Differential Effects of Angiostatin, Endostatin and Interferon-Α1 Gene

Gene Therapy (2002) 9, 867–878  2002 Nature Publishing Group All rights reserved 0969-7128/02 $25.00 www.nature.com/gt RESEARCH ARTICLE Differential effects of angiostatin, endostatin and interferon-␣1 gene transfer on in vivo growth of human breast cancer cells

S Indraccolo1,2, E Gola2, A Rosato2, S Minuzzo2, W Habeler2, V Tisato2, V Roni2, G Esposito2, M Morini3, A Albini4, DM Noonan3, M Ferrantini5, A Amadori2 and L Chieco-Bianchi2 1IST-Viral and Molecular Oncology Section-Padova, Padova, Italy; 2Department of Oncology and Surgical Sciences, University of Padova, Padova, Italy; 3IST-Modulo di Progressione Neoplastica, Genova, Italy; 4IST-Laboratorio di Biologia Molecolare, Genova, Italy; and 5Laboratory of Virology, Istituto Superiore di Sanita`, Roma, Italy

The administration of different angiogenesis inhibitors by interfere with the expression of angiogenic factors. However, gene transfer has been shown to result in inhibition of tumor primary endothelial cell proliferation and migration in vitro growth in animal tumor models, but the potency of these were inhibited by supernatants of the transduced cells con- ␣ genes has been only partially evaluated in comparative stud- taining angiostatin, endostatin, and IFN- 1. Stable gene ␣ ies to date. To identify the most effective anti-angiogenic transfer of the IFN- 1 cDNA by retroviral vectors in both molecule for delivery by retroviral vectors, we investigated MCF7 and MDA-MB435 cells resulted in a marked and long- ␣ the effects of angiostatin, endostatin and interferon(IFN)- 1 lasting inhibition of tumor growth in nude mice that was gene transfer in in vivo models of breast cancer induced associated with reduced vascularization. Endostatin reduced neovascularization and tumor growth. Moloney leukemia the in vivo growth of MDA-MB435, but not MCF7 cells, virus-based retroviral vectors for expression of murine despite similar levels of in vivo production, and angiostatin ␣ angiostatin, endostatin and IFN- 1 were generated, charac- did not impair the in vivo growth of either cell line. These terized, and used to transduce human breast cancer cell findings indicate heterogeneity in the therapeutic efficacy of lines (MCF7 and MDA-MB435). Secretion of the recombi- angiostatic molecules delivered by viral vectors and suggest ␣ nant proteins was confirmed by biological and Western blot- that gene therapy with IFN- 1 and endostatin might be useful ting assays. Their production did not impair in vitro growth for treatment of breast cancer. of these breast cancer cells nor their viability, and did not Gene Therapy (2002) 9, 867–878. doi:10.1038/sj.gt.3301703

Keywords: angiostatin; endostatin; interferon; retroviral vectors; gene therapy

Introduction effective extracellularly, which circumvents the need to transduce all tumor cells. Anti-angiogenic therapy represents a promising Originally isolated from the serum of Lewis lung carci- approach to cancer treatment because most solid tumors noma-bearing mice, angiostatin is a 38-kDa internal frag- cannot grow without the necessary supply of oxygen and ment of plasminogen that spans the first four kringle nutrients ensured by the formation of new blood vessels. domains generated by cleavage of plasminogen by a Moreover, metastatic spread of solid tumors depends on macrophage-derived metalloelastase or other matrix met- the vascularization of the primary mass, indicating that alloproteinases.3 Endostatin is a 20-kDa COOH-terminal a blockage of tumor angiogenesis will also block tumor fragment of collagen XVIII.4 Both angiostatin and endo- metastasis. A wide range of angiogenesis inhibitors has statin have been reported to inhibit specifically endo- been tested, many of which showed efficacy against a thelial cell proliferation and migration in vitro. Further- variety of solid tumor types, in particular the endogenous more, both inhibited experimental primary tumor growth inhibitors, such as angiostatin, endostatin, serpin as well as angiogenesis-dependent growth of metastases antithrombin, the soluble form of the vascular endothelial in mice, inducing tumor dormancy without detectable growth factor (VEGF) receptor 1, and some members of toxicity or development of resistance.5,6 IFN-␣ and IFN-␤ the interferon (IFN) family (reviewed by Kong and Crys- are multifunctional regulatory cytokines involved in the 1 2 tal and Cao ). These proteins are good candidates for control of cell proliferation and viral infections that also gene delivery because they are produced physiologically, show anti-angiogenic activity both in vitro and in vivo.7,8 indicating that they are probably not immunogenic, and Frequent systemic administration of low-dose IFN-␣ and IFN-␤, or the introduction of their genes in some tumor or stromal cells has produced therapeutic effects against Correspondence: S Indraccolo, IST-Viral and Molecular Oncology Section 9–11 and Department of Oncology and Surgical Sciences, University of Padova, many tumor types in several animal models. via Gattamelata 64, 35128-Padova, Italy Angiostatic therapy appears to require long-term Received 20 September 2001; accepted 15 February 2002 administration of the inhibitor to ensure tumor growth Angiogenesis inhibitors gene transfer in breast cancer cells S Indraccolo et al 868 suppression in vivo.1 Long-term systemic delivery of recombinant molecules is expensive, time-consuming for the patient, and may be insufficient to obtain high local concentrations of the therapeutic molecule in the tumor mass. The delivery of the molecule through a gene therapy approach could be a solution, as it would lead to constantly high local levels of the anti-angiogenic protein. Although a number of studies address the therapeutic activity of angiogenesis inhibitors in tumor models, few compare their efficacy when administered by gene transfer.12 The identification of an ‘optimal’ candidate for gene therapy studies clearly involves many steps, among others a definition of the level required for therapeutic effects, and in this regard, some marked differences among the angiostatic molecules might exist. Indeed, it is clear that anti-angiogenic therapy with recombinant angiostatin and endostatin demands a high dose of proteins, in the range of 10–20 mg/kg/day in mice, but it is largely unknown what levels should be reached within the tumor to observe a therapeutic effect. On the other hand, IFN-␣ seems more active as an angiostatic drug 9 when administered systemically at relatively low doses. Figure 1 Angiostatin and endostatin expression vector design and in The issue of dose–response effects is particularly relevant vitro expression studies. (a) Schematic representation of the constructs to gene therapy approaches, because the available viral used in this study: details of molecular cloning are reported in the and non-viral gene transfer systems have a limited capa- Materials and methods. The HA tag was fused to the C-terminus of the bility to express the transgene in vivo. Therefore, we stud- angiostatin fragment. The human B29 leader sequence was fused to the N-terminus of endostatin cDNA to allow its extracellular secretion. SD ied which angiogenesis inhibitor might be the most effec- and SA indicate the splice donor and the splice acceptor sites of the MFG tive when delivered by retroviral vectors, one of the few retroviral vector; ⌿ is the packaging signal of the vector. Restriction sites approved vector systems that could confer long-term relevant to cloning are indicated. The SV40-Neo box in the LMuIFN␣1SN expression of angiostatic molecules in patients. We gener- vector indicates the neomycin phosphotransferase gene driven by the SV40 ated MLV-based retroviral vectors for murine angiosta- promoter. (b) Expression of angiostatin and endostatin by retroviral vec- tin, endostatin and IFN-␣ , and used the vectors to tor-transduced MDA-MB435 and MCF7 cells as determined by Western 1 blotting. In each panel, supernatants from MFG-mAST-, MFG-endo-, deliver these genes to two different breast cancer cell ␣ IFN- 1-, and control LE-transduced cells are analyzed. Murine HA- lines in parallel. We observed that stable gene transfer of tagged angiostatin generated a band of 58 kDa; endostatin and IFN-␣ ␣ 1 IFN- 1 and endostatin cDNAs, but not angiostatin generated bands of 20 kDA. cDNAs resulted in delayed tumor growth in nude mice. These findings confirm that endogenous inhibitors of angiogenesis administered by a gene therapy approach the cells transduced by the LE vector in parallel experi- could be useful for cancer treatment, and clearly indicate ments, and was >90% for each cell line (data not shown). that the therapeutic effects of these agents delivered by Angiogenesis inhibitor production by the genetically viral vectors depend on the tumor type. modified cells was subsequently analyzed by immunoblotting analysis of conditioned medium from the transduced cells (Figure 1b). Both MCF7 and MDA-MB435 Results cells released detectable amounts of angiogenesis inhibitors into the culture medium. Immunoblotting of super- Generation and characterization of retroviral vectors natants indicated that the MFG-mAST vector conferred expressing angiogenesis inhibitors angiostatin expression at relatively high-levels in vitro The Moloney murine leukemia virus (Mo-MLV)-based (Figure 1b). Its apparent molecular weight was about 58 MFG retroviral vector served as the basic vector to derive kDa, which is higher than that reported for murine the murine angiostatin- and endostatin-expressing vec- angiostatin,3 partly because of the presence of an HA tag tors used in this study (Figure 1a). A retroviral vector attached to the C-terminus as a fusion protein, as pre- expressing the green fluorescent protein as the reporter viously described.14 No immunoreactive bands were gene, termed LE, was used to evaluate the efficiency of detected in the supernatants of the LE-transduced cells. ␣ gene delivery to the breast cancer cells. The IFN- 1- Radioimmunoprecipitation analysis with the anti-HA expression vector, termed LMuIFN␣1SN, was previously mAb confirmed these findings (data not shown). Since a described by others and is based on the LXSN backbone murine angiostatin-specific quantitative ELISA is not (Figure 1a).13 Retroviral vector-containing supernatants available, we estimated angiostatin levels by Western were generated by transient transfections of 293T packag- blotting, and found that transduced MCF7 and MDA- ing cells, and used to transduce both MCF7 and MDA- MB435 cells generated similar levels of angiostatin (5– MB435 breast cancer cells, and fibroblastic NIH-3T3 cells. 10 ␮g/ml). As some of the vectors used did not contain a selectable Transduction of both MCF7 and MDA-MB435 cells by marker, gene transfer was repeated several times to max- the MFG-endo retroviral vector resulted in the pro- imize the fraction of transduced cells. The percentage of duction of murine endostatin by the breast cancer cells, genetically modified cells was estimated by measuring clearly visible as a 20 kDa band of similar intensity in the enhanced green fluorescent protein (EGFP) expression in supernatants obtained from each cell line upon immuno-

Gene Therapy Angiogenesis inhibitors gene transfer in breast cancer cells S Indraccolo et al 869 blotting. The production of endostatin in vitro was quant- human bFGF and VEGF expression by RT-PCR. We ified by an ELISA assay, which indicated concentrations observed that MCF7 cells expressed only VEGF in vitro, of 325 ng/ml and 310 ng/ml in the 24 h-conditioned while MDA-MB435 cells expressed both VEGF and bFGF medium of retroviral-vector transduced MCF7 and (Figure 2c). RT-PCR with VEGF-specific primers ampli- MDA-MB435 cells, respectively. Background levels of fied two bands of 543 and 403 bp in all samples, corre- Ͻ endostatin ( 2 ng/ml) were detected in the supernatants sponding to the VEGF121 and VEGF165 isoforms, respect- of breast cancer cells transduced by the LE vector and in ively. On the other hand, RT-PCR with bFGF-specific the parental cell supernatants. Repeated sampling of the primers amplified one band of 239 bp in all samples. No supernatants showed that angiostatin and endostatin significant alteration in the amount of VEGF or bFGF production by MCF7 and MDA-MB435 transduced cells PCR products was observed in the transduced human was stable, with no apparent reduction even after 45 days cell lines producing angiostatin, endostatin, or murine ␣ in vitro culture (data not shown). IFN- 1, as compared with the levels detected in the con- ␣ Interferon production by the IFN- 1 retroviral vector trol cells (LE) or parental cell lines (Figure 2c), suggesting transduced cells was determined by Western blotting no significant alterations in transcription of these (Figure 1b) and quantified by an IFN titration assay, mRNAs. which indicated levels of 256–512 IU/ml in supernatants of both MCF7 and MDA-MB435 cells. Effects of angiogenesis inhibitors produced by breast cancer cells on endothelial cells in vitro In vitro growth kinetics of the genetically modified cells Filtered conditioned culture medium was collected from and analysis of basic fibroblast growth factor (bFGF) each cell line, and layered over P4-P8 passage human and VEGF expression umbilical vein endothelial cells (HUVEC) to examine We next determined whether the expression of the angi- directly the effects on HUVEC proliferation using a [3H]- ogenesis inhibitors, or perhaps the repeated cycles of thymidine incorporation assay. We observed that both ␣ gene transfer by retroviral vectors, might have affected IFN- 1 and endostatin dramatically inhibited HUVEC the in vitro growth kinetics of the breast cancer cell lines. proliferation, while angiostatin had a less pronounced In vitro tumor cell growth impairment has never been effect (Figure 3a). ␣ reported for angiostatin and endostatin, while IFN- 1 HUVEC readily migrated in response to conditioned ␣ produced by IFN- 1 retroviral vector genetically modi- medium from NIH-3T3 cells in chemotaxis assays. When ␣ fied cells might have direct antiproliferative effects. The pre-incubated with supernatants of IFN- 1 expressing proliferation of wild-type MCF7 and MDA-MB435 cells MCF7 cells, an approximately 50% reduction in HUVEC ␣ in the presence of IFN- 1-containing medium was ident- migration was observed as compared with controls ical to that of control cells (data not shown), suggesting (Figure 3b). Angiostatin- and endostatin-containing that the former are not directly sensitive to IFNs and that supernatants did not significantly impair HUVEC IFN gene transfer would not lead to the selection of IFN- migration under the same experimental conditions. How- resistant breast cancer cells. Proliferation assays demon- ever, when produced at higher levels by 293T cells transi- strated that neither angiostatin, endostatin nor interferon ently transfected by the same vectors, whose conditioned at the levels detected in this study, significantly affected medium contained about five-fold more factor than con- the proliferation of breast cancer cells in vitro. The growth ditioned medium from transduced MCF7 cells (data not ␣ kinetics of the angiostatin, endostatin and IFN- 1-produc- shown), angiostatin and endostatin reduced endothelial ing cells were similar to those of cell lines transduced cell migration by 60% and 47%, respectively (Figure 3c). with the EGFP-encoding vector or the parental cells (Figure 2a). To determine whether gene delivery by MLV In vivo tumor growth inhibition vectors may induce alterations in the cell cycle profiles Genetically modified breast cancer cells were implanted of the target cells, we stained them with propidium iod- s.c. in nude mice, and the tumors generated were meas- ide and analyzed the cell cycle by flow cytometry. We ured over time. In the case of MCF7 cells, neither angio- found that a significant fraction of parental MCF7 cells statin nor endostatin significantly reduced tumor growth growing in vitro were cycling cells, as the G0/G1 and the as compared with the EGFP-expressing retroviral vector S/G2 cell populations were 29% and 71%, respectively. transduced control cells (Figure 4a). In contrast, the IFN- ␣ Furthermore, analysis of the transduced cells disclosed 1-producing MCF7 cells showed much slower tumor patterns similar to those of the parental cells (data not growth than controls (Figure 4a), demonstrating a shown), thus indicating that retroviral vector gene deliv- marked therapeutic effect of murine IFN. The tumors for- ery did not grossly change the growth characteristics of med by MDA-MB435 cells were generally smaller than the cells. Similar results were obtained with MDA- those formed by MCF7 cells, and grew slower in nude MB435 cells. mice. Tumors formed by the different experimental To investigate whether repeated transductions may groups over the first 6 weeks were similar in size, after ␣ affect cell viability, which in turn might influence tumor which the tumors generated by endostatin- or IFN- 1- growth in vivo, we verified it by eosin staining and a two- producing MDA-MB435 cells underwent a marked color fluorescence viability assay; in all cases >99% of the reduction in size as compared with controls (Figure 4b). cells were alive, with no appreciable differences among Angiostatin did not significantly reduce tumor growth in the different samples. The results of a representative this system (Figure 4b). The divergent effects of endosta- analysis on MCF7 cells are shown in Figure 2b. tin on the growth of these two breast cancer cell lines To evaluate whether the production of angiostatic mol- were not due to significant differences in the amount of ecules might interfere with the expression of angiogenic endostatin produced in vitro, as determined in the con- molecules by the tumor cell lines, and thus possibly with ditioned medium by immunoblotting and confirmed by their capacity to form tumors in vivo, we evaluated ELISA (see above).

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Figure 2 In vitro effects of the angiogenesis inhibitors on breast cancer cell proliferation, viability and bFGF or VEGF expression. (a) In vitro growth ␣ rates of parental (wt), angiostatin-, endostatin-, IFN- 1-, or EGFP-producing MDA-MB435 (left panel) and MCF7 (right panel) cells as determined by [3H]-thymidine incorporation. No significant differences in proliferation rate were found among the different cell populations. (b) Flow cytometric viability assay using a cell viability/cytotoxicity kit (Molecular Probes). Parental or retroviral vector-transduced MCF7 cells were stained with calcein AM and ethidium homodimer-1 as detailed in the Materials and methods. After 5 min, flow cytometry analysis was carried out with excitation at 488 nm. The fluorescence emission was acquired separately at 530 nm and 585 nm for the live-cell (bottom panels) and the dead-cell (top panels) population, respectively. Ethanol-fixed MCF7 cells were used to define the dead cell fraction. (c) Expression of human bFGF and VEGF by the parental and the transduced cell lines in vitro by RT-PCR. MDA-MB435 cells express both angiogenic factors, while MCF7 express only VEGF in vitro. Angiogenesis inhibitor production did not interfere with bFGF and VEGF expression.

␣ Histological studies 1-producing tumors (Figure 5h) compared with controls The histology of the tumors formed by the different (Figure 5e). No appreciable differences in the number of transduced MDA-MB435 cells after 14 days of growth in blood vessels were found between control MDA-LE and vivo was similar, with the tumor cells arranged in nests MDA-angio or MDA-endo cells (Figure 5e–g). Microves- and solid patterns (Figure 5a–d). However, the tumors sel density counts in MDA-MB435 tumors indicated 132, ␣ generated by the IFN- 1-producing cells showed exten- 140, 131 and 84 microvessels in 10 representative fields of sive areas of ischemic coagulative necrosis formed of cell tumors formed by control MDA-LE, MDA-endo, MDA- ␣ debris, bordered by viable tumor cells with several angio, and MDA-IFN- 1 cells, respectively. Similar find- mitotic figures (Figure 5d). ings were obtained following histological and immunohi- Immunohistochemical analyses with anti-CD31 mAb stochemical analysis of the different MCF7 tumors (data clearly indicated a reduced vascularization in the IFN- not shown).

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Figure 4 Patterns of in vivo growth of tumors by breast cancer cells producing angiogenesis inhibitors. MCF7 (panel a) and MDA-MB435 cells (panel b) were transduced ex vivo by retroviral vectors to express either ␣ angiostatin (open circles), endostatin (filled squares), IFN- 1 (open Figure 3 Effects of the angiogenesis inhibitors on the proliferation and squares) or EGFP (control vector) (filled circles). The genetically modified migration of endothelial cells. (a) Proliferation of HUVEC following incu- cells were subsequently mixed with matrigel and injected s.c. into the ␣ bation with conditioned medium from endostatin-, IFN- 1-, and, to a flanks of nude mice (six mice/group). Nodule dimensions were used to lesser extent, angiostatin-producing MCF7 cells, as compared with incu- compute tumor volumes. Statistically significant differences between the bation with supernatants from MCF7 cells transduced with the control experimental groups and controls (Student’s test) are indicated by aster- vector (LE) or DMEM (medium). The assay was performed in collagen isks. The experiment was repeated with another set of animals (six I-coated 96-multiwell plates as detailed in the Materials and methods and mice/group) with identical results. repeated twice. Bars, s.d. (b) Effects of conditioned medium from transduced MCF7 cells (MCF7-CM) on chemotaxis of HUVEC. NIH-3T3- conditioned medium was used as chemoattractant; serum-free medium were analyzed for expression of transgene-encoded (SFM) with 0.1% BSA was used as a negative control to evaluate random mRNAs by RT-PCR. Tumor RNA was treated with migration. To evaluate the effects of the different angiogenesis inhibitors DNase I to remove any residual genomic DNA that could on endothelial cell migration, HUVEC were pre-incubated with identical amounts of conditioned medium from MCF7 cells transduced with the generate PCR products related to transgene presence, different retroviral vectors, as detailed in Materials and methods. NIH- rather than expression. Expression of angiostatin, endos- ␣ 3T3, migration to supernatant of NIH-3T3 cells; SFM, migration to tatin and IFN- 1 was detected exclusively in tumors gen- ␣ serum-free medium; LE, mAST, endo, IFN- 1 columns: migration of erated by cells transduced with the corresponding retro- HUVEC to supernatant of NIH-3T3 cells following pre-incubation with viral vector, as shown in Figure 6. This experiment conditioned medium from MCF7 cells transduced by the LE, angiostatin, ␣ indicated that the transgenes were transcriptionally endostatin, and IFN- 1 retroviral vectors, respectively. The assay was performed in triplicate in Boyden chambers and repeated twice. Bars, s.d. (c) active in vivo, suggesting that the viral promoter was not Effects of conditioned medium from transiently transfected 293T cells silenced in this system. Although angiostatin did not (293T-CM) on chemotaxis of HUVEC. The assay was performed as delay tumor growth, it was expressed in both the MDA- detailed in (b). Vectors and transfected cells are indicated as above. angio and the MCF-angio tumors, as confirmed by both RT-PCR analysis of tumor samples (Figure 6) and immunohistochemistry for the HA tag attached to the angiosta- Determination of in vivo expression of the angiogenesis tin fusion protein (Figure 7a–d) which showed strong, inhibitors diffuse positivity in the mAST-transduced tumors. To Different approaches were employed to confirm that gen- rule out that angiostatin may undergo cleavage in vivo etically modified cells produced the angiogenesis inhibi- with loss of function, we analyzed tumor lysates by West- tors in vivo. Following the death of the animals, tumors ern blotting with the anti-HA-specific antibody. As

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Figure 5 Histological and immunoistochemical analysis of the growing tumors 14 days after injection of 5 × 105 MDA-MB435 transduced cells. Panels × ␣ (a–d), H&E staining, original magniﬁcation: 20. Only the tumor sample obtained from IFN- 1-producing cells (d) showed a large area of ischemic coagulative necrosis, while control MDA-LE cells (a), MDA-endo (b), and MDA-angio (c) injected mice show tumor masses composed of viable cells. Panels (e–h), immunohistochemical analysis with anti-CD31 mAb showed a reduced microvessel density in IFN-producing tumors (h) compared with control MDA-LE (e), MDA-angio (f), and MDA-endo (g) tumors.

shown in Figure 7e, full-length angiostatin was detected duction in vivo, murine splenocytes were analyzed for the in tumors formed by MDA-MB435 and MCF7 cells. expression of the Ly-6c marker, which is up-modulated To investigate whether interferon was locally pro- by this cytokine. These data (not shown) indicated that ␣ duced, we recovered the tumor cells 3 weeks after IFN- 1 was produced in vivo by the tumor cells and implantation, cultured them in vitro, and assayed cyto- released systemically. ␣ kine levels in the culture medium. IFN- 1 levels of 256– As assays to detect endostatin in serum are available, 512 U/ml were detected in these supernatants, indicating we investigated systemic endostatin expression. In ani- ␣ that the tumor cells continued to produce IFN- 1 for at mals which had received endostatin-producing cells least 3 weeks after implantation. To conﬁrm IFN pro- serum endostatin levels increased with increasing tumor

Gene Therapy Angiogenesis inhibitors gene transfer in breast cancer cells S Indraccolo et al 873 both HUVEC proliferation and migration in vitro, and showed maximal anti-tumor activity in vivo. Despite a lack of efficacy on breast tumors, angiostatin produced by the retroviral vector was biologically active in vitro, even though compared with IFN its effects on HUVEC proliferation and migration were less pronounced. The angiostatin used was fused to the HA epitope. This was necessary to detect the protein, as specific anti-murine angiostatin antibodies are not available. Although a comparison with native murine angiostatin is lacking, this HA-tagged murine angiostatin inhibited capillary endothelial cell proliferation and suppressed the growth of T241 fibrosarcoma cells.14 We recently showed that Figure 6 Angiogenesis inhibitor expression within the tumors. RT-PCR angiostatin delivery by the same vector significantly analysis with transgene-specific primers confirmed angiostatin, endostatin delayed the growth of Kaposi’s sarcoma cells in vivo, ␣ and IFN- 1 expression in the tumors formed by cells transduced with the transduced by the same vector.21 Furthermore, other respective vectors. groups have used HA-tagged angiostatin for gene therapy purposes, with evidence of anti-angiogenic effects.19 size, and 8 weeks after MDA-MB435 and MCF7 cell This points to the conclusion that the tag does not implantation were on average 287 ng/ml and 670 ng/ml, interfere with angiostatin activity. respectively (Figure 8). Interestingly, an increase was also Since all the angiostatic molecules used in this study observed in animals that had received control cells. This were active on endothelial cells in vitro, we suggest that might represent endogenous production of endostatin in the heterogeneous anti-tumor effects observed in vivo tumor-bearing animals (Figure 8). Indeed, basal levels in might be related to the levels of angiostatin and endosta- the different animal groups were similar, and about 30– tin. As expression levels can vary greatly among different 70 ng/ml as observed by others,15 and did not signifi- tumors following gene delivery, they should be con- cantly increase over time, unless the animals were sidered as a potential limiting factor for such approaches. injected with tumor cells (data not shown). In fact, the levels of angiostatin produced by transduced Kaposi’s cells, whose growth was inhibited by angiosta- 21 Discussion tin, were approximately 10-fold higher than those produced by breast cancer cells, likely due to the different Attention for angiostatic genes has recently been height- activity of the Mo-MLV promoter/enhancer driving ened by the possibility that they may be delivered by transgene expression in the two cell lines. Thus, it is poss- gene transfer methods. The goal of angiostatic gene trans- ible that critical angiostatin levels must be achieved for fer is to utilize some host cells as the ‘cell factories’ of significant anti-tumor effects to be observed. the therapeutic molecule, thus circumventing the critical The similar growth rate of endostatin-producing MCF7 problems associated with long-term administration of tumors compared with controls, and angiostatin’s lack of protein-based drugs. Angiogenesis seems to represent an effect on the growth of both cell lines did not appear to be important prognostic factor in breast cancer,16,17 suggest- due to gene silencing in vivo, which has been occasionally ing that its inhibition could play a role in controlling this observed in previous studies.22 Indeed, transgene disease, and leading us to test model systems of breast expression and local full-length angiostatin production cancer. Previous reports indicated that selected angios- were demonstrated in the tumors. Furthermore, serum tatic molecules delivered by gene transfer methods act as endostatin levels up to 1 ␮g/ml were observed in ani- effective inhibitors of mammary tumor growth in mice.18–20 mals that received endostatin-transduced MCF7 cells. However, a direct comparison of different inhibitors was Interestingly, endostatin showed divergent effects on lacking. Our objective was to determine the sensitivity of the two breast cancer cell lines studied. These two lines two different mammary tumor systems to three endogen- differ in many genetic and phenotypic aspects. We ous inhibitors of angiogenesis delivered by viral vectors. observed that parental MCF7 cells grow much faster than This should help to define which agent might be more MDA-MB435 cells in nude mice. Rapidly growing tumors effective in this setting. may generate ischemic areas within the tumor mass at Our findings indicate that mammary tumor cell lines early time points, leading to up-regulation of pro-angio- implanted s.c. in nude mice were most sensitive to IFN- genic ischemia-induced genes, such as VEGF that could ␣ 1. Endostatin significantly retarded the growth of MDA- favor tumor growth. Endostatin therapy, which in our MB435-derived tumors, but had little effect on MCF7- study delayed the growth of MDA-MB435 but not MCF7 derived tumors, while angiostatin did not exert any effect cells, might be particularly effective in slowly growing on tumors derived from either cell type. Interestingly, tumors. In keeping with these findings, a recent study of Kuo et al12 also observed heterogeneous inhibition of the effects of angiogenesis inhibitors on multistage pan- tumor growth by anti-angiogenic genes. These investi- creas carcinogenesis in mice showed that AGM-1470, gators used recombinant adenoviruses carrying the angiostatin, BB-94 and endostatin had distinct efficacy angiostatin, endostatin and soluble VEGF receptor genes profiles depending on the stage of tumor development to increase systemic levels of the angiostatic molecules in and probably the kinetics of cell growth.23 In future stud- different tumor models. ies, it will also be interesting to evaluate the possible cor- Evaluation of the in vitro biological activity of the angio- relation between status of estrogen receptors on breast genesis inhibitors was based on endothelial cell prolifer- cancer cells and response to endostatin. ␣ ␣ ation and migration. Interestingly, only IFN- 1 reduced Our findings also indicated that IFN- 1 is the most

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Figure 7 Angiostatin expression in MCF7 and MDA-MB435 tumors. (a–d) Immunohistochemical analysis of tumor sections with anti-HA staining (mAb anti-HA, hematoxylin counterstain, original magniﬁcation ×20). HA immunostaining in one MCF7-angio tumor (a) and in one MDA-angio tumor (c). No speciﬁc staining was detected following incubation of the samples only with the secondary antibody (b and d). (e) Expression of angiostatin in MDA-MB435 and MCF7 tumors by Western blotting. Lysates from tumors formed by angiostatin- (mAST) and EGFP- (LE) transduced cells were analyzed: mAST-transduced tumors contained full-length angiostatin (58 kDa).

active single agent against the two breast cancer cell lines including macrophages and NK cells, might contribute to studied. IFN-␣ is used for the treatment of more than 14 the observed anti-tumor effect in these mice. In fact, type types of cancer, including some hematological malig- I IFNs exert multiple regulatory activities on host nancies and certain solid tumors, such as melanoma, immune cells that are particularly relevant to the overall renal carcinoma and Kaposi’s sarcoma.7,24 Its anti-tumor antitumor response.25,26 We did not detect a more intense ␣ activity can be attributed, at least in part, to its direct lymphoid infiltrate in IFN- 1-producing tumors com- effects on tumor cells. In fact, in some cases IFN-␣ can pared with controls or angiostatin- or endostatin-produc- directly inhibit the proliferation of normal and tumor ing tumors. Furthermore, we tested the susceptibility of cells in vitro and in vivo by modulating the expression of MCF7 and MDA-MB435 cells to NK-mediated lysis in oncogenes or tumor suppressor genes, and it can increase vitro, and found that neither was efficiently killed by acti- MHC class I expression, thus favoring immune recog- vated murine NK cells (data not shown). It might be nition.24 Our findings rule out a direct anti-proliferative possible that some host cells contribute differently to the ␣ ␣ effect of IFN- 1 on MCF7 and MDA-MB435 cells. How- anti-tumor effect of IFN- 1, ie by producing soluble fac- ever, the possibility exists that some other cell types, tors that reduce tumor growth, or by affecting endothelial

Gene Therapy Angiogenesis inhibitors gene transfer in breast cancer cells S Indraccolo et al

␣ 875 vector encoding murine IFN- 1, a cytokine known to exhibit some species specificity.32 However, we also analyzed the expression of murine bFGF in vivo by RT-PCR, and found similar levels of expression in all the tumors examined (data not shown) So, within the limitations of our PCR assay, we conclude that the angiogenesis inhibition observed in this system was not associated with marked down-regulated expression of angiogenic molecules in vivo. Since IFNs might exert angiostatic effects at low, con- stant doses, which are readily obtained by current gene delivery systems as shown here, it is tempting to specu- late that local IFN-␣ production by genetically modified cells, either tumor cells as in this study or host non-neo- plastic cells, might represent an effective way to delay tumor growth by inhibition of angiogenesis. The inclusion of this gene in lentiviral vectors will also enable us to test this hypothesis on established mammary tumors. Materials and methods ␣ Molecular cloning of angiostatin, endostatin, and IFN 1- encoding retroviral vectors The pMFG-mAST retroviral vector was generated by cloning a 1.5 kb-long NcoI/BamHI restriction fragment obtained from the digestion of pCMV-mAST,14 which encodes murine angiostatin with an HA tag attached as a fusion protein to the C-terminus, in the corresponding cloning sites of the MFG retroviral vector (Figure 1).37 Similarly, murine endostatin cDNA fused to the human B29 leader sequence was released from pB29ENDO,38 and cloned in the NcoI/BamHI sites of pMFG. These constructs were verified by PCR, restriction analysis, and sequencing before their further use. The LE retroviral Figure 8 Serum endostatin levels in nude mice following s.c. injection vector used as control is a derivative of the LXSN retrovi- of endostatin-transduced cells. Serum samples were analyzed at regular ral vector,39 that carries an EGFP gene driven by the Mo- intervals for endostatin content by an ELISA assay (Cytimmune) after MLV LTR.40 The murine IFN-␣ -expressing retroviral × 5 1 injection of 5 10 endostatin- or LE-transduced (control) cells in nude vector was produced by a transfected GP+env Am12 mice. Endostatin levels were higher in the groups of mice that received 13 endostatin-producing cells as compared with controls. An increase in packaging cell line, as previously described. endostatin serum levels was detected with both MDA-endo (a) and Cell lines and transfections MCF-endo (b) cells. 293T human kidney cells, MCF7 and MDA-MB435 human breast cancer-derived cell lines were obtained

27 from ATCC. All cell lines were grown in DMEM sup- cell function, as recently reported by Yao et al for IL- plemented with 10% fetal calf serum (FCS) and 1% L- 12-activated murine NK cells. glutamine. Infectious particles carrying the angiostatin, The inhibition of tumor-induced angiogenesis, and the the endostatin, and the control LE retroviral genomes induction of extensive ischemic necrosis in the tumor were generated by a transient three-plasmid vector-pack- area by type I IFNs are well documented phenomena in aging system, as previously described.41 Briefly, 293T 28,29 ␣ animal models. Moreover, IFN- was the first of the cells were seeded in 100-mm-diameter Petri dishes at 5 × known angiogenesis inhibitors to be evaluated clinically 106cells/dish, and transfected overnight according to a and to show its therapeutic potential in angiogenic dis- calcium-phosphate protocol using 12 ␮g of a plasmid eases, such as childhood hemangioma and Kaposi’s sar- 30,31 DNA encoding the different retroviral vector genomes, 6 coma. Its anti-angiogenic activity has been well estab- ␮g of a Mo-MLV gagpol expression construct, and 0.3 lished by a number of in vitro studies indicating that it ␮ 8,32 g of a VSV-G expression plasmid, which encodes the can block endothelial cell migration, and also down- envelope used by the vectors in this study. Thirty-six regulate angiogenic factor expression, such as bFGF, hours after transfection, culture medium was replaced IL-8, MMP-2 and MMP-9.33–36 ␣ with fresh DMEM without FCS. Twenty-four hours later IFN- also down-regulates bFGF expression in human the retroviral vector-containing supernatants were har- carcinomas.34 However, we did not observe reduced ␮ ␣ vested, passed through 0.45- m filters, and stocked at expression of bFGF mRNA in the IFN- 1-producing –80°C until further use. MDA-MB435 cell line, neither in vitro (Figure 2c) nor in vivo (data not shown), as evaluated by RT-PCR experi- Transduction of cells with retroviral vectors ments. This finding might be related to the fact that we To assess the ability of the virions to transduce breast transduced human breast cancer cells with a retroviral cancer cells, 1 ml of filtered supernatant was layered over

Gene Therapy Angiogenesis inhibitors gene transfer in breast cancer cells S Indraccolo et al 876 MCF7 and MDA-MB435 target cells that had been seeded filters with 12 ␮m pores for HUVEC cells coated with 5 into six-well culture plates at 2 × 105 cells per well the day ␮g/ml of gelatin. 5 × 104 HUVECs in serum-free medium before infection. Protamine sulphate (8 ␮g/ml) (Sigma, St (SFM) containing 0.1% bovine serum albumin were Louis, MO, USA) was added to the wells, and the cells placed in the upper compartment; the lower compart- were kept in a total volume of 2 ml. After 6–9 h at 37°C, ment was filled with either SFM as a negative control, or 3 ml of medium were added to dilute the protamine sul- conditioned medium from NIH-3T3 cells as chemoat- phate; 36 h later, the cells were split 1:4 in 6-cm-diameter tractant. To test the inhibitory activity of recombinant Petri dishes, and kept in culture until they underwent a proteins, HUVEC were preincubated for 30 min with dif- new transduction cycle 24 h later. This procedure was ferent amounts of DMEM, conditioned medium from repeated five times. The percentage of EGFP+ cells after MCF7 and MDA-MB435 cells transduced with the endo- ␣ each transduction cycle with the EGFP-encoding vector statin, angiostatin, IFN- 1, or the control retroviral vec- was determined by FACS analysis, and used as a para- tors. The chambers were then incubated at 37°Cin5%

meter to estimate the genetically modified cell fraction CO2 for 6 h. Cells remaining on the upper surface of the which, after the fifth transduction cycle, was >90%. filter were then mechanically removed, and five to 10 random fields of cells which had migrated to the lower sur- Western blotting face of each filter were counted after staining. Assays Angiostatin and endostatin expression in supernatants were performed in triplicate, and repeated at least was determined by Western blotting, according to stan- three times. dard protocols. For immunoblotting analysis, 40 ␮l FCS- free supernatant was electrophoresed on 10% polyacryla- In vitro proliferation assays mide gels, and separated proteins were blotted for 2 h ␣ The effects of angiostatin, endostatin and IFN- 1 on the at 400 mA on to a nitrocellulose membrane. In a set of in vitro proliferation of breast cancer and endothelial cells experiments with endostatin, the supernatant was con- were tested using [3H]thymidine proliferation assays. In centrated 10-fold on Ultrafree columns (Millipore, a set of experiments, MCF7 and MDA-MB435 cells, trans- Bedford, MA, USA) with 10 kDa cutoff. The membrane duced with the different vectors, were plated in 96-multi- was then saturated with PBS 1% non-fat dry milk (Sigma) well plates at 1 × 104 and 5 × 103 cells/well, respectively. ° as blocking buffer for 3 h at RT, and then stored at –20 C Cell proliferation was assessed at 24 h intervals over 5 until use. Immunoprobing was performed with a 1:8000 consecutive days. At the appropriate time points, 1 dilution of a murine mAb against the hemagglutinin ␮Ci/well of [3H]thymidine was added to the cultures and (HA) tag (BabCo, Richmond, CA, USA), or a 1:5000 incorporation was quantified 24 h later. In experiments dilution of a rabbit antiserum against murine endostatin with endothelial cells, HUVEC were plated in collagen I- (Neosystem, Meylan, France), followed by hybridization coated 96-multiwell plates at 800 cells/well. After over- with a 1:5000 diluted anti-mouse or anti-rabbit HRP-con- night incubation at 37°C, the medium was aspirated and jugated antibody (Amersham-Pharmacia, Little Chalfont, replaced with equal volumes (50 ␮l) of the conditioned UK). Antigens were identified by luminescent visualiz- medium from MCF7 and MDA-MB435 cells transduced ation using the SuperSignal kit (Pierce, Rockford, IL, with the different vectors. Eight replicates of each super- USA). natant were tested. After 30 min incubation at 37°C, 100 ␮ Angiostatin and endostatin measurements l of M199 medium containing 10% FCS and 10 ng/ml bFGF were added. After 72 h incubation at 37°C, HUVEC Angiostatin levels in the supernatants of the transduced proliferation was analyzed by [3H]thymidine labelling (1 breast cancer cell lines were estimated by Western blot- ␮Ci/well) and incorporation was quantified 24 h later. ting and compared with an HA-tagged ␤-gal protein which was used as an internal standard. Endostatin levels Tumor growth in vivo in conditioned supernatants and serum were measured 5x105 retroviral vector-transduced MCF7 and MDA- with a commercially available ELISA kit (Cytimmune MB435 cells in Matrigel (10 mg/ml) were injected s.c. in Sciences, College Park, MD, USA). the flanks of female nude (nu/nu) mice (Charles River Cell viability assays Breeding Laboratories Italia, Calco, Italy). Tumor size We analyzed the viability of the genetically modified cells was measured regularly over time with skin calipers. before in vivo experiments by conventional eosin staining, When the tumors reached pre-set dimensions according and a two-color fluorescence cell viability assay to current ethical practice, or 3 months after the begin- (LIVE/DEAD Kit, Molecular Probes, Leiden, The ning of the experiment, the animals were killed and Netherlands), that allows quantitative analysis of live and tumors were collected, fixed in formalin, paraffin-embed- dead cells by flow cytometry. To this end, 1 × 106 MDA- ded, sectioned, and stained with H&E. Procedures MB435 and MCF7 cells were trypsinized, washed in PBS involving animals and their care conformed with insti- and stained for 10 min at room temperature in a 200 ␮l tutional guidelines that comply with national and inter- volume of 1 ␮M calcein AM and 8 ␮M ethidium homodi- national laws and policies (EEC Council Directive mer-1. Following incubation, cell samples were analyzed 86/609, OJ L 358, 1, December 12, 1987; NIH Guide for by flow cytometry with excitation at 488 nm. The fluor- the Care and Use of Laboratory Animals, NIH Publi- escence emission was acquired separately at 530 nm and cation 85-23, 1985). 585 nm for the live-cell and the dead-cell population, respectively. RT-PCR Total RNA was isolated from cell lines and tumors using In vitro migration assays the TRIzol reagent (Gibco BRL, Gaithersburg, MD, USA) Chemotaxis assays were performed in Boyden chambers according to the manufacturer’s instructions. One as previously described,42 using pvp-free polycarbonate microgram of total RNA was digested with RNase-free

Gene Therapy Angiogenesis inhibitors gene transfer in breast cancer cells S Indraccolo et al 877 DNase I (Roche, Milan, Italy) and used for the synthesis a monoclonal anti-CD31 (Becton-Dickinson, Franklin of first-strand cDNA using oligo-dT primers and MLV Lakes, NJ, USA) to highlight vessels, or a monoclonal reverse transcriptase (Applied Biosystems, Foster City, anti-HA tag (BabCo) to localize the HA-angiostatin CA, USA) following the manufacturer’s protocol. DNase fusion protein. Parallel negative controls without primary treatment was necessary to remove any residual genomic antibodies were run in all cases. Microvessel areas were DNA from the RNA sample, which would have gener- quantified by manual counting of hotspots in sections. ated spurious bands at subsequent PCR analysis. Ampli- Ten representative fields for each tumor were counted. fication was performed using the following primers: Statistical analysis 5’-GGAACCCAGATGGAGAAACT-3’ (mAST-for) The significance of the difference in tumor size was 5’-GGCTAGCGTAATCCGGAACAT-3’ (mAST-HA-rev) determined by Student’s t test. 5’-TGAAGATCTGTGACCATGGCCAGGCTGGCGTTGT-3’ (endo-for) 5’-CTATTTGGAGAAAGAGGTCATGAA-3’ (endo-rev) Acknowledgements 5’-GGCTCTGTGCTTTCCTGATGG-3’ (IFN-for) We thank Dr E Lechman for the MFG retroviral vector; 5’-CTCTTCTCTCAGTCTTCCCAGC-3’ (IFN-rev) Dr E Shewach for the anti-Ly-6C antibody; Dr V Tosello 5’-CGAAGTGGTGAAGTTCATGGATG-3’ (VEGF-for) for cytofluorimetric analysis; Mr P Gallo for artwork and 5’-TTCTGTATCAGTCTTTCCTGGTGAG-3’ (VEGF-rev) Ms P Segato for help in the preparation of the manu- 5’-ATGGCAGCCGGGAGCATCACC-3’ (bFGF-for) script. This work was supported by grants from the 5’-CACACACTCCTTTGATAGACACAA-3’ (bFGF-rev) MURST 40% and 60%, the Italian Association for Cancer 5’-ACCATTGGCAATGAGCGGTT-3’ (act-for) Research (AIRC), the Ministero della Sanita` Programma 5’-TCCTGCTTGCTGATCCACAT-3’ (act-rev) Nazionale Ricerca sull’AIDS, the Italian Foundation for PCR was performed for 35 cycles at 95°C30s,56°C30 Cancer Research (FIRC), the Fondazione Cassa di Rispar- s, 72°C 1 min. The amplified products were resolved by mio di Padova e Rovigo, the CNR PF Biotecnologie. 1.5% agarose gel electrophoresis, and stained with ethidium bromide. No bands were obtained following PCR References analysis of DNase-treated RNA samples amplified without RT. 1 Kong HL, Crystal RG. Gene therapy strategies for tumor antian- giogenesis. J Natl Cancer Inst 1998; 90: 273–286. IFN titration 2 Cao Y. Endogenous angiogenesis inhibitors and their thera- IFN was titrated on murine L929 cells as described pre- peutic implications. 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