or ctivted humn HSCs treted with either or r-βgl t n MOI of.1 for 12 hours. Antiodies specific for αsma or the housekeeping protein β-tuulin were used. The imge shown is representtive of three independent experiments. (d) Geltin zymogrphy ws performed from superntnts from quiescent or ctivted primry humn HSCs (top pnel) or LX-2 cells (ottom pnel) fter 8-hour tretment with or r. Negtive nds indicte mtrix metlloproteinses-2 ctivity vol. 21 no. 11 nov. 213

4 The Americn Society of Gene & Cell Therpy Antifirotic Properties of for HCC nd Firosis inctivtion or senescence, rther thn cell deth. Quntittive rel-time PCR using primers specific for humn αsma reveled significnt reduction in αsma mrna expression in ctivted HSCs 12 hours postinfection with (Figure 2), nd western lot nlysis demonstrted similr reduction in αsma t the protein level t the sme time point (Figure 2c). Mtrix metlloproteinse-2, n enzyme which degrdes type IV collgen, is known to e secreted from ctivted HSCs, nd levels re upregulted in firotic liver tissue. 19,2 Geltin zymogrphy demonstrted n increse in mtrix metlloproteinse-2 ctivity in the superntnts of ctivted versus quiescent primry HSCs nd LX-2 cells, which ws rogted fter 12-hour infection with (Figure 2d). Whether these events represent true shift towrd inctivtion, or they re mere consequence of the generl shutdown of host trnsltion, remins to e determined. Specificity of repliction for ctivted HSCs is ssocited with cell-cycle progression Becuse interferon (IFN) signling defects hve een identified s mechnism which llows oncolytic viruses to replicte in tumor cells, 21 we investigted whether this sme mechnism could explin the specificity of for ctivted HSCs. Reporter ssys for the IFN-β nd IFN-stimulted response element promoters were performed to ssess IFN induction nd response, respectively. For these experiments, we utilized mutnt r vector (M51R), which hs n enhnced ility Fold-induction IFN-β promoter Fold-induction ISRE promoter TCID5/ml IFN protection TCID5/ml pl:c M51R Growth curves Q + Q + M51R A + A + M51R Time postinfection (hours) TCID5/ml 1 pl:c M51R , IFN conc. (IU/ml) 1 1 Virl titers DMSO MNK Rosco Rp CDK4 Ly29 Aphi % Of cells 1 5 Cell cycle nlysis DMSO MNK Rosco Rp CDK4 Ly29 Aphi G1 S G2 c Cyclin B1 Topo 2α Actin B1 scr Topo sirna % Of cells Cell cycle nlysis Virl titers G1 1 5 S 1 4 G Control scr Cyc B1 Topo2α scr Cyclin B1 Topo-2α TCID5/ml sirna Figure 3 Mechnism of r specificity for ctivted heptic stellte cells (HSCs). () IFN-β nd ISRE promoter ctivtion in primry humn HSCs ws quntified using luciferse reporter plsmids. or ctivted cells were cotrnsfected with pifnβ-luc or pisre-luc nd prl (constitutively ctive Renill), nd stimulted 24 hours post-trnsfection with pi:c or IFN, nd nd (M51R). After overnight incution, firefly luciferse expression levels were normlized to Renill expression nd clculted s fold-induction in comprison with mock-stimulted cells. IFN protection ssy results re presented in the right pnel. or ctivted HSCs were pretreted overnight with incresing doses of IFN s indicted nd infected with r t n MOI of.1 for 24 hours. Aliquots of superntnt were sujected to TCID5 nlysis for the determintion of virl titers. Multicycle growth curves re shown for quiescent (Q) or ctivted (A) primry HSCs infected with or (M51R) t n MOI of.1. Virl titers were determined y TCID5, nd dt re presented s the men of three experiments ± stndrd devition. () LX-2 cells were treted with pnel of cell-cycle inhiitors or DMSO for 24 hours prior to overnight infection with r t n MOI of.1. TCID5 nlysis of virl titers re shown (left pnel). P vlue for Rosco versus DMSO <.5. cells treted with DMSO or cell cycle inhiitors for 24 hours were stined with propidium iodide (PI) for FACS nlysis of percentge of cells in ech phse of the cell cycle (right pnel). (c) LX-2 cells were trnsfected with sirnas trgeting cyclin B1 or topoisomerse-2α, or with scrmle sirna, nd then infected with t n MOI of.1 overnight. Cell lystes were sujected to western lot nlysis to confirm knock-down (left pnel), nd PI-stined cells were nlyzed y FACS to determine cell cycle (middle pnel). Aliquots of the medium were nlyzed y TCID5 to determine virl titers (P <.5 for sirna versus scr). Men vlues of ll dt re presented + SD of triplicte experiments. Moleculr Therpy vol. 21 no. 11 nov

5 Antifirotic Properties of for HCC nd Firosis The Americn Society of Gene & Cell Therpy to trigger IFN responses due to n mino cid sustitution in the endogenous mtrix protein. Although oth quiescent nd ctivted LX-2 cells demonstrted induction of IFN-β nd IFNstimulted response element in response to polyi:c or IFN nd r (M51R) stimultion, there ws slight impirment of IFN signling in the ctivted cells (Figure 3). To further ddress this issue, multicycle growth curves of r nd r(m51r) were compred in quiescent versus ctivted primry HSCs. In line with the reporter ssys, we oserved n ttenution of the M51R virus in comprison with in oth quiescent nd ctivted cells; however, the titers of M51R t 24 nd 48 hours postinfection were t lest 1-log higher in the ctivted cells, indicting tht IFN signling in ctivted cells ws only prtilly responsive or tht perhps there is n dditionl mechnism in plce which llows the virus to replicte in ctivted HSCs, despite functionl IFN signling pthwy. Further investigtion y IFN protection ssy reveled tht reltively low concentrtions of IFN were sufficient to inhiit repliction in ctivted HSCs, nd no significnt differences in titers were detected mong ctivted nd quiescent cells regrdless of the dose of IFN (Figure 3). Tken together, we concluded tht IFN signling is perhps not the mjor determinnt of specificity of for ctivted cells. To explore the role of cell prolifertion on repliction, ctivted LX-2 cells were pretreted with pnel of cell cycle inhiitors prior to infection. While most of the inhiitors hd no ffect on virus growth, pretretment with roscovitine cused significnt ttenution (Figure 3). FACS nlysis of propidium iodide-stined cells fter 24 hours of tretment with ech cell cycle inhiitor reveled tht roscovitine ws unique in tht it cused significnt cellulr ccumultion in G2 phse (Figure 3). To confirm the role of G2 rrest on repliction, we pplied sir- NAs ginst cyclin B1 nd topoisomerse-2α, which re ssocited with G2-S progression. After verifying tht the expression of ech protein ws inhiited y the respective sirna nd tht G2 cell cycle rrest ws chieved, we mesured virl titers in superntnts of LX-2 cells fter overnight infection in sirna-treted cells. Indeed, ws significntly ttenuted in cells treted with sirna trgeting cyclin B1 nd topoisomerse-2α in comprison with the scrmle control (Figure 3c). replictes in HCC tumors in firotic setting nd lso loclizes to ctivted HSCs To investigte the effect of liver function on therpy for HCC, rts hroring HCC in the setting of helthy versus firotic liver were rndomized for injection of or r-βgl vi the heptic rtery. Anlysis of tissue extrcted fter 24 hours of tretment reveled tht mintins its ility to ccess nd replicte in tumors growing in the context of heptic firosis, without cusing significnt chnges in intrheptic titer (Figure 4). Immunofluorescent stining of tissue sections reveled tht, lthough the mjority of protein ws loclized within tumors, some coloclized with stellte cells within firotic liver, s demonstrted y costining for desmin (Figure 4). Together, these dt indicte tht the repliction of within ctivted HSCs in vivo is proly limited, yet sufficient to produce therpeutic effect. Of note, no protein ws detected in norml heptocytes, nd histologicl exmintion of liver tissue reveled no oservle toxicity ssocited with r therpy. Morphometric nlysis of tumor sections demonstrted tht r tretment resulted in comprle tumor responses, irrespective of liver firosis; however, tumors generlly grew to lrger size in the firotic setting, resulting in higher percentge of seline necrosis (Figure 4). This phenomenon, in which HCC tumors grow more ggressively in firotic environment, is in line with previous report compring the invsiveness of HCC tumors implnted in thiocetmide-induced firotic, versus norml, liver setting. 22 Serum chemistries for glutmic pyruvic trnsminse, lood ure nitrogen, nd cretinine demonstrted only trnsient elevtions, which returned to norml levels on dy 3 nd did not differ significntly mong tretment groups, indicting no significnt impirment of heptic or renl function in response to therpy (Figure 4c). In vivo therpy results in improved liver stging nd reduction of heptic firosis Liver sections were sujected to histologicl exmintion nd Desmet scoring to determine the firotic stges in response to or tretment. While -treted sections were ssigned men score of 3 4 due to the presence of portoportl nd portocentrl ridges, r-treted sections received men score of 2, representing significnt improvement in firotic stge (Figure 5). As quntittive mesure of firosis, the intrheptic collgen contents of versus r-treted nimls were compred y Sircol ssy, which demonstrted significnt reduction in solule collgen contents in response to r therpy (P <.5) (Figure 5). Trnsrteril infusion of r cuses reduction of αsma expression y HSCs Immunohistochemicl stining for αsma demonstrted cler reduction in the intensity nd numer of positively stined cells on dy 1 post-r dministrtion s compred with the controls (Figure 6). Semiquntittive western lot nlysis similrly demonstrted significnt reduction of αsma in firotic livers in response to r therpy (P =.1) (Figure 6). Furthermore, quntittive rel-time PCR reveled tht the modultion of αsma occurs t the mrna level, with significnt decrese (P <.5) in αsma mrna expression in firotic livers treted with r s compred with (Figure 6c). These results indicte tht cuses either reduction in ctivted HSC numer or dedifferentition into n inctivted or senescent stte. r tretment is ssocited with the poptosis of ctivted HSCs To identify poptotic-ctivted HSCs, we performed costining for TUNEL nd αsma in firotic liver tissue. Hyrid imges reveled striking colocliztion of poptotic nuclei with αsmapositive cells in -treted livers (Figure 7), suggesting n importnt mechnism for the reversl of firosis. Of note, the numer of poptotic heptocytes ws similr mong tretment groups, implying tht there ws no significnt compromise to the norml liver prnchym ssocited with therpy in this model vol. 21 no. 11 nov. 213

6 The Americn Society of Gene & Cell Therpy Antifirotic Properties of for HCC nd Firosis TCID5/mg tissue Virl titers Control TAA % Necrosis Morphometric nlysis Tumor re (cm 2 ) Tumor re 1 Liver Tumor Control Control TAA TAA. Control TAA Desmin DAPI Merge Firotic liver Tumor c GPT (U/l) GPT Cretinine (mg/dl) Cretinine BUN (mg/dl) BUN Control- TAA- Control- TAA- Dy 1 Dy 3 Time post-tretment (dys). Dy 1 Dy 3 Time post-tretment (dys) Dy 1 Dy 3 Time post-tretment (dys) Figure 4 Specificity nd sfety of vesiculr stomtitis virus () for heptocellulr crcinom (HCC) with underlying heptic firosis. () Rts hroring unifocl HCC lesions implnted into helthy livers versus livers with thiocetmide-induced heptic firosis were treted y heptic rteril infusion of or r-βgl (n = 5). On dy 1 post-tretment, smples of liver nd tumor were snp-frozen nd nlyzed y TCID5 to determine virus titers (left pnel). Morphometric nlysis of necrotic res ws performed on H/E preprtions of tumor sections excised on dy 3 post-tretment using ImgeJ softwre (middle pnel). Tumor res were determined y mesuring the length nd width of ech tumor prior to fixtion (right pnel). Men vlues + SD re shown in ech pnel. () Additionl smples otined on dy 1 post-tretment were fixed overnight nd prffin-emedded. (red) nd desmin (green) were visulized y immunofluorescent stining. Nuclei were detected y DAPI stining (lue). Representtive imges of tumor nd firotic liver re extrcted from -treted liver re shown t 63 nd 4 mgnifiction, respectively.white rrows indicte res of colocliztion. Scle r = 2 μm in tumor tissue nd 5 μm in liver. (c) Serum smples from control nimls nd nimls with thiocetmide-induced heptic firosis were otined on dy 1 nd 3 postheptic-rteril dministrtion of r or. Men vlues + SD re shown. Nturl killer (NK) cell ccumultion corresponds with r tretment in firotic livers Incresing evidence demonstrtes tht NK cells inhiit heptic firosis vi direct killing of ctivted HSCs nd y induction of IFNγ, justifying the clinicl development of NK cell therpy for heptic firosis. 23,24 Since we hve previously oserved striking intrtumorl infiltrtion of NK cells to sites of repliction, 25,26 we performed immunohistochemicl stining of firotic liver tissue to quntify the heptic ccumultion of NK cells in response therpy. Of note, we oserved significnt increse in the numer of NK cells in - versus -treted livers (Figure 7), which ccumulted long the connective tissue. We speculte tht virus-induced ctivtion of NK cells could potentite ystnder cell killing, cusing poptosis even in those HSCs not directly infected y. The precise role of NK cells in -medited ntifirotic therpy is n importnt topic of ongoing investigtions. In ddition, to further chrcterize the immune cell infiltrte in response to ntifirotic therpy, we performed immunohistochemicl stining for myeloperoxidse-positive grnulocytes, CD68+ mcrophges, nd CD3+ T-lymphocytes; however, no significnt differences in cell numers were detected within firotic liver tissue treted with versus (dt not shown). Moleculr Therpy vol. 21 no. 11 nov

7 The Americn Society of Gene & Cell Therpy Antifirotic Properties of for HCC nd Firosis Norml liver Firosis/ Firosis/ Tuulin Figure 5 Vesiculr stomtitis virus () therpy cuses improvement in liver stging nd reduction in heptic collgen content. () Rts hroring thiocetmide-induced heptic firosis were treted y intrheptic rteril infusion of either or r nd scrificed on dy 1 post-tretment. Prffin-emedded liver sections were visulized y H/E stining, nd representtive fields of view were photogrphed t 2 or 1 mgnifiction. Stge 4 firosis (prole or definite cirrhosis) is exemplrily shown in the -treted upper pnel, s evidenced y firous ridges (rrows) connecting portl trcts (PT) nd centrl veins (V) with formtion of multiple nodules. The -treted ottom pnel is representtive of stge 2 firosis, in which connective tissue links neighoring portl trcts, ut the overll rchitecture is preserved. () A commercilly ville kit ws used to determine the concentrtions of cid-pepsin solule collgen in firotic livers treted with or for 24 hours. Men concentrtions + SD re shown. P <.5. tretment results in therpeutic modultion of key genes ssocited with firotic progression To investigte the effects of r therpy on relevnt mrna expression, we performed quntittive rel-time PCR. Consistent with repression of the firotic condition, we oserved significnt decreses in TGF-β, collgen 1, nd TIMP-1 mrna, s well s n increse in IFN-α mrna in response to therpy fter 24 nd 72 hours; representtive dt from 24 hours re shown (Figure 8). Of prticulr importnce, TGF-β hs een identified s potentil nti-inflmmtory nd ntifirotic trget, nd therpies imed t inhiition of TGF-β signling re under development.27 Therefore, -medited inhiition of TGF-β expression could provide significnt indirect mechnism of ntifirotic ctivity. In ddition, the upregultion of IFNα mrna is noteworthy, s it ws recently demonstrted tht the dministrtion of Control qpcr Fold-mRNA conc. 3 Reltive intensity Collgen μg/mg liver tissue αsma Figure 6 Vesiculr stomtitis virus () therpy leds to decresed αsma expression in vivo. () Rts hroring thiocetmide-induced firotic livers were treted with either or r, nd prffin sections were prepred from tissues hrvested 24 hours post-tretment. Immunohistochemicl stining ws performed with n ntiody specific for αsma, nd representtive sections were imged t 5 (left pnel) or 2 (right pnel) mgnifiction. The lck r indictes 1 μm. () Homogentes of snp-frozen sections of norml liver or thiocetmide-induced firotic liver hrvested 24 hours fter intr-heptic rteril infusion of or were sujected to western lot nlysis for αsma expression. Blots were stripped nd reproed with n ntiody for tuulin to control for loding vritions. Semiquntifiction of nd intensities normlized to tuulin ws performed using ImgeJ softwre, nd vlues re expressed s reltive intensity with respect to the norml liver control. P-vlue for -treted smples versus is <.5. Three representtive liver homogentes from ech tretment group re shown. (c) mrnas extrcted from firotic liver treted with or for 24 hours were reverse-trnscried nd used s templte for quntittive rel-time PCR (qpcr) to determine the reltive SMA expression level fter tretment. Levels were normlized to GAPDH, nd -treted smples re presented in terms of their reltive expression level with respect to the controls. Mens + SD re shown. P <.5. IFNα meliortes firosis vi reduction in TGFβ nd TIMP-1 expression.28 DISCUSSION The rising incidence of cirrhosis nd susequent onset of HCC, coupled with the ovious deficiency of currently ville therpies to mnge this complex clinicl scenrio, highlight the urgent need for lterntive nd effective tretment modlities. A mjor wekness of mny preclinicl investigtions into novel HCC therpies is tht they rely on inpproprite niml models, which poorly reflect the clinicl setting. We hve previously reported in vol. 21 no. 11 nov. 213

8 The Americn Society of Gene & Cell Therpy Antifirotic Properties of for HCC nd Firosis αsma TUNEL DAPI Merge NK cells 3 Cells/ 1 field 2 1 Figure 7 Incresed TUNEL stining nd NK cells in firotic livers following vesiculr stomtitis virus () therpy. Rts hroring thiocetmide-induced heptic firosis were treted with or r vi trnsrteril infusion of the heptic rtery. () On dy 3 post-tretment, nimls were euthnized, nd liver sections were prffin-emedded nd sujected to coimmunofluorescent stining to loclize terminl deoxynucleotidyl trnsferse dutp nick end leling (TUNEL), ppering in yellow, nd αsma, in red. Sections were counterstined with DAPI for locliztion of nuclei. Representtive sections re shown t 4 mgnifiction with oil (white r indictes 1 μm). () Liver sections otined on dy 3 fter tretment with (left pnel) or (right pnel) were sujected to immunohistochemicl stining for nturl killer cell mrker (ANK61). Representtive sections re shown t 2 mgnifiction (scle r = 1 μm). The numer of positively stined cells per 1 field of view ws quntified nd is shown s men + stndrd devition. P vlue <.1. TGF-β 2. Collgen Fold-mRNA conc Fold-mRNA conc IFN-α 2. TIMP-1 Fold-mRNA conc Fold-mRNA conc Figure 8 Modultion of mrna expression following r dministrtion. Thiocetmide-induced firotic livers in rts were treted y trnsrteril infusion of or r vi the heptic rtery. Liver smples snp frozen 24 hours post-tretment were sujected to mrna purifiction nd susequent cdna preprtion using commercilly ville kits. Aliquots of cdna were used for the quntifiction of relevnt genes ssocited with firotic progression y rel-time PCR, s indicted. vlues were set to 1, nd vesiculr stomtitis virus vlues were clculted s fold-expression with respect to. Mens from ech tretment group re shown, with error rs representing stndrd devitions. P vlues <.5 were considered significnt. Moleculr Therpy vol. 21 no. 11 nov

9 Antifirotic Properties of for HCC nd Firosis The Americn Society of Gene & Cell Therpy n immune-competent rt model of orthotopic HCC tht heptic rteril infusion of recominnt results in significnt tumor responses nd susequent prolongtion of survivl. 16,29,3 Here, we investigte the use of in more chllenging nd cliniclly relevnt model of HCC with underlying heptic firosis. We demonstrte tht heptic rteril infusion of in this setting is not only sfe nd effective s n HCC therpy, ut it lso possesses inherent ntifirotic properties. In prticulr, dministrtion resulted in reduction of ctivted HSCs, decresed firotic content in the liver, nd qulittive improvement in liver stging s determined y Desmet score. The dt presented here demonstrte tht the ntifirotic effects of re potentilly the result of three distinct, lthough potentilly interconnected, mechnisms: (i) direct cell killing of infected HSCs, (ii) recruitment of ctivted NK cells, nd (iii) modultion of gene expression in fvor of firotic resolution. Whether the modultory effects on NK cells nd gene expression truly represent direct response to infection, or they re merely consequence of the reduction of ctivted HSCs, remins to e seen nd is topic of ongoing investigtions. However, regrdless of the mechnism, we speculte tht these indirect spects of therpy re crucil in providing fvorle therpeutic outcome, s it is improle tht the virus cn homogenously infect HSCs throughout the liver, especilly in the firotic context, where lrge mounts of extrcellulr mtrix provide physicl rrier to efficient virus spred. Furthermore, since these studies focused on the therpy of HCC in cirrhotic context, we did not investigte the efficcy of therpy for firosis in the sence of heptic tumors. Therefore, we cn only presume tht the ntifirotic properties of re not dependent on the presence of HCC; however, since the tretment of the firotic condition prior to the onset of HCC would e highly desirle ppliction for future therpy, these investigtions re wrrnted nd re ongoing. As criticl endpoint, we closely exmined the sfety of therpy for HCC in the firotic setting. Becuse of the correltion etween repliction nd cell cycle progression in ctivted HSCs, it ws n ovious concern tht the virus could indvertently replicte in other proliferting cells in the setting of chronic liver injury; however, we sw no indiction tht this ws the cse in our model. Intrheptic virl titers were comprle in firotic versus helthy livers, nd creful exmintion of histology nd immunofluorescent stining filed to revel ny signs of toxicity or virl repliction in heptocytes in the firotic setting. This could e in prt due to the fct tht heptocyte prolifertion is thought to occur only s first-line defense, fter which time if the injury ecomes chronic, heptocytes lose their ility to replicte, nd the mjority of liver regenertion occurs vi differentition of heptic progenitor cells (lso known s ovl cells). 31 Furthermore, we hve previously shown tht in HCC cells, prolifertion does not ply role in determining the ility of to replicte. 32 Therefore, we speculte tht cell cycle progression is merely component of HSC permissiveness to, nd tht other spects of the ctivtion process surely contriute to this phenomenon. Further investigtions into the mechnism of specificity for ctivted HSCs re ongoing. Due to significnt improvements over the lst decde in the tretment of heptitis C virus, it is now possile to fully resolve the infection; 33 however, in the sence of n effective ntifirotic therpy, those ptients with estlished cirrhosis nevertheless fce high risk of developing HCC. It is therefore crucil tht novel therpies re developed to sfely nd effectively tret heptic firosis. Here, we hve shown tht replictes selectively oth in tumor cells nd ctivted HSCs, providing n idel gent for the simultneous tretment of oth HCC nd underlying firosis. Furthermore, if is dministered t the erly stges of firosis development, it is possile tht the progression of the disese cn e interrupted, nd the onset of HCC nd other complictions of end-stge liver disese cn e delyed or potentilly voided. However, we cknowledge tht further modifictions of the pltform will surely e necessry, s the long-term survivl enefit provided y the wild-type vector my e minor, if ny. Therefore, the current studies re ment to report our novel findings s proof-of-principle for future long-term survivl studies, which will employ optimized vectors nd comintion therpies, s well s dditionl niml models of heptic firosis, to comprehensively ssess the potentil clinicl enefit of s n ntifirotic nd oncolytic gent. In conclusion, the dt presented here support the development of vectors s sfe nd effective tools for the clernce of ctivted HSCs nd susequent regression of firosis. This work suggests tht the use of, nd possily other negtive-strnd RNA viruses, is not limited to cncer therpy, ut cn potentilly e pplied to dditionl therpeutic trgets. The simultneous therpy of HCC nd underlying heptic firosis with single gent provides significnt therpeutic dvntge over the current stte of the rt, nd therefore, hs the potentil to e developed into powerful oncolytic nd ntifirotic gent for future clinicl ppliction. MATERIALS AND METHODS Virus nd cell lines. Production of r-lcz nd r (M51R) nd propgtion of BHK-21 cells, the rt HCC cell line, McA-RH7777 (ATCC, Mnsss, VA), nd HepG2 cells ws performed s descried. 3,32 Unless otherwise stted, infections were performed t multiplicity of infection (MOI) of.1 for 24 hours, nd virl titers were quntified y TCID5 nlysis on BHK-21 cells. Multicycle growth curves were performed in quiescent nd ctivted humn HSCs y infection with viruses t n MOI of.1 for 1 hour, followed y wshing nd replcement with complete medium. Aliquots of superntnt were hrvested t vrious time points postinfection (, 16, 24, nd 48 hours) nd nlyzed y TCID5. LX-2 cells, kind gift from Scott Friedmn (Mount Sini School of Medicine, NY), were either serum-strved or supplemented with 1% FBS nd 2 ng/ ml TGFβ (R&D Systems, Minnepolis, MN) for 48 hours for differentition into quiescent or ctivted phenotypes. Cryopreserved ctivted primry humn HSCs were cultured in DMEM (ATCC) supplemented with 1% FBS nd plted for either 2 or 1 dys, to chieve either quiescentlike or ctivted sttus, respectively. Lctte dehydrogense ssys (Roche Applied Science, Indinpolois, IN) were performed s recommended, or cells were incuted with 1 μg/ml Hoechst (Sigm-Aldrich, St. Louis, MO) nd visulized y fluorescence microscopy for the detection of nucler leing. Propidium iodide (Sigm-Aldrich)-stined cells were nlyzed for cell cycle phses or su-g1 events using flow cytometry (FACS cliur with Cell Quest Softwre; Becton Dickinson, Sn Jose, CA) s previously descried. 34 Geltin zymogrphy. LX-2 nd primry HSCs were mock-treted or infected with r for 12 hours prior to loding liquots of conditioned 24 vol. 21 no. 11 nov. 213

10 The Americn Society of Gene & Cell Therpy Antifirotic Properties of for HCC nd Firosis Tle 1 Rel-time polymerse chin rection primer sequences Gene Forwrd primer Reverse primer αsma 5 -CACCAACTGGGACGACATGG-3 5 -CCATCTCCAGAGTCCAGCAC-3 TGFβ 5 -AAGAAGTCACCCGCGTGCTA-3 5 -TGTGTGATGTCTTTGGTTTTGTCA-3 Collgen1 5 -GCGGAGAGTACTGGATCGACCCT-3 5 -CCTCGGTGGACATCAGGCG-3 IFNα 5 -GCTATCCCTGTCCTGCATGAGCTG-3 5 -CAGGGCTCTCCAGACTTCTGC-3 TIMP1 5 -GCCTACACCCCAGCCATGGAG-3 5 -TGGATTCCGTGGCAGGCAGGC-3 GAPDH 5 -CAACGACCCCTTCATTGACCTC-3 5 -CACCAGCATCACCCCATTTG-3 medium onto polycrylmide gels contining.1% geltin (Sigm- Aldrich). Gels were developed nd stined with 5% coomssie, ccording to estlished protocols 35 nd scnned using n Odyssey infrred imger (LI-COR Biosciences, Lincoln, NE). IFN ssys. Luciferse reporter ssys were performed s previously descried. 36 Cells were either mock-treted, or stimulted with poly I:C (2.5 μg/ml), universl type I IFN (5 IU), or r-lcz, or r(m51r)- GFP t MOI of 1 overnight. For IFN protection ssys, cell monolyers were mock-treted or treted overnight with Universl type I IFN (PBL Interferon Source, Pisctwy, NJ), followed y infection t MOI of 1. Virl titers were quntified fter 24 hours. Cell cycle inhiitors nd sirna. HSCs were treted overnight with DMSO or the following chemicl cell cycle inhiitors prior to infection with r-lcz: MNK1 (4 μmol/l), Roscovitine (5 μmol/l), Rpmycin (1 nmol/l), CDK4 (25 nmol/l), LY2942 (4 μmol/l), or Aphidicolin (2.5 μg/ml) (Cliochem-Merck, Gistown, NJ). Inhiitor doses were chosen sed on tht shown to produce the strongest inhiition in the sence of toxicity in preliminry dose response experiments in ctivted HSCs. In ddition, LX-2 cells were trnsfected with sirnas (Dhrmcon, Wlthm, MA), using Lipofectmine RNAiMAX (Invitrogen, Crlsd, CA). After 48 hours, the trnsfected cells were infected overnight with r-lcz. Animl studies. All procedures involving nimls were performed ccording to the guidelines of the institution s niml cre nd use committee, nd the locl government. Six-week-old mle Bufflo rts (Hrln Winkelmnn; Borchen, Germny) received either norml drinking wter or.1% solution of thiocetmide continuously over the course of the experiment. After 19 weeks, 1 6 McA-RH7777 cells suspended in 2 μl of DMEM were implnted orthotopiclly in the liver. After 1 dys, when HCC nodules reched.5 1 cm in dimeter, or 1 7 pfu of r-lcz (n = 1) ws infused vi the heptic rtery, nd nimls were euthnized on dy 1 or 3 fter tretment (n = 5). Serum chemistry mesurements were performed t the Institute for Clinicl Chemistry nd Pthoiochemistry t the Klinikum rechts der Isr. Tumor nd liver sections were flsh frozen or fixed overnight in 4% prformldehyde. Histology, immunohistochemistry, nd immunofluorescence. 3 μm thin prffin sections were stined with hemtoxylin eosin or immunohistochemiclly using ntiodies for αsma (Sigm-Aldrich) or the NK cell mrker ANK61 (Snt Cruz Biotechnology, Snt Cruz, CA). Douleimmunofluorescent stinings were performed using the following comintions of ntiodies: desmin (Thermo Scientific, Wlthm, MA) nd -G (Alph Dignostic, Sn Antonio, TX) in conjunction with FITCconjugted ntirit nd Cy3-conjugted ntimouse ntiodies (Jckson ImmunoReserch Lortories, West Grove, PA); αsma (Sigm-Aldrich) nd Alex Fluor 568 conjugted got nti-mouse IgG (Invitrogen) together with the Apoptosis Detection Kit (Millipore, Billeric, MA). ImgeJ softwre (Ntionl Institutes of Helth, Bethesd, MD) ws used for morphometric nlysis of tumor necrosis. Firotic stges were ssessed on 4 scle ccording to the criteri of Desmet 37 y pthologist who ws lind to the study groups. Collgen ssy. Snp-frozen liver sections otined on dy 1 post-tretment were sujected to nlysis of cid pepsin solule collgen content using the Sircol Collgen Assy (Biocolor, Crrickfergus, UK) ccording to the mnufcturer s instructions. Western lot nd rel-time PCR. Nitrocellulose memrnes (Amershm, Buckinghmshire, UK) were proed with the following ntiodies: αsma (Sigm-Aldrich), cyclin B1 (Cell Signling, Boston, MA), topoisomerse 2α (Cell Signling), nd tuulin (Sigm-Aldrich) nd ntimouse or rit secondry ntiodies conjugted with horserdish peroxidse (Jckson ImmunoReserch Lortories). The ECL chemiluminescence kit (Amershm) ws used for detection. ImgeJ softwre (NIH) ws used for semiquntifiction. RNA extrcted from snp-frozen tissue using the RNesy Mini Kit (QIAGEN, Vlenci, CA) ws reverse trnscried with Quntitect Reverse Trnscriptse (QIAGEN) nd mplified y quntittive rel-time PCR (Roche, Indinpolois, IN) using the KAPA SYBR Fst LightCycler 48 kit (PEQLAB, Biotechnology GmH, Germny) nd the primers listed in Tle 1. To tke into ccount ny vrition due to heterogeneity of RNA expression in different res of the liver, we smpled tissue from two different loes of ech liver, such tht in totl 1 events were nlyzed for ech tretment group. Expression levels of the gene of interest were normlized to tht of the internl housekeeping control (GAPDH). Sttisticl nlysis. Dt were nlyzed for sttisticl significnce using GrphPd Prism 5. (GrphPd Softwre, Sn Diego, CA). Individul dt points were compred y pplying two-sided Student t-test, nd P vlues of less thn.5 were considered sttisticlly significnt. ACKNOWLEDGMENTS We would like to thnk Scott Friedmn (Mount Sini School of Medicine, NY) for shring LX-2 cells nd protocols. In ddition, we re thnkful to Johnnes Schwrz nd Dniel Dietel (Comprehensive Pneumology Center, Munich, Germny) for performing poptosis stining nd to Brr Lindner nd Srin Goldmnn for excellent technicl ssistnce. This work ws supported in prt y the Germn Reserch Aid (Mx-Eder Reserch Progrm), the Federl Ministry of Eduction nd Reserch (Grnt 1GU55) nd the SFB 824 (DFG Sonderforschungsereich 824), Germn Reserch Foundtion, Bonn, Germny. With regrds to disclosure of finncil conflicts of interest, this work forms the scientific sis of n ppliction for ptent, with JA nd OE eing the co-inventors. REFERENCES 1. Prkin, DM, Bry, F, Ferly, J nd Pisni, P (21). Estimting the world cncer urden: Gloocn 2. Int J Cncer 94: Okud, H (27). Heptocellulr crcinom development in cirrhosis. Best Prct Res Clin Gstroenterol 21: Mzznti, R, Grmntieri, L nd Bolondi, L (28). Heptocellulr crcinom: epidemiology nd clinicl spects. Mol Aspects Med 29: Colomo, M (1993). Heptocellulr crcinom in cirrhotics. Semin Liver Dis 13: Lefton, HB, Ros, A nd Cohen, M (29). Dignosis nd epidemiology of cirrhosis. Med Clin North Am 93: , vii. 6. Lim, YS nd Kim, WR (28). The glol impct of heptic firosis nd end-stge liver disese. Clin Liver Dis 12: , vii. 7. Hernndez-Ge, V nd Friedmn, SL (211). Pthogenesis of liver firosis. Annu Rev Pthol 6: Moleculr Therpy vol. 21 no. 11 nov

11 Antifirotic Properties of for HCC nd Firosis The Americn Society of Gene & Cell Therpy 8. Povero, D, Buslett, C, Novo, E, di Bonzo, LV, Cnnito, S, Pternostro, C et l. (21). Liver firosis: dynmic nd potentilly reversile process. Histol Histopthol 25: Friedmn, SL nd Bnsl, MB (26). Reversl of heptic firosis fct or fntsy? Heptology 43 (2 Suppl. 1): S82 S Kisselev, T nd Brenner, DA (27). Role of heptic stellte cells in firogenesis nd the reversl of firosis. J Gstroenterol Heptol 22 (Suppl. 1): S73 S Elshrkwy, AM, Okley, F nd Mnn, DA (25). The role nd regultion of heptic stellte cell poptosis in reversl of liver firosis. Apoptosis 1: Poynrd, T, McHutchison, J, Dvis, GL, Esten-Mur, R, Goodmn, Z, Bedoss, P et l. (2). Impct of interferon lf-2 nd rivirin on progression of liver firosis in ptients with chronic heptitis C. Heptology 32: Nowtzky, J, Knorr, A, Hirth-Dietrich, C, Siegling, A, Volk, HD, Limmer, A et l. (213). Inctivted Orf virus (Prpoxvirus ovis) elicits ntifirotic ctivity in models of liver firosis. Heptol Res 43: Li, YL, Wu, J, Wei, D, Zhng, DW, Feng, H, Chen, ZN et l. (29). Newcstle disese virus represses the ctivtion of humn heptic stellte cells nd reverses the development of heptic firosis in mice. Liver Int 29: Eert, O, Shinozki, K, Hung, TG, Svontus, MJ, Grcí-Sstre, A nd Woo, SL (23). Oncolytic vesiculr stomtitis virus for tretment of orthotopic heptocellulr crcinom in immune-competent rts. Cncer Res 63: Shinozki, K, Eert, O, Kournioti, C, Ti, YS nd Woo, SL (24). Oncolysis of multifocl heptocellulr crcinom in the rt liver y heptic rtery infusion of vesiculr stomtitis virus. Mol Ther 9: Eert, O, Hrrn, S, Shinozki, K nd Woo, SL (25). Systemic therpy of experimentl rest cncer metstses y mutnt vesiculr stomtitis virus in immunecompetent mice. Cncer Gene Ther 12: Kwi, S, Enzn, H, Hyshi, Y, Jin, YL, Guo, LM, Miyzki, E et l. (23). Vinculin: novel mrker for quiescent nd ctivted heptic stellte cells in humn nd rt livers. Virchows Arch 443: Benyon, RC, Iredle, JP, Goddrd, S, Winwood, PJ nd Arthur, MJ (1996). Expression of tissue inhiitor of metlloproteinses 1 nd 2 is incresed in firotic humn liver. Gstroenterology 11: Tkhr, T, Furui, K, Yt, Y, Jin, B, Zhng, LP, Nmu, S et l. (1997). Dul expression of mtrix metlloproteinse-2 nd memrne-type 1-mtrix metlloproteinse in firotic humn livers. Heptology 26: Stojdl, DF, Lichty, B, Knowles, S, Mrius, R, Atkins, H, Sonenerg, N et l. (2). Exploiting tumor-specific defects in the interferon pthwy with previously unknown oncolytic virus. Nt Med 6: Kuriym, S, Ymzki, M, Mitoro, A, Tsujimoto, T, Kikukw, M, Tsujinoue, H et l. (1999). Heptocellulr crcinom in n orthotopic mouse model metstsizes intrhepticlly in cirrhotic ut not in norml liver. Int J Cncer 8: Rdev, S, Sun, R, Jrug, B, Nguyen, VT, Tin, Z nd Go, B (26). Nturl killer cells meliorte liver firosis y killing ctivted stellte cells in NKG2D-dependent nd tumor necrosis fctor-relted poptosis-inducing lignd-dependent mnners. Gstroenterology 13: Muhnn, N, Au Tir, L, Doron, S, Amer, J, Azzeh, M, Mhmid, M et l. (211). Ameliortion of heptic firosis y NK cell ctivtion. Gut 6: Altomonte, J, Wu, L, Chen, L, Meseck, M, Eert, O, Grcí-Sstre, A et l. (28). Exponentil enhncement of oncolytic vesiculr stomtitis virus potency y vectormedited suppression of inflmmtory responses in vivo. Mol Ther 16: Altomonte, J, Wu, L, Meseck, M, Chen, L, Eert, O, Grci-Sstre, A et l. (29). Enhnced oncolytic potency of vesiculr stomtitis virus through vector-medited inhiition of NK nd NKT cells. Cncer Gene Ther 16: Dooley, S nd ten Dijke, P (212). TGF-ß in progression of liver disese. Cell Tissue Res 347: Hung, KW, Hung, YC, Ti, KF, Chen, BH, Lee, PH nd Hwng, LH (28). Dul therpeutic effects of interferon-lph gene therpy in rt heptocellulr crcinom model with liver cirrhosis. Mol Ther 16: Shinozki, K, Eert, O nd Woo, SL (25). Erdiction of dvnced heptocellulr crcinom in rts vi repeted heptic rteril infusions of recominnt. Heptology 41: Altomonte, J, Brren, R, Schulz, S, Mrozin, S, Rummeny, EJ, Schmid, RM et l. (28). Synergistic ntitumor effects of trnsrteril viroemoliztion for multifocl heptocellulr crcinom in rts. Heptology 48: Greenum, LE nd Wells, RG (211). The role of stem cells in liver repir nd firosis. Int J Biochem Cell Biol 43: Mrozin, S, De Toni, EN, Rizzni, A, Altomonte, J, Junger, A, Schneider, G et l. (21). Cell cycle progression or trnsltion control is not essentil for vesiculr stomtitis virus oncolysis of heptocellulr crcinom. PLoS ONE 5: e Birerdinc, A nd Younossi, ZM (21). Emerging therpies for heptitis C virus. Expert Opin Emerg Drugs 15: Nicoletti, I, Migliorti, G, Pglicci, MC, Grignni, F nd Riccrdi, C (1991). A rpid nd simple method for mesuring thymocyte poptosis y propidium iodide stining nd flow cytometry. J Immunol Methods 139: Hrtl, I, Schneider, RM, Sun, Y, Medvedovsk, J, Chdwick, MP, Russell, SJ et l. (25). Lirry-sed selection of retroviruses selectively spreding through mtrix metlloprotese-positive cells. Gene Ther 12: Mrozin, S, Altomonte, J, Stdler, F, Thsler, WE, Schmid, RM nd Eert, O (28). Inhiition of the IFN-et response in heptocellulr crcinom y lterntive spliced isoform of IFN regultory fctor-3. Mol Ther 16: Desmet, VJ, Gerer, M, Hoofngle, JH, Mnns, M nd Scheuer, PJ (1994). Clssifiction of chronic heptitis: dignosis, grding nd stging. Heptology 19: vol. 21 no. 11 nov. 213