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[Cancer Research 61, 5441-5446, July 15, 2001]
© 2001 American Association for Cancer Research

Experimental Therapeutics

Effects of Angiostatin Gene Transfer on Functional Properties and in Vivo Growth of Kaposi’s Sarcoma Cells1

Stefano Indraccolo, Monica Morini, Eleonora Gola, Fabio Carrozzino, Walter Habeler, Simona Minghelli, Leonardo Santi, Luigi Chieco-Bianchi, Yihai Cao, Adriana Albini and Douglas M. Noonan2

Istituto Nazionale per la Ricerca sul Cancro, Biotechnology Section-Padova, 35128 Padova, Italy [S. I.]; Modulo di Progressione Neoplastica [M. M., D. M. N.] and Laboratorio di Biologia Molecolare, 16132 Genova, Italy [A. A.]; Department of Oncology and Surgical Sciences, University of Padova, 35128 Padova, Italy [S. I., E. G., W. H., L. C-B.]; Advanced Biotechnology Center, 16132 Genova, Italy [F. C., S. M., L. S.]; Departments of Oncology, Biology, and Biotechnology, University of Genova, 16132 Genova, Italy [L. S.]; and Laboratory of Angiogenesis Research, Microbiology and Tumor Biology Center, Karolinska Institute, S171 76 Stockholm, Sweden [Y. C.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gene transfer delivery of endogenous angiogenesis inhibitors such as angiostatin would circumvent problems associated with long-term administration of proteins. Kaposi’s sarcoma (KS), a highly vascular neoplasm, is an excellent model for studying tumor angiogenesis and antiangiogenic agent efficacy. We investigated the effects of angiostatin gene transfer in in vitro and in vivo models of KS-induced neovascularization and tumor growth. A eukaryotic expression plasmid and a Moloney leukemia virus-based retroviral vector for expression of murine angiostatin were generated harboring the angiostatin cDNA with cleavable leader signals under the control of either the strong cytomegalovirus promoter/enhancer or the Moloney leukemia virus long terminal repeat. Angiostatin secretion was confirmed by radioimmunoprecipitation and Western blot analysis. Supernatants of angiostatin-transfected cells inhibited endothelial cell migration in vitro. Stable gene transfer of the angiostatin cDNA by retroviral vectors in KS-IMM cells resulted in sustained angiostatin expression and delayed tumor growth in nude mice, which was associated with reduced vascularization. These findings suggest that gene therapy with angiostatin might be useful for treatment of KS and possibly other highly angiogenic tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expansion of the tumor mass and metastatic dissemination of most solid tumors appears to depend on the vascularization of the primary mass. This suggests that blockage of tumor angiogenesis will block tumor growth and metastasis and is a promising approach to cancer treatment. Several angiogenesis inhibitors have shown efficacy against solid tumors, in particular, the endogenous inhibitors of angiogenesis, such as angiostatin (1) , endostatin (2) , and tissue inhibitors of metalloproteinases (3 , 4) , are effective in animal models and harbor several useful features. They are produced physiologically, suggesting that they are safe for patients, and they are effective extracellularly, circumventing the need to transduce all tumor cells, and can be administered by a gene therapy approach.

Angiostatin was originally isolated from the serum of Lewis lung carcinoma-bearing mice and contains a Mr 38,000 internal fragment of plasminogen (amino acids 98–440), spanning the first four Kringle domains (1 , 5) . It is generated by cleavage of plasminogen by macrophage-derived metalloelastase (6) or other matrix metalloproteinases (5 , 7) . In vitro, angiostatin inhibits endothelial cell proliferation and migration (8) , induces cell cycle arrest and apoptosis (9 , 10) , and reduces endothelial cell invasion and morphogenesis (10) . In vivo, it inhibited growth of primary tumor and metastases without detectable toxicity or development of resistance (11) .

As for most angiostatic factors, the molecular pathway by which angiostatin exerts its effects is as yet unknown. Angiostatin binds the {alpha}/ß subunit of ATP synthase on the surface of endothelial cells (12) , suggesting that it could affect endothelial cell proliferation by making them more sensitive to hypoxia. It has been shown to down-regulate endothelial cell M-phase phosphoproteins and increase the apoptotic rate (13) . Angiostatin has been reported to diminish extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2 mitogen-activated protein kinase activation by basic fibroblast growth factor and vascular endothelial growth factor (14) , suggesting that the antiangiogenic effects may be linked to attenuated mitogen signaling.

Angiostatic therapy requires a long-term administration of the inhibitor to insure suppression of the tumor cells in vivo (15) . Long-term systemic delivery of recombinant molecules is expensive, time-consuming for the patient, and could 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 fascinating solution, producing chronic, locally high levels of the antiangiogenic protein (16 , 17) .

KS3 is a highly vascularized mesenchymal neoplasm associated with human immunodeficiency virus and human herpes virus 8 (18 , 19) . KS lesions are characterized by spindle-shaped cells, an inflammatory infiltrate, and abundant new vascularization. We isolated an immortal cell line from a KS lesion (KS-IMM) that retains most of the properties of KS cells (20) , including induction of endothelial cell migration and invasion in vitro (20 , 21) and strong angiogenic responses in vivo (21, 22, 23) . The intense vascularization of KS makes it an excellent model of angiogenic tumors, we used this system to test the potential of angiostatin gene transfer to inhibit tumor vascularization in KS.

Expression vectors for murine angiostatin including a eukaryotic expression plasmid and an MLV-based retroviral vector were generated. Angiostatin produced by transfected cells inhibited endothelial cell migration, whereas stable gene transfer of the angiostatin expression construct into KS-IMM cells only partially inhibited migration and Matrigel invasion in vitro. Although stable gene transfer of the angiostatin cDNA by retroviral vectors in the KS-IMM cell line did not affect growth in vitro, it resulted in delayed tumor growth by these cells in nude mice. These data confirm that the use of some endogenous inhibitors of angiogenesis, administered by a gene therapy approach, could be useful for treatment of a highly vascularized neoplasm such as KS and subsequently extended to other vascular tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Molecular Cloning of Angiostatin.
The murine angiostatin expression plasmid pCMV-mAST is a pRC/CMV-derived expression plasmid (Stratagene) containing the sequence of murine angiostatin under the CMV promoter/enhancer (24) with a HA tag attached as a fusion protein to the COOH terminus (Fig. 1)Citation . The pCMV-mAST(AS) construct, a negative control, was generated by cloning in the antisense orientation in the HindIII/XbaI sites of pRC/CMV. The pMFG-mAST retroviral vector was generated by cloning a 1.5-kb-long NcoI/BamHI restriction fragment obtained from digestion of pCMV-mAST in the corresponding cloning sites of the MFG retroviral vector (gift of Dr. E. Lechman, University of Pittsburgh, Pittsburgh, PA; Ref. 25 ; Fig. 1Citation ). The LE retroviral vector used as control is a derivative of the LXSN retroviral vector (26) that carries an EGFP (3) gene driven by the Moloney murine leukemia virus long terminal repeat (27) . These constructs were verified by PCR, restriction analysis, and sequencing prior to their use in additional experiments.



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  		Fig. 1. Angiostatin expression vector design and in vitro expression. A, schematic representation of the angiostatin expression constructs. HA, HA tag present in each construct; SD and SA, splice donor and splice acceptor sites, respectively; {Psi}, packaging signal of the vector. Cloning restriction sites are indicated. B, expression of angiostatin by transiently transfected 293T and KS-IMM cells. 35S-Labeled supernatants of cells transfected with pCMV-mAST, pCMV-mAST(AS), and pMFG-mAST were analyzed by RIPA in Lanes 1–3, respectively, of each panel. C, expression of angiostatin by retroviral vector-transduced NIH3T3 and KS-IMM cells by RIPA. In each panel, supernatants from MFG-mAST- and LE-transduced cells are analyzed in Lanes 1 and 2, respectively.

 
Cell Lines and Transfections.
The 293T human kidney cells were obtained from American Type Culture Collection. KS-IMM cells are a human KS-derived cell line (23) , and the Eahy926 cells are an endothelial-like hybrid line (28) that maintains endothelial characteristics (29) . These cell lines were grown in DMEM supplemented with 10% FCS (3) and 1% L-glutamine. Primary HUVECs were isolated by standard procedures and cultured in 199 medium supplemented with 10% FCS, 1% L-glutamine, 100 µg/ml heparin, 10 ng/ml epidermal growth factor, acidic fibroblast growth factor, basic fibroblast growth factor, and 10 µg/ml hydrocortisone. For transient transduction, cells were seeded in 100-mm-diameter Petri dishes at 106 cells/dish and transfected overnight according to a calcium-phosphate protocol using 20 µg of plasmid DNA (30) . Thirty-six h after transfection, culture medium was replaced with fresh DMEM without FCS; 24 h later, the supernatant was harvested and analyzed for angiogenesis inhibitor content. Transfection efficiency was determined by cell lysis in 100 mM potassium phosphate (pH 7.8), 0.2% Triton X-100, and 1 mM DTT, protein concentration measurement with a Bio-Rad Dc Protein Assay kit (Bio-Rad, Hercules, CA), followed by analysis in triplicate using the Galacto-Light Plus kit (Tropix, Bedford, MA), and a plate luminometer. ß-gal activity was expressed as cps/µg protein in the sample.

Transduction of Cells with Retroviral Vectors.
Infectious particles were generated by a transient three-plasmid vector-packaging system, as described previously (31) , and passed through 0.45-µm-pore-size filters. To assess the ability of the virions to transduce KS-IMM cells, serial dilutions of the filtered supernatants were layered over KS-IMM target cells that had been seeded into six-well culture plates the day before infection at 1.0 x 105 cells/well. Protamine sulfate (8 µg/ml; Sigma Chemical Co., St. Louis, MO) was added to the wells, and the cells were kept in a total volume of 2 ml. After 6–9 h at 37°C, 3 ml of medium were added to dilute the protamine sulfate; 36 h later, the cells were split 1:10 in 6-cm-diameter Petri dishes and kept in culture until they underwent a new transduction cycle 24 h later. This procedure was repeated seven times; the percentage of EGFP+ cells after each transduction cycle was determined by fluorescence-activated cell sorter analysis and used as a parameter to estimate the genetically modified cell fraction which, after the 7th transduction, was >90%.

Western Blotting and RIPA.
Angiostatin expression in cell lysates and supernatants was determined by both Western blotting and RIPA, according to standard protocols (32) . For RIPA, transfected cells were metabolically labeled for 6 h with a mixture of [35S]methionine and [35S]cysteine (Amersham-Pharmacia, Little Chalfont, United Kingdom) and lysed in RIPA buffer (140 mM NaCl, 8 mM Na2HPO4, 2 mM NaH2PO4, 1% NP40, 0.5% sodium deoxycholate, and 0.05% SDS), as described (30) . Angiostatin was immunoprecipitated overnight at 4°C from the cell lysates with a mouse mAb directed against the HA tag (BabCo, Richmond, CA); bound proteins were released by boiling in SDS-PAGE sample buffer in the presence of 2-mercaptoethanol and separated by SDS-PAGE through 12.5% polyacrylamide gels. To reduce nonspecific binding to cellular proteins, the antibodies were combined with the protein A-Sepharose matrix and preadsorbed for 2 h at 4°C with an unlabeled cell lysate of nontransfected cells. Angiostatin levels in the conditioned medium of transfected and transduced cells were estimated by Western blotting and compared with a HA-tagged ß-gal protein standard loaded in the same gel in known amounts.

In Vitro Migration and Invasion Assays.
Chemotaxis and invasion assays were performed in Boyden chambers as described previously (33) using polyvinylpyrrolidone-free polycarbonate filters with 8-µm pores for KS-IMM cells and with 12-µm pores for primary HUVECs and EAhy926 cells coated with 5 µg/ml of gelatin. Cells (1.5 x 105) in SFM containing 0.1% BSA were placed in the upper compartment, and the lower compartment was filled with either SFM, as a negative control, or cell supernatants. The cell supernatants used for chemotaxis of endothelial cells were from 293T cells transiently transfected with the angiostatin expression construct or the control vector or from the stable retrovirally transduced KS-IMM cells. For chemotaxis of the stably transfected KS-IMM cells, conditioned medium from either NIH3T3 cells or a primary culture from an iatrogenic KS, KS-cap, were used as chemoattractants with similar results. The chambers were then incubated at 37°C in 5% CO2 for 6 h. Cells remaining on the upper surface of the filter were then mechanically removed, and 5–10 random fields were of those migrated to the lower surface of each filter counted after staining. Assays were performed in triplicate and repeated at least three times. The chemoinvasion assay (33) was performed in the same manner as for chemotaxis, except that the filters were coated with 25 µg/ml of Matrigel (33 , 34) .

Cell Growth on Matrigel.
A 24-microwell plate that had been prechilled at -20°C was carefully filled with 300 µl/well of Matrigel (10 mg/ml) with a cold pipette. The Matrigel was then polymerized for 1 h at 37°C (34) . Stably transfected KS-IMM cells harboring either a murine angiostatin expression construct, an antisense angiostatin construct, or vector alone (70.000 cells/well) were layered on top of the polymerized gel and incubated at 37°C. The wells were photographed every 24 h on a Leitz DM-IRB inverted microscope with CCD optics and a digital analysis system.

In Vitro Proliferation Assays.
The effects of angiostatin on the growth of KS-IMM were tested using the MMT metabolic growth assay or crystal violet staining and computer-assisted counting. KS-IMM cells transfected with an angiostatin expression construct or control cells transfected with a vector plasmid were plated in a 96-multiwell plate at 800 cells/well. The cell number was then assessed at 24-h periods over 4 consecutive days. For MTT assays, at the indicated time 50 µl of 5 mg/ml MMT were added to each well and incubated at 37°C for 4 h. The MMT reduced by living cells into a blue formazan product was solubilized in DMSO, cleared by centrifugation, and read with a multiwell scanning spectrophotometer at 540 nm. In alternative, cells were washed with PBS, stained with crystal violet (0.7% in 8.64% formaldehyde, 32% ethanol, and 200 mM NaCl), and counted with an image analysis system (Image-Pro Plus).

KS-IMM Tumor Growth in Vivo.
The effects of angiostatin on tumor growth were determined by a series of different experiments by s.c. injection of the 3 x 106 KS-IMM cells in Matrigel (10 mg/ml) in the flanks of nude (nu/nu) mice. The tumor size was measured regularly over time, the animals were sacrificed, and tumors were collected when the largest tumors reached preset dimensions, according to current ethical practice. The tumors were fixed in formalin, paraffin embedded, sectioned, and stained. In initial experiments, KS-IMM cells (5 mice), or KS-IMM cells transiently transfected with the angiostatin (5 mice), or control (5 mice) expression plasmids were implanted. A second series of experiments compared growth of KS-IMM cells transduced with the angiostatin encoding retroviral vector to KS-IMM cells transduced with the control vector. This experiment was repeated twice (for a total of 12 mice per group), and identical results were obtained.

RT-PCR.
Total RNA was isolated from tumors using the TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer’s instructions. Reverse transcription was performed with oligo-dT primers and Superscript II (Life Technologies, Inc.) as described previously (35) . Amplification was performed using the following primers, 5'-TGGCGGAGAACAGCAAGACTTC-3' (forward) and 5'-TGTCACAGTATTCCCAGCGTTTG-3' (backward), for 40 cycles at 95°C for 30 s, 68°C for 30 s, and 72°C for 1 min. The 549-bp amplified angiostatin product was resolved in gel electrophoresis and stained with ethidium bromide. Controls were samples amplified without RNA and samples amplified without reverse transcriptase.

Histology and Immunohistochemistry.
Four-µm sections were rehydrated and stained with either H&E or processed for immunohistochemistry by standard procedures. Rehydrated sections were blocked with irrelevant serum, followed by incubation with primary antibody, alkaline phosphatase-linked secondary antibody, and visualized with the DAKO Envision system AP (DAKO, Carpinteria, CA). Sections were then lightly counterstained with hematoxylin and mounted in Glycergel. Primary antibodies used were a monoclonal anti-factor VIII (DAKO) or a PHA-L lectin (Sigma Chemical Co.) to highlight vessels or a polyclonal anti-HA tag (BabCo) to localize the HA-angiostatin fusion protein. Parallel negative controls without primary antibodies were performed in all cases. Images were acquired as above.

Vessel densities were estimated by counting the vessels in anti-factor VIII antibody-stained sections. Random fields were selected for sections of several different tumors derived from either the mAST or control-transfected KS-IMM cells, and all of the clearly visible vessels in the unit field were counted. The counts were done blindly as to the origin of the tissue section.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of Angiostatin Expression Vectors.
The expression vectors used were the eukaryotic expression plasmid pCMV-mAST (24) and a MLV-based retroviral vector derived from the retroviral vector MFG (25) , termed pMFG-mAST (Fig. 1A)Citation . These vectors conferred expression of angiostatin (Kringles 1–4 of plasminogen) with a HA tag attached to the COOH terminus as a fusion protein. RIPA of cell lysates and supernatants indicated that both pCMV-mAST and the MFG-based retroviral vector conferred angiostatin expression at relatively high levels in vitro (Fig. 1B)Citation . The apparent molecular weight was Mr ~58,000, higher than that reported for murine angiostatin (1) , because of the presence of the HA tag, as described previously. The presence of the HA tag has been shown previously to have no effect on the angiostatic properties of the recombinant angiostatin protein (24) . No anti-HA-binding proteins were detected with RIPA from cells transfected with the antisense orientation negative control pCMV-mAST(AS) plasmid. Western blot analysis with the anti-HA mAb confirmed these findings (data not shown). Densitometric analysis of the RIPA indicated that transfection of 293T cells with the pCMV-mAST plasmid generated 5–10 times more angiostatin than pMFG-mAST retroviral vector transfection. Because ß-gal analysis indicated that the transfection efficiency was similar (4.4 x 104 and 3.7 x 104 CPS/µg cell protein for the pCMV-mAST- and pMFG-mAST-transfected cells, respectively), the difference in angiostatin production in 293T cells appears to depend on promoter activity. Retroviral vector-containing supernatants were used to transduce murine NIH3T3 fibroblasts and KS-IMM cells, conferring high levels of angiostatin production in these cells (Fig. 1C)Citation . Murine angiostatin levels in the conditioned medium of the transfected 293T, KS-IMM, or retrovirus-transduced KS-IMM cells were estimated to be 20, 3.5, and 50 µg/ml, respectively, by Western blot with a known standard. These different vectors were used subsequently to address the effects of angiostatin on KS cells in this study.

In Vitro Effects of Angiostatin.
Chemotaxis assays using endothelial cells and the Eahy926 endothelial cell-like line as target cells demonstrated that endothelial cells showed strongly impaired migration when exposed to supernatants of angiostatin-expressing cells as compared with controls (Fig. 2)Citation . This was observed using supernatants from both the transiently transfected 293T cells (Fig. 2, A and B)Citation and the stable retroviral transduced KS-IMM cells (Fig. 2C)Citation . The angiostatin-transfected KS-IMM cells themselves showed partially reduced migration as compared with control KS-IMM transfectants (Fig. 3A)Citation . Similarly, the angiostatin transfectants showed a partial, although significant, reduction of invasion (Fig. 3B)Citation in the chemo-invasion assay (33) . The extent of inhibition of invasion of these cells (Fig. 3B)Citation was similar to that of chemotaxis (Fig. 3A)Citation , implying that angiostatin affected migration. Invasive growth of KS-IMM cells on Matrigel in vitro was also affected by angiostatin transfection, with the formation of smaller clumps by the angiostatin-expressing cells (Fig. 3C)Citation . No differences in cell proliferation rates in vitro were observed between the angiostatin or control-transfected KS-IMM cells for transient or stable, as well as plasmid or retrovirus, transfected cells (data not shown).



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  		Fig. 2. In vitro effects of angiostatin on the chemotaxis and invasion of endothelial and KS cells. A, chemotaxis of Eahy926 endothelial cells to conditioned medium from transiently transfected 293T cells (293T-CM). Vector, supernatants from pCMV-mAST(AS)-transfected cells; mAST, supernatants from pCMV-mAST-transfected cells. SFM with 0.1% BSA and KS-IMM conditioned medium (KS-CM) was used as negative and positive controls. Bars, SD. Eahy293 cell chemotaxis to angiostatin-transfected cell supernatants was significantly less than that to vector-transfected controls (P < 0.01 using Student’s t test). The assay was performed in triplicate and repeated twice. B, chemotaxis of HUVECs to conditioned medium from transiently transfected 293T cells (293T-CM). Vector and transfected cells are indicated as above. SFM with 0.1% BSA and NIH-3T3 conditioned medium (3T3-CM) was used as negative and positive controls. Bars, SD. Endothelial cell chemotaxis to angiostatin-transfected cell supernatants was significantly less than that to vector-transfected controls (P < 0.0001 using Student’s t test). The assay was performed in triplicate and repeated three times. C, chemotaxis of HUVECs to conditioned medium from stably retrovirally transduced KS-IMM cells (KS-IMM-CM), as indicated above. Vector, supernatants from LE-transduced cells; mAST, supernatants from pMFG-mAST-transduced cells. SFM with 0.1% BSA was used as the negative control. Bars, SD. Endothelial cell chemotaxis to angiostatin-transduced cell supernatants was significantly less than that to vector-transduced controls (P < 0.0001 using Student’s t test). The assay was performed in triplicate and repeated three times.

 


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  		Fig. 3. In vitro effects of angiostatin on invasive growth of KS cells. A, chemotaxis of KS-IMM cells stably transfected with an angiostatin expression vector or vector alone-transfected KS-IMM cell controls. The chemotaxis of angiostatin-transfected KS-IMM cells was significantly less than that of vector-transfected KS-IMM cells (P < 0.02 using Student’s t test). Conditioned media from either NIH3T3 cells (shown) or a primary KS culture were used as chemoattractants with similar results. The assay was carried out in triplicate and repeated twice. Bars, SD. B, invasion of KS-IMM cells stably transfected with the pCMV-mAST angiostatin expression vector or vector alone-transfected KS-IMM cell controls. Conditioned media from either NIH3T3 cells or a primary KS culture (shown) were used as chemoattractants with similar results. The invasion of angiostatin-transfected KS-IMM was significantly less than that of vector-transfected KS-IMM cells (P < 0.038 using Student’s t test). The assay was carried out in triplicate and repeated twice. Bars, SD. C, the effect of angiostatin expression on KS-IMM cell growth in Matrigel in vitro after 48 h. Stably transfected KS-IMM cells were seeded on a Matrigel gel in 24-well polystyrene plates. Mouse antisense angiostatin- or vector alone-transfected KS-IMM cells were used as controls, as indicated.

 
In Vivo Growth of Angiostatin-producing KS-IMM Cells.
Two different in vivo experiments were used to evaluate the effects of angiostatin on KS tumor growth. Transiently transfected KS-IMM tumor cells were used to determine the effects of high-level, short-term angiostatin expression on tumor growth. Angiostatin production was confirmed by immunoblotting analysis of supernatants, whereas efficiency of transfection was measured by quantitative ß-gal assessment of a cotransfected ß-gal expression plasmid at the time of injection. High angiostatin levels were produced, whereas intersample variations were limited to 10–20%. No significant differences in growth in vivo of the different transfected cells were observed, suggesting that transient expression of the angiostatin gene in the early phases of tumor growth was not sufficient to reduce tumor growth (data not shown).

Genetically modified KS-IMM cells were obtained by repeated transduction of the KS-IMM cell line with the MFG-mAST retroviral angiostatin expression vector or a control EGFP-coding retroviral vector, termed LE. Western blotting analysis showed high angiostatin production by MFG-mAST KS-IMM cells, similar to that observed after transient transfection, with no apparent reduction after 45 days in vitro (data not shown). Although no differences in proliferation rates were observed in vitro, the angiostatin-producing MFG-mAST KS-IMM cells showed much slower tumor growth than the controls (Fig. 4)Citation , demonstrating a marked therapeutic effect of murine angiostatin on human KS cell growth in vivo. Histological (Fig. 5, a and b)Citation immunohistochemical (Fig. 5, c and d)Citation analyses clearly indicated a reduced vascularization in the angiostatin-producing samples compared with controls. Vessel counts showed a significantly (P < 0.0001, t test) reduced microvessel density in the tumors formed by the mAST-transduced cells (5.7 ± 1.7) as compared with those of the control cells (16.9 ± 5.3). Expression of angiostatin in the tumors was confirmed by both RT-PCR of tumor samples (data not shown) and by immunohistochemistry for the HA tag attached to the angiostatin fusion protein (Fig. 5f)Citation , which demonstrated strong, diffuse positivity in the small mAST-transduced KS-IMM tumors but not in the controls. These data confirm that the gene was transcriptionally active at the end of the experiment, ruling out silencing of the viral promoter in vivo in this system. Our data indicate that retroviral transduction of KS-IMM cells resulted in long-term expression of angiostatin that led to reduced vascularization and tumor growth in vivo as compared with vector-alone transduced or wild-type KS-IMM cells.



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  		Fig. 4. Inhibition of tumor growth by transduction of KS-IMM cells with an angiostatin-expressing retroviral vector. KS-IMM cells were transduced with either the pMFG-mAST(S) angiostatin expression vector (•) or the corresponding control vector LE ({blacksquare}). The transduced cells were mixed with Matrigel and injected s.c. into the flanks of athymic nude mice. Nodule dimensions were used to compute tumor volume. The differences between the groups were statistically significant after day 16, with P < 0.021 on day 16, P < 0.011 on day 18, P < 0.023 on day 20, P < 0.009 on day 23, and P < 0.005 on day 25 using Student’s test, and the curves were statistically different with two-way ANOVA. The experiment was repeated with identical results; bars, SD.

 


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  		Fig. 5. Histology of tumors formed by angiostatin-transduced pMFG-mAST cells (b, d, and f) as compared with those formed by the LE vector-alone cells (a, c, and e). Bar, 50 µm. a and b, histological staining of tumors for the control (LE, a) and angiostatin-transduced (pMFG-mAST) cells by H&E. c and d, immunohistochemical staining of tumors for the control (LE, c) and angiostatin-transduced (pMFG-mAST, d) cells using anti-factor VIII monoclonal antibodies to highlight microvessels. The control tumors show much more extensive vascularization than mAST-transduced KS-IMM tumors. e and f, immunohistochemical detection of the HA tag attached to the transduced angiostatin as a fusion protein tag. Tumors formed by LE-transduced KS-IMM cells showed no staining (e), whereas extensive staining is observed in the small tumors formed by pMFG-mAST-transduced KS-IMM cells (f).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of this study was to determine the sensitivity of KS cells to the angiogenesis inhibitor angiostatin delivered via gene transfer. Following the original description of angiostatin and its activity as an inhibitor of neoangiogenesis (1 , 11) , several studies have reported its activity against different tumor types in murine models (24 , 36, 37, 38, 39, 40, 41, 42, 43) . Studies have also shown angiostatin activity against highly vascular tumors (44) ; however, no studies on KS have been conducted to date. Because angiostatin administration has been reported to be devoid of side effects, at least in mice (1 , 11 , 44) , it would be reasonable to also use it to treat severely immunodeficient patients, who make up the majority of KS patients. This is unlike other therapeutic options, which, although effective in vitro on KS cells, are hampered by severe toxicity in vivo, especially in AIDS patients (45) . Several reports have indicated that many AIDS-KS patients show regression of KS under HAART (46 , 47) . However, at present HAART is not an option for aggressive iatrogenic or endemic KS, and there is high probability for KS recurrence in AIDS patients failing HAART (48 , 49) . Our findings show a clear-cut effect of angiostatin on KS-IMM tumor growth in vivo. No direct antiproliferative effects of angiostatin on KS cells were observed, in agreement with previous findings with murine hemangioendothelioma (44) , and limited effects on migration and invasion of these cells in vitro were observed. The major effects in vitro were on endothelial cells, indicating that angiogenesis was the preferential target. Our data show that the mechanism for tumor growth inhibition in vivo is mainly attributable to suppression of angiogenesis, rather than a primary effect on the KS-IMM cells.

Our study also demonstrates that angiostatin gene transfer effectively delays growth of KS cells in mice. A gene therapy approach to deliver antiangiogenic genes to cancer patients would be an effective alternative to protein infusion, especially in view of the problems associated with rapid clearance of angiostatin from the circulation (1 , 11) and industrial-scale recombinant angiostatin production.

Short-term expression of angiostatin in vivo using transient transfection of KS-IMM cells was not sufficient to inhibit tumor growth, in agreement with the necessity for chronic administration of angiostatic drugs. Long-term, high-level, angiostatin-expressing KS-IMM cells grew significantly slower in vivo than the controls, forming small tumors in nude mice, similar to those described previously (24) . The negative findings with transient transfectants suggest that both the level and duration of expression are critical parameters in determining the outcome of angiostatin therapy.

KS cells and the KS-IMM cell line have a mixed endothelial-macrophage-mesenchymal phenotype (20 , 50) , and it has been suggested that these cells may be primitive vascular precursor cells (51) . The KS lineage reinforces the rationale for using angiostatin in this tumor, because it might act on both the normal endothelium that supports tumor growth and in part directly on the tumor cells. Although angiostatin did not affect proliferation of KS-IMM cells in vitro, it reduced KS-IMM chemotaxis and invasion through the basement membrane extract Matrigel, suggesting that it may directly interact with these cells. RT-PCR and histological expression of the angiostatin fusion mRNA and protein were detected clearly in the small tumors formed by the angiostatin-transduced cells, demonstrating long-term expression. Counts of the vessels in the tumors showed that the vascular structures formed in the angiostatin-transduced tumors were significantly less than the extensive vascularization in tumors formed by control cells. Angiostatin gene therapy significantly reduced KS tumor mass and appears to be suitable for therapy of KS, possibly in combination with classic chemotherapy and/or HAART in the case of AIDS KS.


   ACKNOWLEDGMENTS
 
We thank T. Cai (Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy) for technical assistance; P. Gallo (University of Padova, Padova, Italy) for artwork; Dr. E. Piovan (University of Padova, Padova, Italy) and M. Barabino (Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy) for precious help in the preparation of the manuscript; and Drs. R. Benelli and F. Del Grosso (Advanced Biotechnology Center, Genova, Italy) for helpful discussions.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from Ministero del’Università e Ricerca Scientifica e Tecnologica 40%, Associazione Italiana Ricerca sul Cancro, Compagnia di San Paolo, Ministero della Sanità Programma Nazionale Ricerca sull’ AIDS, Fondazione Italiana Ricerca Cancro, and the Fondazione Cassa di Risparmio di Padova e Rovigo. Back

2 To whom requests for reprints should be addressed, at Modulo di Progressione Neoplastica, Istituto Nazionale per la Ricerca sul Cancro (IST), c/o Centro di Biotecnologia Avanzate, Largo Rosanna Benzi, n. 10, 16132 Genova, Italy. Phone: 39-010-5737367; Fax: 39-010-573736; E-mail: noonan{at}cba.unige.it Back

3 The abbreviations used are: KS, Kaposi’s sarcoma; MLV, Moloney leukemia virus; HUVEC, human umbilical vein endothelial cell; CMV, cytomegalovirus HA, hemagglutanin antigen; EGFP, enhanced green fluorescent protein; ß-gal, ß-galactosidase; RIPA, radioimmunoprecipitation analysis; SFM, serum-free medium; MMT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide; RT-PCR, reverse transcription-PCR; HAART, highly active antiretroviral therapy. Back

Received 2/ 1/01. Accepted 5/16/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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