Suppression of Metastatic Hemangiosarcoma by a Parvovirus

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Suppression of metastatic hemangiosarcoma by a parvovirus MVMp vector transducing the IP-10 chemokine into immunocompetent mice Nathalia A Giese,1 Zachary Raykov,1 Luisa DeMartino,1 Annunciata Vecchi,2 Silvano Sozzani,2 Christiane Dinsart,1 Jan J Cornelis,1 and Jean Rommelaere1 1Applied Tumor Virology Program F0100 and INSERM U375, Deutsches Krebsforschungszentrum, Heidelberg 69120, Germany; and 2Laboratory of Inflammation and Signal Transduction, IRF Mario Negri, Milan 20157, Italy.

We have previously shown that the growth of human tumor xenografts in immunodeficient mice can be efficiently suppressed upon infection with the autonomous parvovirus H-1 or with cytokine-transducing derivatives thereof. To further evaluate the benefits of implementing parvoviruses in cancer gene therapy, we have created a new recombinant vector, MVMp/IP-10, transducing the immunoactive, antiangiogenic chemokine IP-10, and used this virus to treat syngeneic tumors grown in immunocompetent mice. Intratumoral/intraperitoneal administration of only 3Â107 replication units of MVMp/IP-10 per animal strongly inhibited the progression of established H5V cell–induced vascular tumors, a highly malignant mouse model for human cavernous hemangioma and Kaposi’s sarcoma. Retardation of recurrent tumor growth and suppression of life-threatening metastatic dissemination to internal organs were accompanied by a striking delay in hemangioma-associated mortality. Parental MVMp did not have a significant effect under these conditions up to the dose of 1010 infectious units/animal, but had strong antihemangiosarcoma activity when used to infect H5V cells ex vivo prior to implantation. In all cases, virus therapy was very well tolerated. Virus-induced suppression of hemangiosarcoma was dependent on host T cells and associated with intratumoral persistence of IFN -expressing cytotoxic lymphocytes, and led to the reduced expression of hepatic plasminogen activator inhibitor-1 (PAI-1), a metastasis-linked marker. This proof of principle study demonstrates that MVMp/IP-10 can aid the treatment of vascular tumors and that autonomous parvovirus-based vectors can be considered potent tools for cancer gene therapy purposes. Cancer Gene Therapy (2002) 9, 432– 442 DOI: 10.1038/sj/cgt/7700457 Keywords: hemangiosarcoma; metastasis; parvovirus MVMp; IP-10; IFN ; PAI-1

irus-based anticancer therapies involve the use of to generate progeny virions, they retain their genuine Vviruses either as replicating oncolytic agents, or as parvoviral promoters and replication origins, and encode recombinant vectors for gene transfer.1 The autonomous the pivotal nonstructural protein, NS1.7 These features allow parvoviruses MVMp and H-1 belong to a group of small amplification of the viral genome, efficient expression of (5 kb) nonintegrating single-stranded DNA viruses. Their inserted transgenes, and limited oncotoxicity.7,8 Parvovirus oncotropic and oncotoxic properties make them good vectors are expected to be relatively safe, being poorly candidates for both types of applications.2 Malignant expressed in quiescent cells and most nontransformed transformation of a number of rodent and human cells cells.3,8 Furthermore, the MVMp and H-1 viruses are correlates with their increased capacity to amplify and/or weakly immunogenic and rarely pathogenic to adult animals: express parvoviral DNA, and with hypersensitivity to the infections they cause can be abortive or persistent, but parvovirus-induced killing.3 These features contribute to they are usually clinically nonapparent.9,10 We have recently the intrinsic oncosuppressive capacity of these viruses, but demonstrated that wild-type H-1 virus can reduce the they are often insufficient to promote full inhibition of tumorigenicity of HeLa xenografts in immunodeficient mice, oncogenesis.4,5 Therefore, we are developing strategies to and that recombinant derivatives transducing various reinforce the antineoplastic activity of parvoviruses through cytokines (IL-2, MCP-3) are more efficient antineoplastic incorporation of therapeutic transgenes.6 Although the agents than the parental virus.11,12 Our current focus is on MVMp- and H-1–based vectors produced so far are unable assessing the antitumor activity of autonomous parvovirus vectors in immunocompetent hosts, and on identifying target malignancies for parvovirus-based anticancer therapy. Received February 11, 2002. Address correspondence and reprint requests to: Dr Nathalia A Giese, In the present work, MVMp, known to target transformed Applied Tumor Virology Program F0100 and INSERM U375, Deutsches cells of both human and murine origin, and recombinant Krebsforschungszentrum, Heidelberg 69120, Germany. MVMp derivatives were tested for their ability to protect E-mail: [email protected] mice against a very aggressive mouse hemangiosarcoma. Hemangiosarcoma suppression by parvovirus vector MVMp/IP-10 NA Giese et al 433 Antiangiogenic therapy is increasingly recognized as a gift from J Farber, NIH) between the SacI and Klenow powerful strategy for impeding the growth of various types polymerase-filled MluI restriction sites of pdBMVÁ800. of tumors and for circumventing acquired resistance to MVMp/GFP contains the 730-bp ‘‘humanized’’ GFP coding anticancer drugs. Vascular tumors represent an extreme case sequence, placed under the control of the MVMp P38 of neovascularization and comprise lesions ranging from promoter.12 Infectious particles were produced by cotrans- Kaposi’s sarcoma associated with human herpesvirus-8 fecting 293T/17 cells with the corresponding recombinant (HHV-8) to infantile hemangiomas of unknown etiol- molecular clone and a helper plasmid encoding the ogy.13,14 Kaposi’s sarcoma is a major cause of mortality in parvoviral structural proteins.7 The wild-type and recombi- AIDS patients, while hemangiomas, the most common nant MVMp viruses, purified by CsCl gradient centrifuga- tumors of childhood, are disturbingly disfiguring and may tion followed by G25 Sephadex gel filtration, were diluted in induce life-threatening complications. Because conven- MEM (Life Technologies, Eggenstein, Germany) prior to tional treatments of vascular tumors such as irradiation and use. Recombinant viruses were titrated by infected cell surgery are unsatisfactory, an intensive search for new hybridization assays on A9 cells,7 using an NS1- or IP-10– therapeutics is in progress.14 Murine hemangioma models specific DNA probe. Recombinant parvovirus titers rou- have recently been developed, allowing the evaluation of tinely reached 3–5Â107 replication units (RU)/mL. new antiangiogenic compounds.15 - 17 One of the most potent anticancer cytokines, IL-12, has been shown to Cell cultures suppress progression of experimental hemangiomas.18,19 IP-10 (interferon-inducible protein with molecular weight The cell lines 293T/17 (transformed human embryonic of 10 kDa, recently classified as CXCL10) has been kidney cells; American Type Culture Collection, ATCC), identified as a major IL-12–induced downstream effector A9 (mouse fibroblasts, ATCC), and H5V [polyoma virus middle T antigen (PymT)–transformed mouse endothelial of antitumor reactions, and found to inhibit the growth of 17 several experimental tumors with varying efficacy, depend- cells] were maintained in DMEM (Life Technologies) ing on the model and method of delivery.20 - 26 Although supplemented with 2 mM L -glutamine, 100 g/mL the mechanism of IP-10–induced tumor suppression has penicillin, 100 U/mL streptomycin, and 10% (293T and not yet been fully elucidated, both antiangiogenic and H5V cells) or 5% (A9 cells) fetal calf serum. Prior to immune system-mediated effects appear to be essential to infection, A9 or H5V cells were seeded at a density of either 27,28 2Â103 cells/well into 96-well plates (for survival tests and the antitumor activity of this chemokine in vivo. 4 Interestingly, IP-10 can act as an inverse agonist of the enzyme-linked immunosorbent assays, ELISAs), 4Â10 cells/well into six-well plates (for FACS analyses), or G-protein–coupled receptor homologue encoded by 6 Kaposi’s sarcoma–associated HHV-8, and thereby inhibit 3Â10 cells/plate into 10-cm Petri dishes (for animal the constitutive signaling occurring in transformed cells29 experiments), and incubated for 24 hours. After infection These facts prompted us to attempt to combine, in a with MVMp, MVMp/GFP, or MVMp/IP-10, the cells and recombinant vector MVMp/IP-10, the immunomodulat- culture supernatants were harvested at the indicated time ing/angiostatic properties of IP-10 with the oncotropic/ points and analyzed for cell survival or transgene expres- oncostatic features of MVMp, and to test the protective sion. The multiplicity of infection (MOI) is expressed in effect of MVMp/IP-10 against malignant hemangiosar- PFU (wild-type virus) or RU (recombinant vectors) per coma (H5V) implanted into syngeneic immunocompetent cell. mice. In the present preclinical study, wild-type MVMp was found to inhibit H5V tumor progression, provided Determination of cell survival that infection was carried out ex vivo to ensure that most The tetrazolium salt, 3-(4,5-dimethylthiazol-2yl)-2,5- cancer cells were hit. When administered in vivo,MVMp diphenyltetrazolium bromide (MTT), test for identifying did not protect effectively against H5V, in contrast to its living cells (Sigma-Aldrich Chemie, Deisenhofen, Ger- recombinant derivative MVMp/IP-10. The latter was many) was used to compare the growth kinetics of able to slow down the growth of relapsing primary tumors uninfected and infected cultures. The extent of cell death and to drastically suppress the formation of multiple in treated cultures was assayed by measuring the release of metastases. lactate dehydrogenase (LDH) according to the CytoTox961 test (Promega Deutschland, Mannheim, Germany).

Materials and methods Measurement of IP-10 production Viruses To determine levels of secreted murine IP-10, an ELISA was Minute virus of mice (prototype strain MVMp) was established using goat antirat Ig (Becton Dickinson, produced and titrated by plaque assay using A9 indicator Heidelberg, Germany) to coat the plates, purified rat cells as described previously. 30 Virus titers are expressed antimouse IP-10 monoclonal antibodies cocktail A102-5/ in plaque-forming units (PFU) per milliliter. Vector DNA A102-6 (Becton Dickinson) to capture IP-10, and biotiny- clone pdBMVÁ8007 was used to produce the recombinant lated goat antimouse IP-10 polyclonal antibodies BAF466 derivatives MVMp/GFP and MVMp/IP-10. The molecular (R&D Systems, Wiesbaden, Germany) to detect IP-10. clone of MVMp/IP-10 was obtained by inserting the 752-bp Recombinant murine IP-10 protein (Becton Dickinson) was EcoRV – SacI fragment of pBluescript mIP-10 (a generous used as a standard.

Cancer Gene Therapy Hemangiosarcoma suppression by parvovirus vector MVMp/IP-10 NA Giese et al 434 FACS analysis lioma cells/animal were subcutaneously implanted into the right hind flanks of syngeneic female C57Bl/6 mice For detection of GFP expression, MVMp/GFP–infected (CRWiga, Sulzfeld, Germany). Disease progression was cells were harvested by trypsinization and washed in PBS containing 2% FCS, prior to analysis with a FACScan flow monitored on the basis of the appearance of cavernous hemangiomas at the injection site. Tumor size was cytometer (Becton Dickinson). measured with a metric electronic caliper and calculated according to the formula 0.785ÂwidthÂlength. Sudden Reverse transcription polymerase chain reaction (RT-PCR) death occurred frequently in tumor-bearing mice, being due analysis to metastases and internal hemorrhage. Therefore, the mice Total RNA was prepared from samples of isolated tissues were regularly checked for life-threatening symptoms such 1 (kept at À808C) using Trizol (Life Technologies) as a weakness, a drop in body temperature, and shivering, according to the manufacturer’s instructions. DNAseI- and moribund animals were killed. At necropsy or autopsy, treated RNA was converted to cDNA, amplified, and the internal organs were inspected visually for the analyzed according to RT-PCR protocols detailed previ- presence of metastatic foci and processed for histopatho- ously.31,32 The primers used for HPRT, CD4, CD8, NK 1.1, logical (hematoxylin–eosin staining of formaldehyde- IL-2, IFN , IL-12p40, VEGF, and bFGF cDNA amplifica- fixed tissue sections) or RT-PCR analysis. Virus (MVMp, tion have been described elsewhere.31,33,34 The primers used MVMp/IP-10) or mock (dilution medium) treatment of for other markers were: PAI-1 (511 bp): 50 -GCT GGT GAA animals was initiated in two ways: either H5V cells were TGC CCT CTA CTT-30 and 50 -CAC TGT GCC GCT CTC infected with virus and then injected into mice (ex vivo GTT TAC-30; PAI-2 (320 bp): 50 -AGG CAC AAG CAG approach), or mice with established tumors were exposed to GAG ATA AAA-30 and 50 -GAC AGC ATT CAC CAG daily intratumoral, then intraperitoneal, injections (in vivo CAC CAT-30; uPA (600 bp): 50 -CTA GGC CTG GGG approach). The virus doses and treatment regimens are AAA CAC AAT-30 and 50 -GTC TGA ACC AAA CGG described in the main text. The animals were kept in laminar AGC ATC-30; tPA (679 bp): 50 -GTA CTG CTG CTT TGT flow safety cabinets (BDK Luft- und Reinraumtechnik, GGA CTG G-30 and 50 -AGG AGG CCT GGG ATG TGG Sonnenbu¨hl, Germany). Procedures for animal handling, TGA G-30; CXCR3 (290 bp): 50 -CCT GAG CAG CAC treatment, and care were in compliance with institutional GGA CAC CTT C-30 and 50 -GCA GAC AGA GAC CCC and governmental guidelines. ATA CAA C-30; Granzyme B (253 bp): 50 -ACT CAA ACA CGC TCA AAG A-30 and 50 -ATC CAG GAT AAG AAA 0 0 CTC G-3 ; CD40L (282 bp): 5 -ATC TGT GCT TTT TGC Results TGT GT-30 and 50 -TTA CTG TTG GCT TCG CTT AC-30; CTLA4 (438 bp): 50 -ATT CAC CAT CAC ACA ACA CT- In vitro susceptibility of transformed endothelial H5V cells 30 and 50 -GGG GCA TTT TCA CAT AGA CC-30;IP-10 to infection with MVMp and its recombinant derivatives (381 bp): 50 -TGA GCA GAG ATG TCT GAA TC-30 and Wild-type MVMp was first tested for its ability to infect and 50 -TCG CAC CTC CAC ATA GCT TAC AG-30; viral kill H5V cells in vitro. H5V cultures proved competent transcripts (511 bp, unspliced RNA; 414 bp, the major form for MVMp uptake and viral gene expression, accumulating of spliced NS1 RNA): 50 -ACG CTC ACC ATT CAC GAC 2Â106 transcripts/g total RNA within 24 hours after virus ACC GAA A-30 and 50 -ATC ATA GGC CTC GTC GTG inoculation (MOI=10 PFU/cell), as measured by QRT- CTC TTT-30.The metastasizing H5V cells were detected PCR. Using the MTT assay to identify living cells, we found using PymT-specific primers (439 bp): 50 -TTC TGA GCA that MVMp infection led to a dose-dependent reduction of ACC CGA CCT AT-30 and 50 -CTT CTT AGG TGG CGT the rate of H5V culture growth (Fig 1A). To determine the TGC AT-30 ) and probe: 50 -GCC ACT CCT ATC CCC CAA role of cell death in this inhibition, the LDH content of the CCC-30. PCR products were analyzed by 2% gel electro- culture medium was measured upon infection. Cytoplasmic phoresis and visualized either by direct staining with LDH is freed by dying cells and constitutes a reliable marker ethidium bromide or SybrGreenII (Roche Diagnostics, of cytolysis. As shown in Figure 1A, increased release of Mannheim, Germany) or by Southern blot hybridization LDH was detected in infected H5V cultures, indicating that and ECL detection (Amersham Pharmacia Biotech Europe, MVMp is toxic to these cells. Yet, this cytotoxicity was Freiburg, Germany), depending on the level of expression. pronounced only at high MOIs (100 PFU/cell), possibly PCR signals were measured31,32 and ranked as described in reflecting a low capacity of H5V cells for MVMp multi- the legend to Figure 4, with hypoxanthine phosphoribosyl plication. In fact, the output/input virus ratio corresponded transferase (HPRT) expression serving as a reference for to 0.2 in MVMp-infected H5V cultures (as compared to 105 equalizing the cDNA input for PCR. Viral transcripts in in the highly sensitive A9 producer cell line; data not H5V cultures and tissues of infected animals were also shown). Furthermore, wild-type MVMp inhibited the 2 quantitated using LightCycler real-time QRT-PCR growth of H5V cultures to about the same extent as the technology (Roche Diagnostics).35 recombinant vectors MVMp/GFP and MVMp/IP-10, which lack the capsid genes and are unable to yield progeny viruses (Fig 1B). Though defective, the recombinant Tumor model and virus treatments parvoviruses used in this study retain the pivotal NS1 The mouse H5V hemangiosarcoma model has been gene and are proficient in DNA replication and gene described previously.17 A total of 5Â106 H5V endothe- expression.6-8,11 We conclude that the H5V cell populations

Cancer Gene Therapy Hemangiosarcoma suppression by parvovirus vector MVMp/IP-10 NA Giese et al 435 A days after infection with MVMp/GFP at MOI of 1 RU/cell, MVMp the proportion of GFP-expressing cells in H5V cultures 100 100 reached 10±3%. Stained cells were detected up to 7 days 75 75 after infection (data not shown). As illustrated in Figure 1C, MVMp/IP-10–treated H5V cultures produced substantial 50 50 amounts of the chemokine, even when infected at a low multiplicity (1 RU/cell). Endogenous production of IP-10 25 25 protein was below the level of detection in mock-, MVMp- and MVMp/GFP–infected H5V cultures ( <1 ng/mL, as

0 0 %] LDH Release [ MTT Reduction [ %] MTT Reduction [ 10 0 10 2 10 4 determined by ELISA), although the corresponding mRNA Multiplicity of Infection (pfu/cell ) was detected by RT-PCR (see below, Fig 4A). Interestingly, transduced IP-10 did not increase the intrinsic toxic effect of MVMp on H5V cells as growth inhibition was the B MVMp MVMp/GFP MVMp/IP-10 same whether induced by MVMp/IP-10 or MVMp/GFP (Fig 1B). This finding is in keeping with our failure to detect 100 expression of the IP-10 receptor CXCR3 in H5V cells by RT-PCR (see below, Fig 4A). These various results prompted us to use H5V endothelioma as a model for IP- 75 10 transduction and assessment of the chemokine’s ability to potentiate MVMp-based treatment of vascular tumors. MTT Reduction [ %] 50 Reduced tumorigenicity of H5V cells infected ex vivo with 0 24 6MVMp Days post Infection We first examined whether MVMp could reduce the tumorigenic potential of H5V cells in vivo. H5V hemangiosarcoma is an immunogenic MHC class I + (H-2b ) tumor. C no virus MVMp/IP-10 Progression of the disease in terms of remission, relapse, and 60 metastasis depends strongly on the number of engrafted cells.17 Implants of up to 2Â105 cells appear to be fully 40 rejected, but as the number of inoculated cells increases, so does the fatal recurrence of tumors among treated mice. In 6 20 the present study, 5Â10 cells were inoculated per mouse,

IP-10 [ng/ml ] resulting in highly malignant hemangiosarcomas in 100% of the animals. As shown in Figure 2, A–C, H5V-injected mice 0 0 1 2 3 did not succumb to the primary tumors, which developed at Days post Infection the injection site and subsequently regressed. Instead, the animals died later from local relapse and development of Figure 1 Susceptibility of H5V cells to infection by wild-type and metastases at multiple distant sites. Wild-type MVMp was recombinant MVMp viruses. A,B: Cell viability was assayed using tested for the ability to interfere with this sequence of events MTT and LDH tests. Cultures were analyzed on day 3 (A) or at different times (B)following injection with indicated viruses. The MOI when administered ex vivo into H5V cells (MOI=50 PFU/ was varied (A) or set at 1 RU/cell (B). Results of MTT reduction cell) prior to their subcutaneous injection into syngeneic measurements are expressed as percentages of the value obtained C57Bl/6 mice. Figure 2B shows the effects of this treatment for the uninfected control at the corresponding time point. Results of on tumor formation at the implantation site. The treatment LDH assays (amounts of enzyme released in the culture medium) did not prevent the appearance of primary hemangiomas: all are expressed as percentages of the total LDH content of the animals implanted with virus-infected cells developed corresponding well. LDH release by uninfected cells was less than tumors, albeit of smaller size than those derived from 5%. These data were confirmed using different culture conditions and mock-treated cells. These primary tumors regressed in both alternative methods for assessing viability. C: IP-10 production by treated and mock-treated animals. In contrast, a striking MVMp/IP-10–transduced cells was determined by ELISA. Culture supernatants were harvested at the indicated times following infection difference was observed between the two groups at the (MOI=1 RU/cell), and their IP-10 contents were measured. The subsequent stage of tumor relapse. Viral treatment led to a data shown are mean±SEM from three independent experiments. significantly longer remission period: 43±7 days for MVMp-exposed mice, compared to 15±1 days for mock- treated mice (Wilcoxon rank-sum test, P<.001). In the showed little ability to support a productive MVMp control group, furthermore, relapsing tumors grew aggres- infection, but that even at low MOIs, viral metabolism takes sively, whilst in the MVM-infected group, they stagnated at place in at least a fraction of infected cells, causing growth to the site of implantation (Wilcoxon rank-sum test, P<.01). be significantly delayed in treated cultures. Recurrent tumor nodules from MVMp-infected H5V cells In agreement with this view, H5V cell cultures infected regressed occasionally, and three of five animals displayed with the recombinant vectors MVMp/GFP or MVMp/IP-10 no discernable hemangioma by the end of a 3-month were able to support NS1 and transgene expression. Two observation period, though H5V cell-containing tumor

Cancer Gene Therapy Hemangiosarcoma suppression by parvovirus vector MVMp/IP-10 NA Giese et al 436 A Mock MVMp tions, all control mice succumbed to hemangiomas within 3 months postimplantation, whereas only one of six mice in 100 the MVMp group was found dead on day 74 (Fig 2A, Mantel–Haenszel test, P<.01). In summary, ex vivo 75 MVMp infection at a moderate multiplicity drastically 50 reduces the tumorigenicity of H5V cells, as shown by the more effective host control of tumor growth, suppression of

Survival [ %] Survival [ 25 metastatic expansion of primary hemangiomas, and pre- vention of mortality. 0 0255075 100 Treatment of established tumors with MVMp and Days post Implantation MVMp/IP-10 The strong antineoplastic effect of MVMp in ex vivo experiments encouraged us to test the efficacy of the virus B Mock MVMp against established H5V tumors. A total MVMp dose of 1010 200 ]

2 PFU/mouse was administered in vivo to tumor-bearing animals according to the following regimen: starting on day 150 6 postimplantation, a single intratumoral injection each day

100 for 5 days, then a 2-day pause, followed by a single intraperitoneal injection each day for 5 days, another 2-day 50 pause, then a final 5-day course of intraperitoneal injections. The reason for changing the route of administration in the Tumor Area [mm 0 course of the experiment was to take into account the 75 100 02550 regression of the primary tumors and the spread of H5V cells Days post Implantation to distal organs. Under these conditions, the wild-type virus had no significant effect on tumor growth, formation of metastases, and disease outcome (data not shown). This C result prompted us to generate the recombinant vector

%] MVMp/IP-10 and determine whether the antineoplastic 80 effect of the parvovirus was reinforced through the additional 60 action of transduced IP-10. Treatment of established hemangiomas was initiated 5 days after implantation of 40 H5V cells into C57Bl/6 mice, and consisted of a total dose of 3Â107 RU of MVMp/IP-10 per mouse, administered 20 according to the above-mentioned regimen. For comparison, other groups of mice received an equivalent inoculum of 0 wild-type MVMp or medium alone. Whereas the wild-type Metastasis-free Mice [ Mock MVMp virus had little effect on hemangioma progression, MVMp/ Figure 2 Reduction of the tumorigenicity of H5V endothelioma cells IP-10 provided significant antitumor protection. As illus- as a result of their ex vivo infection with MVMp. C57Bl/6 mice were trated in Figure 3A, mock-treated mice survived the tumor injected subcutaneously with 5Â106 mock-treated or MVMp-infected cell implant no longer than 15 weeks, while half of the (MOI=50 PFU/cell) H5V cells. Disease progression and outcome MVMp/IP-10–infected mice responded to the therapy and were determined as described in Materials and methods by assessing stayed alive significantly longer (Mantel–Haenszel test, the survival of implant-receiving animals (Kaplan-Meier survival P<.05). Strikingly, one third of MVMp/IP-10–treated curves, A), the growth of tumors (average tumor area±SEM, B)at the implantation site, and the absence of metastases at autopsy (as animals survived until the end of the experiment at 6 months determined by macroscopic and histopathological examination of postimplantation, i.e., at least twice as long as controls. As tissues, C). This experiment included five and six mice in the control shown in Figure 3B, the growth of recurring hemangiomas in and MVMp-treated groups, respectively. the long-term survivors was more restrained than in control mice (Wilcoxon rank-sum test, P<.01 between MVMp/IP- 10 and mock groups; P >.1 between MVMp and mock remnants were detected at autopsy (data not shown). Even groups), indicating a potent antitumor effect of the more remarkably, the histological analysis of internal organs recombinant virus. Another remarkable feature of the after autopsy showed that all mice implanted with mock- MVMp/IP-10 treatment was the significant suppression treated H5V cells carried multiple deforming cavernous of metastases (Fig 3C, chi-square test, P<.05 between metastases in the liver, spleen, uterine tubes, peritoneal mock- and MVMp/IP-10–infected groups by 3 months cavity, and ovaries, whereas only one mouse implanted with postimplantation; and Fig 3D). We conclude from these MVMp-infected cells carried a single metastatic nodule in experiments that MVMp/IP-10 gene therapy has a clear-cut the peritoneal cavity (Fig 2C). This represents very therapeutic effect on hemangioma-bearing mice, even when significant MVMp-dependent suppression of metastases relatively low doses of vector are administered in vivo. (chi-square test, P<.01). In keeping with these observa- Interestingly, the treatment of hemangiosarcomas with

Cancer Gene Therapy Hemangiosarcoma suppression by parvovirus vector MVMp/IP-10 NA Giese et al 437 recombinant MVMp/IL-2 was unsuccessful (data not Potential mechanism of MVMp and MVMp/IP-10 action shown), pointing to the specificity of IP-10 interference against hemangiosarcoma with hemangioma progression. It should also be mentioned MVMp vectors fail to alter the expression of malignancy- that macroscopic and histological examinations of mice associated molecules in H5V cells. The parvovirus-mediated revealed no deleterious side effects attributable to parvo- 10 antihemangioma effects described above might involve virus-based treatments, even at the high dose of 10 multiple mechanisms ranging from direct action of the virus PFU/animal and after multiple injections (data not on H5V cells (cytotoxicity or changes in the expression shown). profile of surface and secreted proteins) to indirect interference with disease development. To approach this question, we first investigated whether MVMp or MVMp/ IP-10 could directly affect the capacity of H5V cells to produce known markers of vascular tumor progression.36 - 38 A Mock MVMp MVMp/IP-10 To this end, RT-PCR analyses were carried out using H5V 100 cell cultures to measure the expression of plasminogen activator proteolytic system (PAS) components (tissue-type 75 and urokinase-type plasminogen activators, tPA and uPA, 50 and their inhibitors, PAI-1 and PAI-2), as well as angiogenic markers basic fibroblast growth factor (bFGF) and vascular Survival [%] 25 endothelial growth factor (VEGF). As illustrated in Figure 4A (left column), all these markers were produced 0 0 50 100 150 200 constitutively by H5V cells, but differed in their level of expression — high (PAI-1, uPA), low to moderate (VEGF, Days post Implantation bFGF, PAI-2), or extremely low (tPA), as reproduced in six independent experiments. Importantly, infection of H5V cell

B Mock MVMp MVMp/IP-10 cultures with either MVMp or MVMp/IP-10 did not cause ]

2 150 any significant alteration in this expression profile, as measured 5 or 24 hours postinfection (data not shown). This argues against the hypothesis that these putative 100 determinants of the H5V cell malignant phenotype are targets for the MVMp-mediated antineoplastic activity, 50 although we cannot rule out a direct interference of the virus with the expression of other relevant yet undetermined Tumor Area [mm 0 H5V cell markers of malignancy. 1 4 13 Weeks post Implantation Suppression of H5V tumor formation by MVMp vectors requires T cells. As shown above, (recombinant) MVMp had a direct influence on H5V cell populations, namely the reduction of their growth in culture. This effect might C contribute to inhibiting tumor development, especially under [%] 60 conditions in which most transformed cells become infected (ex vivo approach). Yet, the (recombinant) MVMp-induced 40 suppression of H5V culture growth was limited and transient (Fig 1B), suggesting that it may not be sufficient on its own 20 to account for the protection of mice against hemangioma development. This was confirmed through experiments using T-cell–deficient mice (data not shown). When + + Metastasis-free Mice 0 C57Bl/6 nu /nu mice served as recipients for H5V cell Mock MVMp MVMp/IP-10 implantation, animals inoculated with MVMp-infected cells

MVMp/IP-10 MOCK Figure 3 Protection of mice against hemangiosarcoma progression as a result of their in vivo treatment with MVMp/IP-10. C57Bl/6 mice D were injected subcutaneously with 5Â106 H5V cells. After tumor establishment (5 days postimplantation), the animals were exposed Liver to a regimen of daily intratumoral MVMp/IP-10 injections (5 days, Spleen then a 2-day pause) followed by daily intraperitoneal injections (5 days, a 2-day pause, then again 5 days). The total dose was Uterine tubes 3Â107 RU/mouse. A: Animal survival. B: Tumor growth. C,D: PrimaryTumor Metastases were determined as in Figure 2. Data are compiled from three experiments comparing mock-treated (n=12), MVMp-infected (n=13), and MVMp/IP-10–infected (n=13) animals.

Cancer Gene Therapy Hemangiosarcoma suppression by parvovirus vector MVMp/IP-10 NA Giese et al 438 did develop tumors more slowly than the ones receiving protect nude mice against progression of H5V-type vas- mock-treated implants. Furthermore, in the former group, cular tumors (data not shown). Altogether, these observa- the primary lesions were restricted to small, isolated foci. tions point to the involvement of immune system’ effector However, this inhibition was of short duration, and all mice cells as mediators of the antihemangioma activity of in both groups eventually developed tumors and metastases, (recombinant) MVMp. and succumbed to the disease. This contrasts with the long- Stagnation of tumor growth is associated with long-term term antihemangioma protection given by MVMp in intratumoral persistence of activated lymphocytes, expressing immunocompetent animals under ex vivo conditions IFN . To investigate the immune component of the antineo- (Fig 2). Hence, the direct effects of MVMp on H5V cells plastic activity of MVMp and MVMp/IP-10, tumor speci- (cytotoxicity or still unknown phenotypic changes) may mens derived from treated immunocompetent mice were lead to a delay in tumor development, but have to be further analyzed for the expression of markers of effector cell enforced by host T cells in order that ex vivo infection with recruitment and activation. The tumor expression profile was the parvovirus results in a suppression of hemangioma characterized by RT-PCR for the various transcripts listed in progression. Likewise, the MVMp/IP-10 vector did not Figure 4A. In mock-treated mice, the early phase of primary tumor development was associated with expression of markers indicative of T-cell infiltration (CD4, CD8) and, A undetectable moderate to a variable extent, T and NK cell activation (CXCR3, low high CTLA4, IFN , granzymes A and B) (data not shown). This is in agreement with previous observations showing that host H5V Tumors defense mechanisms involving, more particularly, T cells are in vitro progressing stagnating 17 uPA responsible for the regression of primary H5V tumors. tPA However, this antineoplastic reaction was rapidly exhausted

PAI-1 and failed to eradicate H5V cells, as apparent from the recurrence and progression of mock-treated tumors. As PAI-2 illustrated in Figure 4A, these recurring control tumors VEGF showed an overall reduction of the expression of above- bFGF mentioned immune parameters (in particular CD8, CXCR3, IP-10 granzyme B, and IFN ) and accumulated proteolytic (uPA, CXCR3 tPA, PAI-1, PAI-2) and proangiogenic (VEGF, bFGF) CD4 factor transcripts. Infection with MVMp or MVMp/IP-10 CD8 treatment led to strong virus gene expression in primary

NK 1.1 tumors (Table 1), but apart from that had no significant

CD40L effect on the expression pattern of these tumors during the

CTLA4 initial stage of lesion formation and regression (data not shown). In contrast, the (recombinant) virus treatment was Granzyme A associated with striking changes in immune profile of Granzyme B recurring tumors (Fig 4A), compare the right and middle Perforin columns), although no viral transcripts were detected IFNγ anymore during this later stage (1–2 months after the last IL-2

IL12p40

Figure 4 Expression of markers of immune activation and angiogenesis in parvovirus-infected H5V tumor-bearing mice. C57Bl/6 mice were subjected to ex vivo or in vivo infection protocols described B in the legends to Figures 2 and 3. Two to 3 months after H5V cell Tumor implantation, the mice were autopsied and checked for primary tumor Mock Ex vivo In vivo In vivo No H5V growth and metastases. Organs and tumor remnants were isolated MVMp MVMp MVMp/IP-10 implant and analyzed (three to six specimens per group). A: Expression of PAS components, angiogenic factors, and immune markers was IFNγ measured by RT-PCR in cultured H5V cells (left column) and in H5V tumors that were either progressing (middle column: mock treatment HPRT or infection with MVMp in vivo) or stagnating (right column: infection Area (mm2): 149 26 114 36 with MVMp ex vivo, or MVMp/IP-10 in vivo). Specific PCR products - were obtained, visualized, and subjected to densitometric analysis as Liver described in Materials and methods. Signal intensities were PAI-1 quantitated using NIH Image 1.62 software, and the levels of normalized transcript expression (arbitrary OD units) were ranked as undetectable, low (values <200); moderate (values 201–400), HPRT and high (values >401). B: RT-PCR signals obtained for tumor- Metastasis: associated IFN and liver-associated PAI-1 in control and H5V cell– + - + - - implanted mice treated according to the ex vivo or in vivo protocol.

Cancer Gene Therapy Hemangiosarcoma suppression by parvovirus vector MVMp/IP-10 NA Giese et al 439 Table 1 Parvovirus NS1 gene transcription in H5V tumor-bearing mice

NS1 transcripts (copies/g total RNA)*

Infection Livery Tumory

Ex vivo MVMp (MOI=50 PFU/cell) 250 80,000 In vivoz MVMp (109 PFU/mouse) 600 3,500 MVMp/IP-10 (107 RU/mouse) <1 200 MVMp (107 PFU/mouse) 50 600

*NS1 transcripts were quantitated by QRT-PCR, using Roche LightCycler2 PCR technology. Data are average values from two tissue samples. yMice were sacrificed and organs collected 7 (ex vivo)or11(in vivo) days postimplantation. zTumor-bearing C57Bl/6 mice were injected daily with indicated viruses through the intratumoral/intraperitoneal routes, from days 6 to 10 post-implantation. virus injection) in residual tumors or other organs (data not hand, MVMp and its recombinant derivative had no shown). Virus-induced stagnation of recurring tumors was significant effect on the ability of H5V cell cultures or accompanied by their persistent infiltration with T and NK tumors to produce proangiogenic (VEGF, bFGF) and cells (CD4, CD8, NK 1.1 hallmarks). These cells appeared proteolytic (PAI-1 and 2, uPA, and tPA) molecules (see to be fully activated (CXCR3, CD40L, CTLA4, IL-2) and above and Fig 4A). On the other hand, MVMp gene cytotoxic (granzymes A and B, perforin, IFN ). Especially expression in liver could only be detected at low level soon conspicuous was the high production of IFN transcripts in after virus inoculation (Table 1) and became undetectable at virus-treated tumor remnants, as compared to mock-treated later times, in particular in liver tissues that were free of growing lesions (Fig 4B). Accumulation of IFN can thus metastatic foci (data not shown). Altogether, these data lead be assumed to contribute to suppression of tumor progres- us to assume that the (recombinant) MVMp-dependent sion, this cytokine being both directly cytotoxic to H5V inhibition of hepatic PAI-1 production in tumor-bearing cells39 and capable of stimulating the granzyme/perforin– mice is a consequence rather than a cause of the virus- mediated cytotoxic activity of effector immune cells.40 induced suppression of metastases formation, resulting from Suppression of distant metastases is associated with reduction elimination of malignant H5V cells liable to invade of PAI-1 expression in livers. Hemangioma progression is distal organs. known to involve the recruitment of host endothelial cells into lesions, which has been reported to depend on the release Discussion of proangiogenic and proteolytic factors by transformed and stromal cells.37,38,41 This prompted us to determine whether Over the past years, a number of viruses have been used for the (recombinant) MVMp-induced suppression of meta- cancer gene therapy purposes.1 Rodent parvoviruses typified stases was associated with changes in the expression of these by the MVMp and H-1 viruses are especially promising factors in distant target tissues. To this end, RNA expression bases for the generation of viral vectors with antineoplastic patterns were determined in C57Bl/6 mice carrying hepatic activity, due to their capacity for replicating in transformed metastases (H5V cell–implanted animals subjected, or not, human cells and their oncotropic and oncosuppressive to in vivo treatment with MVMp) or free of metastatic foci properties, which can be demonstrated in the absence of (control mice without H5V cell implantation, and H5V cell– harmful side effects.2- 5,10 This prompted us and others to implanted animals treated ex vivo with MVMp or in vivo with attempt to reinforce the genuine antineoplastic potential of MVMp/IP-10). Among the markers studied, components of these parvoviruses by supplementing them with therapeutic PAS showed a significantly enhanced expression during transgenes.8,11,12 The present work constitutes a proof-of- disease progression. This applied in particular to PAI-1 principle study showing for the first time that a recombinant transcripts, whose continuous accumulation constituted a parvovirus can exert an antitumor activity after being hallmark of hemangioma metastases in the liver (Fig 4B). administered in vivo, under conditions in which the wild- Interestingly, the suppression of hepatic metastases in H5V type virus has little protective effect. This was demonstrated tumor-bearing mice, as a result of parvovirus treatment, in a highly aggressive metastatic hemangiosarcoma mouse correlated with a markedly reduced level of liver PAI-1 model, using a vector based on the autonomous parvovirus transcripts. It is presently unclear whether the source of MVMp to carry a therapeutic transgene IP-10. A number of hepatic PAI-1 overproduction in the presence of H5V properties of this vector system are worth noting. (a) The metastases lays in the tumor cells colonizing the liver or in efficacy of this vector was quite remarkable in that a dose as the hepatic parenchyma reacting to this process. However low as 3Â107 RU of virus per mouse caused at least a that may be, the question arose whether (recombinant) doubling of the survival time in responding animals (about a MVMp inhibited the formation of liver metastases by hundred times less than usual doses of adenoviral vectors, preventing hepatic cells and/or H5V tumor cells, settled in including Ad/IP-10).26 Because recombinant parvoviruses liver, from generating a local microenvironment favorable are comparable with other viral vectors with regard to their to the recruitment of new endothelial cells and cancer efficacy in expressing transgenes, as measured in cell cul- progression. Although it cannot be ruled out, this possibility tures,11,12,20,26,42,43 it can be suggested that the oncotoxicity is not supported by the evidence available to date. On one of parvoviruses may potentiate the effects of transgenes

Cancer Gene Therapy Hemangiosarcoma suppression by parvovirus vector MVMp/IP-10 NA Giese et al 440 carried by their recombinant derivatives (see below). (b) we hypothesize that the parvovirus-mediated toxicity of Neither deleterious effects of MVMp/IP-10 attributable to NS1 for H5V cells stimulated intrinsic immune reaction, and unspecific transgene expression, nor side effects of parental that this response was intensified when the virus expressed MVMp were observed, even after multiple virus injections IP-10 transgene in addition to its own NS1 and NS2 genes. up to a 1000-fold higher dose than the one used for the The present study showed that MVMp delayed the growth of recombinant. (c) Although no total cure was achieved, H5V cell cultures and exerted an oncosuppressive effect in responding to therapy mice stayed alive for more than the absence of transgene, although this effect was only 6 months, which represents an over 100 days increase in apparent when MVMp was administered in vitro to H5V their life expectancy (the equivalent of 10 years of human cells prior to their implantation, i.e., under conditions lifespan). The antitumor protection provided by MVMp/ maximizing the fraction of tumor cells that are hit by the IP-10 is quite impressive because H5V model is an virus. It is thus possible that MVMp was able to reduce the especially aggressive case of experimental hemangiosarco- load of implanted proliferating tumor cells in the ex vivo mas, which are usually refractory to complete cure by protocol. On the other hand, the cytotoxic action of MVMp various treatments,16,39,44,45 with IL-12 therapy being the is likely to be ineffective when the virus is administered in most successful one.19 vivo and reaches only some of the tumor cells, especially The antineoplastic activity of IP-10 is not without because these are scattered between host endothelial cells precedents, although the use of different delivery systems that are recruited into established hemangiomas.15,17 The and tumor models precludes published data from being major parvoviral cytopathic effector, NS1,46 which is quantitatively compared with the present ones. The most encoded by administered vectors, may potentiate effects of powerful protection was observed using plasmocytoma cells immunoactive transgene in following ways: (a) the NS1- stably transfected with an IP-10–expressing plasmid.20 This mediated oncotoxicity might be expected to stimulate the study demonstrated that IP-10 has an antitumor potential release of tumor antigens, and (b) the NS1 ability to which is mediated by T cells, but did not address the question dysregulate cellular gene expression46 might promote an of the in vivo delivery of IP-10 into preexisting malignan- immunogenic phenotype (costimulatory or adhesion mole- cies. Application of IP-10 to established tumors gave cules) of H5V cells. In turn, expression of IP-10 by variable results. When administered directly as IP-10 protein transduced H5V cells could aid more efficient targeting of to immunocompromised mice bearing human tumor xeno- tumor cells by anti-H5V effectors because NK and activated grafts (Burkitt lymphoma, non small cell lung cancer)21,22 or T cells are both known to display the IP-10 receptor CXCR3 as vaccine in the form of tumor antigen/IP-10 fusion protein and to be sensitive to the activating and/or mobilizing to immunocompetent mice bearing syngeneic tumors (38C- properties of the chemokine.47 - 49 13 lymphoma),23 IP-10 induced significant but transient Apart from affecting immune reactions, MVMp/IP-10 antitumor reactions, without complete tumor eradication. could also inhibit neovascularization in different ways, The route of IP-10 delivery had a clear influence because thereby contributing to the suppression of H5V lesions treatment with a plasmid encoding the fusion protein failed development. First, part of the protective effect provided by to induce any protection.23 In another study, an adenovirus- the parvovirus may be related to the decrease of the number based vector carrying IP-10 proved to be more efficient than of the H5V cells able to proliferate and recruit host an equivalent recombinant transducing endostatin, in its endothelial cells, as suggested by the vector-induced ability to extend the life expectancy of B16.F10 melanoma- inhibition of the H5V cells growth in cultures. Second, bearing mice, leading to full tumor eradication in one third of mobilization of activated lymphocytes into tumors could treated animals.26 Two other attempts at using adenovirus- lead to a bystander effect and damage recruited host delivered IP-10 to treat mammary and colorectal adenocar- endothelial cells, besides H5V cells.50 Furthermore, the cinomas and fibrosarcoma showed that IP-10 had moderate MVMp/IP-10 may be endowed with a specific anti– antineoplastic activity on its own, but a striking ability to endothelial activity. This possibility is raised by the recently potentiate antitumor reactions initiated by IL-12 or T-cell reported capacity of human IP-10 for inhibiting endothelial adoptive therapy.24,25 cell growth, although the expression of the IP-10 receptor The latter observation allows to suggest that the ability of CXCR3 by normal and transformed endothelial cells is still a IP-10 to foster ongoing immune reactions could be matter of controversy.27,51,52 H5V cell cultures failed to responsible for MVMp/IP-10–induced suppression of express CXCR3 and were not sensitive to the MVMp- H5V hemangiosarcomas. It is known that lesions arising mediated production of IP-10 (present work and Ref. [27]). from H5V cells are immunogenic and serve as targets for a Hence, the CXCR3 expression detected in H5V tumors in host defense reaction, which allows tumors of a small or vivo can tentatively be assigned to infiltrating activated T and medium size to be eradicated, but becomes overwhelmed NK cells.47 Yet, the IP-10 receptor status of H5V cells and and fails to prevent tumor recurrence when primary implants recruited host endothelial cells in tumors from implanted are too large.17 In particular, anti-H5V hemangiosarcoma animals remains to be determined. responses were found to be potentiated by IFN and IL-12, Apparently, primary tumors constitute likely candidate while they were abrogated by anti–T-cell antibodies.17,19,39 sites for maintaining a lasting immune response, induced Our analysis of H5V lesions expression profiles indicated during parvovirus treatment. To fully understand the that inhibition of disease development in infected mice was mechanism of MVMp/IP-10–mediated protection, a second associated with the persistent infiltration of stagnating site needs to be considered for the virus-induced amplifica- tumors with activated effectors (CTL and NK) cells. Thus, tion of an antitumoral immune response — lymphoid

Cancer Gene Therapy Hemangiosarcoma suppression by parvovirus vector MVMp/IP-10 NA Giese et al 441 organs. The lymph nodes draining primary H5V lesions were 2. Cornelis JJ, Haag A, Kornfeld C, et al. Autonomous parvovirus found to sustain a significant level of virus-driven gene vectors In: Cid-Arregui A, Garcia-Garranca´ A, eds. Viral expression, besides the tumors themselves (data not shown). Vectors: Basic Science and Gene Therapy. Natick, MA: Eaton Whether invading tumor cells and/or components of Publishing. 2000:97–118. lymphoid tissues are responsible for this extratumoral vector 3. Rommelaere J, Cornelis JJ. Antineoplastic activity of parvoviruses. J Virol Methods. 1991;33:233–251. gene expression is currently under investigation. However 4. Faisst S, Guittard D, Benner A, et al. Dose-dependent that may be, the fact that tumor-draining lymph nodes are regression of HeLa cell–derived tumours in SCID mice after targets for MVMp-directed gene expression is especially parvovirus H-1 infection. Int J Cancer. 1998;75:584–589. interesting in the view of using this virus to transduce 5. Guetta E, Graziani Y, Tal J. Suppression of Ehrlich ascites immunomodulating molecules. Lymph nodes constitute tumors in mice by minute virus of mice. J Natl Cancer Inst. gathering sites for the effector cells that cooperate in eliciting 1986;76:1177–1180. an immune response. Thus, should H5V cells express IP-10 6. Russell SJ, Brandenburger A, Flemming CL, et al. Transfor- receptors in vivo (see above), the chemokine produced in mation-dependent expression of interleukin genes delivered by DLN might also promote antitumoral defense by attracting a recombinant parvovirus. J Virol. 1992;66:2821–2828. primary tumor cells to the spot where the immune reaction is 7. Kestler J, Neeb B, Struyf S, et al. Cis requirements for the 53 efficient production of recombinant DNA vectors based on built up. autonomous parvoviruses. Hum Gene Ther. 1999;10:1619– Besides validating the use of parvoviral vectors, this study 1632. substantiates the application of IP-10 to vascular tumor 8. Dupont F, Avalosse B, Karim A, et al. Tumor-selective gene therapy. The chemokine IP-10 is recognized as a powerful transduction and cell killing with an oncotropic autonomous anti–angiogenic agent and major downstream mediator of parvovirus-based vector. Gene Ther. 2000;7:790–796. the potent antitumor cytokine IL-12.27,54,55 Yet, to the best 9. Haag A, Wayss K, Rommelaere J, et al. Experimentally of our knowledge, IP-10 has not been tested to date against induced infection with autonomous parvoviruses, minute virus hemangiomas in clinical or preclinical trials. There is an of mice and H-1, in the African multimammate mouse urgent need for novel therapies of vascular tumors because (Mastomys coucha). Comp Med. 2000;50:613–621. currently applied treatments are not satisfactory or associated 10. Kimsey PB, Engers HD, Hirt B, et al. Pathogenicity of 14,56 fibroblast- and lymphocyte-specific variants of minute virus with substantial side effects. The present work provides of mice. J Virol. 1986;59:8–13. the first indication that hemangiosarcoma suppression can be 11. Haag A, Menten P, Van Damme J, et al. Highly efficient achieved by means of IP-10. This strategy is further transduction and expression of cytokine genes in human tumor supported by the fact that no deleterious side effects cells by means of autonomous parvovirus vectors; generation associated with IP-10 applications have been reported so of antitumor responses in recipient mice. Hum Gene Ther. far (present investigation and Refs. [20–26]). 2000;11:597–609. 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Cancer Gene Therapy