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Experimental Therapeutics |
Cutaneous Biology Research Center and Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Charlestown 02129 [M. S., T. H., K. M., B. L-A., M. D.]; Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston 02114 [A. E. S., J. P. V.]; and Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston 02215 [L. F. B.] Massachusetts
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TSP-24 (reviewed in Refs. 12 , 13 ) is a secreted, Mr 420,000 endogenous angiogenesis inhibitor (14 , 15) . Deficiency of TSP-2 in mice (16) results in more vascularized granulation tissue in full-thickness skin wounds (17) and in enhanced vascularity of cutaneous foreign body reactions (18) . We have identified recently stromal up-regulation of TSP-2 as a novel host antitumor defense mechanism (19) , and TSP-2-deficient mice are characterized by accelerated and enhanced skin carcinogenesis and angiogenesis (19) . Conversely, stable overexpression of TSP-2 in human squamous cell carcinomas potently inhibited orthotopic tumor growth and tumor vascularization (20) .
To achieve efficient inhibition of malignant tumor growth via continuously elevated systemic TSP-2 levels, we developed a systemic, cell-based gene therapy approach for the in vivo production of full-length TSP-2 within the living tumor host. Here, we report that tissue-engineered implants, which contained large numbers of TSP-2-transduced fibroblasts grown on biodegradable polymer scaffolds (21) , efficiently secreted TSP-2 into the blood circulation after i.p. implantation. Bioimplants maintained TSP-2 secretion over prolonged time periods, resulting in an inhibition of tumor growth and angiogenesis of three different, highly aggressive malignant tumors implanted at a distant site. These results establish systemic, antiangiogenic cell therapy as a promising new therapeutic approach for the treatment of cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Transfection.
The retroviral transfection plasmid containing the full-length murine TSP-2 cDNA was prepared by ligation of a 3.6-kbp EcoRI fragment (20)
into the EcoRI site of a pLXSN retroviral transfection vector (Clontech), which contains a neomycin selection cassette. PT67 packaging cells were transfected with the TSP-2/pLXSN retroviral plasmid or with unaltered pLXSN, using the SuperFect transfection reagent (Qiagen, Chatsworth, CA) and 10 µg of each plasmid according to the manufacturer’s recommendations. The cells were selected in medium containing 800 µg/ml G418 (Sigma Chemical Co., St. Louis, MO) for 3–4 weeks, and >60 resistant clones were isolated. Retroviral titers were determined by incubation of NIH 3T3 cells with 10-fold serial dilutions of culture supernatants obtained from TSP-2/pLXSN and pLXSN control transfected clones. PT67 cell clones with a viral titer > 106 cfu/ml were used for additional experiments. For retroviral transfection, NIH 3T3 cells were incubated with virus-containing supernatants in the presence of 8 µg/ml Polybrene (Sigma Chemical Co.) for 6 h on 4 consecutive days and were then subjected to G418 selection.
Preparation of Polymer-Cell Grafts.
Biodegradable polymer grafts were prepared from 1-mm thick sheets of nonwoven fibers of polyglycolic acid (density 70 mg/cm3, fiber diameter 14 µm, average pore size 250 µm; Smith and Nephew, York County, United Kingdom) as described (22)
. Sheets were sectioned into 0.5 cm2 squares, which were placed into 12-well tissue culture plates (Costar, Cambridge, MA), sterilized with 95% ethanol, and washed with PBS. Sterile 1 N sodium hydroxide was added to each well for 60 s to render the polymer hydrophilic. Thereafter, the polymer was washed with distilled water and was coated with 30 µg/ml collagen type I (Vitrogen; Collagen Biomaterials, Palo Alto, CA) for 1 h. Transfected NIH 3T3 fibroblasts were trypsinized, resuspended in culture medium, and 1.5 x 107 cells were seeded onto each polymer square. After 2 h at 37°C and 5% CO2, fresh culture medium was added. Polymer-cell grafts were cultivated in vitro for 14 days before grafting.
Northern Blot Analysis.
Total cellular RNA was isolated from confluent NIH 3T3 cell cultures using the RNeasy kit (Qiagen). RNA (10 µg) were fractionated by electrophoresis on 1% agarose formaldehyde gels and were transferred to Biotrans nylon supported membranes (ICN Pharmaceuticals, Costa Mesa, CA) as described (20)
. 32P-radiolabeled cDNA probes were prepared with a random primed synthesis kit (Multiprime; Amersham, Arlington Heights, IL) using a 4.19-kbp mouse TSP-2 cDNA and a 950-bp mouse VEGF cDNA as templates. A 2-kbp human ß-actin cDNA probe (Clontech) was used as a control for equal RNA loading. Blots were washed at high stringency and were exposed to X-OMAT MR film (Kodak, Rochester, NY) for various times.
Implantation of Polymer-Cell Grafts and Tumorigenesis Assay.
Fourteen days after seeding with TSP-2 secreting or control fibroblasts, 5 x 5 x 1 mm polymer squares were implanted into the right ovarian pedicle of 8-week-old female BALB/c-nu/nu nude mice as described previously (23)
. After induction of anesthesia with a mixture of ketamine (800 µg/10 g body weight; Ketaset; Fort Dodge Laboratories, Fort Dodge, IA) and avertin (0.5 µg/10 g body weight 2,2,2-tribromethanol in 2.5% t-amyl alcohol; Sigma Chemical Co.), a 1-cm horizontal incision was made in the right flank, and the ovarian pedicle was identified and delivered out of the wound. The polymer-cell graft was laid on the ovarian pedicle and sutured on place with 6–0 prolene suture (Roboz Surgical, Rockville, MD). The abdomen was closed with 9-mm autoclips (Becton Dickinson, Sparks, MD). One week after implantation of cell-seeded bioimplants, A431 squamous cell carcinoma cells (2 x 106) or B16F10 malignant melanoma cells (2 x 106) were injected intradermally and LCCs (0.5 x 106) were injected s.c. into both flanks of nude mice (two sites per mouse; five mice per cell line and per type of bioimplant). The smallest and largest tumor diameter were measured weekly, using a digital caliper, and tumor volumes were calculated using the following formula: volume = 4/3 x 
x (1/2 x smaller diameter)2 x 1/2 x larger diameter. Tumor data were analyzed by the two-sided unpaired t test. Mice were sacrificed after 3–4 weeks. In additional experiments, A431 squamous cell carcinoma cells or B16F10 melanoma cells were implanted as above, followed by implantation of control or TSP-2-producing polymer-cell grafts after 5 days when tumors were well established. All of the animal studies were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care.
Western Blot Analysis.
Conditioned media were obtained from confluent fibroblast cultures grown for 48 h in serum-free DMEM. Culture supernatants were also collected from fibroblast-populated polymer scaffolds every 48 h. Serum samples were obtained from mice bearing bioimplants with either TSP-2 overexpressing or control fibroblasts 4 and 5 weeks after implantation. TSP-2 was concentrated as described (20)
, and samples were boiled in denaturing sample buffer. Fifteen µl of each sample were electrophoresed on polyacrylamide gels and were blotted onto polyvinylidene difluoride membranes (Bio-Rad). To verify equal protein loading, membranes were stained with 0.1% Ponceau red (Sigma Chemical Co.) diluted in 5% acetic acid. Membranes were incubated overnight in PBS containing 0.1% Tween 20 and 3% BSA to block nonspecific binding. Membranes were then incubated with anti-TSP-2 antibody (clone N-20; Santa-Cruz Biotechnology, Santa Cruz, CA), washed in PBS/Tween, incubated with horseradish peroxidase-conjugated antigoat IgG (Santa Cruz Biotechnology), and were analyzed by the enhanced chemiluminescence system (Amersham).
Histology, in Situ Hybridization, and Immunohistochemistry.
For routine histology, formalin-fixed tissue was processed and embedded in paraffin for H&E staining and for Gomori’s trichrome staining using anilin blue (24)
. In situ hybridization was performed on 6-µm cryosections of fibroblast-seeded bioimplants 5 weeks after implantation as described (20)
. The sense and antisense single-stranded RNA-probes for mouse TSP-2 were transcribed from a pBluescript II KS+ vector containing a 290-bp fragment of the coding region of mouse TSP-2. Immunofluorescence stainings were performed on NIH 3T3 cells seeded on Falcon culture slides (Becton Dickinson Labware, Franklin Lakes, NJ) and fixed in 4% paraformaldehyde for 10 min. Cells were permealized in PBS/Tween (0.2%) for 5 min and were stained using a polyclonal anti-TSP-2 antibody (Santa Cruz Biotechnology) at 4°C overnight, followed by incubation with an Alexa Fluor 488 conjugated secondary antibody (Molecular Probes, Inc., Eugene, OR) for 30 min. Nuclei were stained with propidium iodide in PBS (2 µg/ml). Goat IgG in the primary step served as negative control. Immunohistochemical and immunofluorescence stainings of sections (6 µm) derived from tumors and polymer-cell grafts were performed as described previously (20)
, using a polyclonal anti-TSP-2 antibody (Santa Cruz Biotechnology), a polyclonal rabbit antimouse TSP-2 antibody (kindly provided by Dr. Paul Bornstein, University of Washington, Seattle, WA), or a rat monoclonal antibody against mouse CD31 (PharMingen). Tumor apoptosis rates were evaluated in six A431 tumors each in mice bearing control implants or TSP-2 expressing implants. Apoptotic cells were identified by TUNEL staining of cryostat sections (6 µm) using the Fluorescein-FragEL DNA fragmentation kit (Oncogene, Cambridge, MA) as described (19)
. Nuclei were stained with propidium iodide in PBS (2 µg/ml). Three x20 fields were evaluated per tumor. Results are expressed as the mean ± SE of TUNEL-positive nuclei per 100 tumor cell nuclei. The two-sided unpaired Student t test was used to analyze differences in apoptosis rates.
Computer-assisted Morphometric Analysis of Blood Vessels.
Representative sections were obtained from five TSP-2- or control-treated tumors for each tumor cell line. Sections were stained with an antimouse CD31 monoclonal antibody and were analyzed using a Nikon E-600 microscope (Nikon, Melville, NY). Images were captured with a Spot digital camera (Diagnostic Instruments, Sterling Heights, MI), and morphometric analyses were performed as described (20)
, using the IP LAB software (Scanalytics, Billerica, MA). The two-sided unpaired t test was used to analyze differences in microvessel density and vascular size and area.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Overexpression of TSP-2 did not affect the mRNA expression of the angiogenesis factor VEGF (data not shown). Western blot analysis of conditioned medium confirmed efficient TSP-2 protein secretion by TSP-2 transfected fibroblasts, whereas in conditioned medium derived from control fibroblasts TSP-2 protein was not detectable (Fig. 1B)
. Consistent with these findings, immunofluorescence analysis of NIH 3T3 cells cultured on glass slides showed strong TSP-2 protein expression in nearly all of the TSP-2-transfected NIH 3T3 cells, whereas TSP-2 protein expression was detected only in a small subset of vector-control transfected NIH 3T3 cells (Fig. 1, C–F)
. The TSP-2-transfected fibroblast clone 50 and the control clone 6 were seeded onto biodegradable polymer scaffolds and were cultured for 14 days before grafting. Culture supernatants were analyzed for TSP-2 expression every 48 h after seeding, and efficient TSP-2 secretion by TSP-2-transfected fibroblasts was detected at 10–14 days after seeding onto the scaffolds (Fig. 1G)
. In contrast, little or no TSP-2 was secreted by control transfected fibroblasts (Fig. 1G)
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. At 2 weeks after implantation, remnants of the biodegradable polymer mesh were still detectable (Fig. 2C)
, whereas no polymer fibers were detected after 5 weeks (Fig. 2, E and F)
, indicating complete dissolution of the temporary cell delivery scaffolds. After degradation of the polymer mesh, the grafts consisted of a firm circumscribed mass of connective tissue reminiscent of a fibroma-like structure (Fig. 2, E and F)
. Importantly, fibroblast-populated grafts remained encapsulated and separated from the adjacent ovary (Fig. 2, D–F)
, and no outgrowth of fibroblasts from the polymer meshes was observed. Serial sections of the peritoneum and of intra-abdominal organs, including liver, spleen, colon, and uterus revealed no traces of the polymer mesh (Fig. 3, A–E)
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, whereas only weak TSP-2 mRNA expression was detected in control polymer-cell grafts (Fig. 4, A and C)
. In accordance with these findings, strong cellular TSP-2 immunoreactivity was found in TSP-2-overexpressing implants (Fig. 4F)
throughout the whole graft as compared with little TSP-2 protein expression in control grafts (Fig. 4E)
. Four and 5 weeks after implantation, Western blot analysis revealed comparable high levels of circulating TSP-2 in the serum of mice bearing TSP-2-overexpressing bioimplants, whereas TSP-2 was barely detectable in the serum of mice transplanted with control polymer-cell grafts (Fig. 5A)
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. In contrast, tumor growth was significantly inhibited (P < 0.001) by >60% in mice bearing TSP-2-secreting bioimplants. A >70%-inhibition of malignant tumor growth by cell-based TSP-2 gene-therapy was observed after implantation of LLC cells (Fig. 6C)
. Differential immunofluorescence staining for TSP-2 and CD31 revealed only sparse TSP-2 expression in the mesenchymal stroma of A431 squamous cell carcinomas in mice bearing control bioimplants (Fig. 5B)
, whereas strong TSP-2 protein expression was detected both in and surrounding tumor blood vessels (Fig. 5C)
of A431 carcinomas in mice bearing TSP-2-secreting bioimplants. We next investigated whether implantation of TSP-2-producing bioimplants might also inhibit the growth of already established malignant tumors. B16F10 melanomas were injected intradermally followed by implantation of control or TSP-2-producing bioimplants after 5 days, when the tumors were well established. Tumor growth was significantly inhibited in mice bearing TSP-2-producing polymer-cell grafts, as compared with control mice (Fig. 7)
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, in B16F10 malignant melanomas (Fig. 8, C and D)
, and in LLCs (Fig. 8, E and F)
. Computer-assisted image analysis of representative digital images revealed that the average vessel size (Fig. 8H)
and the relative tumor area occupied by blood vessels (total vascular area; Fig. 8I
) were significantly reduced (P < 0.01) by circulating TSP-2 in all three of the tumor types, whereas no major changes were found in the average vessel density (Fig. 8G)
. The reduced tumor vascularization observed in mice bearing TSP-2-secreting cell grafts was associated with enhanced rates of tumor apoptosis, as studied in A431 squamous cell carcinomas (Fig. 9, A and B)
. The percentage of apoptotic tumor cells was 2.9 ± 1.2% in control mice, as compared with 5.9 ± 1.4% in mice bearing TSP-2-expressing bioimplants (P < 0.01).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Viral and nonviral experimental gene therapy systems have been developed to achieve prolonged circulating serum levels of endogenous antiangiogenic factors (8) . However, repeated cycles of therapy are required to maintain elevated antiangiogenic protein levels. Moreover, adenoviral therapy may elicit considerable host immune responses after repeated injections, thereby significantly reducing its long-term therapeutic efficiency (28) . On the basis of experiences with experimental corrective gene therapy for monogenic diseases (29) and with tissue engineering techniques (30) , we have established a novel systemic, antiangiogenic, cell-based gene therapeutical approach. We used biodegradable polymer implants that serve as temporary scaffolds capable of supporting the survival of large numbers of genetically modified fibroblasts (31) , thereby establishing a cell factory for the continuous in vivo secretion of the antiangiogenic agent TSP-2.
Our results demonstrate that fibroblasts can be retrovirally transduced to overexpress TSP-2 and that they survive on synthetic biodegradable polymer fibers after i.p. implantation. Synthetic materials such as lactic or polyglycolic acid have been used previously as substrates for mammalian cells in tissue engineering (21 , 30 , 32) , and it has been shown that the biodegradable polyglycolic acid scaffold used in our study provides a sufficient surface area for the attachment of many genetically modified cells without causing a permanent immune reaction because of the biodegradable nature of the polymer fibers (31) . In agreement with these findings, no inflammatory reactions to the implanted polymers were observed. Although this might relate to the immunodeficiency of the host nude mice, previous studies in nonimmunosuppressed animals did not reveal any inflammatory reaction to the identical biodegradable polymer used in our studies (33) .
The transplanted bioimplants formed well-vascularized, encapsulated nodules that maintained TSP-2 overexpression in vivo as determined by in situ hybridization and immunofluorescence analysis and, furthermore, enabled TSP-2 protein to enter the blood stream as shown by Western blot analysis. Moreover, we confirmed increased systemic serum concentrations of TSP-2 that reached biologically active levels as revealed by the significant growth inhibition of three different, highly aggressive tumor cell lines implanted at a distant site. This is consistent with recent findings in an experimental fibrosarcoma model in which xenotransplanted HT1080 cells readily formed tumors, which strongly secreted the angiogenesis inhibitor TSP-1, thereby preventing metastatic foci from inducing neovascularization (34) . We first established, as a proof-of-principle, that implantation of tumor cells into mice bearing TSP-2-secreting cell grafts resulted in potent inhibition of tumor growth. Moreover, tumor growth was also significantly inhibited when cell grafts were implanted into mice bearing established tumors, indicating the potential use of this technology for the systemic treatment of human cancers. TSP-2 expression and serum levels were sustained for at least 5 weeks and were sufficient to significantly inhibit tumor angiogenesis in all three of the tumor models, as measured by a significant reduction of tumor vascularization. Together with the observed accumulation of TSP-2 protein within and adjacent to angiogenic tumor vessels, these findings clearly indicate that bioimplant-derived TSP-2 inhibited tumor growth via inhibition of angiogenesis, associated with enhanced tumor cell apoptosis. These findings are in accordance with our previous findings of enhanced vascularity but reduced tumor cell apoptosis in TSP-2-deficient epithelial tumors (19) . In vitro assays showed no direct effects of TSP-2 on tumor cell survival (20) , suggesting that the TSP-2-mediated induction of tumor cell apoptosis was attributable to inhibition of tumor angiogenesis. This is in support of the concept that tumor angiogenesis may act as paracrine regulator of tumor apoptosis (35) . A recent report demonstrated in vivo mechanisms by which tumors producing the related angiogenesis inhibitor TSP-1 may bypass its inhibitory effects, which, in turn, may raise questions about the long-term effectiveness of antiangiogenic therapy with thrombospondins (36) . In this study, tumor resistance to TSP-1 developed as a result of increased secretion of VEGF by some tumor cell clones and because of resistance to the inhibitory effects of TGF-ß. In contrast, we did not observe previously any major modulations of in vivo VEGF expression in TSP-2-transfected A431 squamous cell carcinoma cells (20) . Moreover, whereas TSP-1 has been shown to activate latent TGF-ß via the unique sequence KRFK found between the first and the second type 1 repeat (amino acids 412–415; Ref. 37 ), the KRFK sequence is absent from the TSP-2 molecule. Accordingly, recombinant TSP-2 does not activate TGF-ß, and active TGF-ß levels are comparable in tissues of TSP-2-deficient and wild-type mice (38) , indicating that TSP-2 inhibits angiogenesis and tumor formation independently from effects on TGF-ß activation.
The detection of TSP-2 in the serum by Western blot analysis was performed over the first 5 weeks after implantation of mesh grafts, with detection of considerable amounts of circulating TSP-2 after 5 weeks. Using normal chondrocytes and osteoblasts seeded onto the identical polymer scaffolds and implanted on the ovarian pedicle of immunosuppressed mice, we found that bioimplants survived for the lifetime of the animals.5 Because retroviral transduction results in stable integration of the expression construct into the fibroblast genome it is conceivable that bioimplants will maintain efficient TSP-2 secretion over prolonged time periods. Future studies using specific TSP-2 ELISA assays currently being developed in our laboratory will allow a more precise evaluation of circulating TSP-2 levels over extended periods of cell graft survival.
To the best of our knowledge, the experiments reported here represent the first experimental proof-of-principle for the feasibility and therapeutic effectiveness of systemic, antiangiogenic gene therapy with cell-seeded biodegradable polymers for the treatment of cancer in vivo. Systemic antiangiogenic therapy, using TSP-2-transduced cell grafts on biodegradable polymer scaffolds, represents a major improvement over the local implantation of cell-seeded alginate beads adjacent to malignant brain tumors reported recently (9 , 10) , because implantation of cell grafts distal to the tumor site potentially allows systemic treatment of the majority of malignant tumors and their metastases. Moreover, it affords better access to the cell factories, including the prospect to remove or replace them (11) . However, potential side effects of systemic cell-based antiangiogenic therapy on other angiogenesis-dependent processes, such as the reproductive cycle or wound repair, remain to be established in future studies. Systemic cell-based antiangiogenic gene therapy may represent a promising new strategy for the treatment of human cancers and other angiogenesis-dependent chronic diseases, using autologous, patient-derived skin fibroblasts transduced to secrete TSP-2 or other angiogenesis inhibitors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by American Cancer Society Program Project Grant 99-23901 (to M. D.), by NIH/National Cancer Institute Grants CA 69184 and CA 86410 (to M. D.), by Department of Defense Grant 1200-202487 (to J. P. V.), by the Dermatology Foundation (to M. S.), by the Deutsche Forschungsgemeinschaft (to T. H., B. L-A.), and by the Cutaneous Biology Research Center through the Massachusetts General Hospital/Shiseido Co. Ltd. Agreement (to M. D.). A. E. S. is supported by a training grant in cancer biology from the NIH (CA71345-04) and by the Marshall K. Bartlett Fellowship from the Department of Surgery, Massachusetts General Hospital. 
2 These authors contributed equally to this work.
3 To whom requests for reprints should be addressed, at Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, Building 149 13th Street, Charlestown, MA 02129. Phone: (617) 724-1170; Fax: (617) 726-4453; E-mail: michael.detmar{at}cbrc2.mgh.harvard.edu
4 The abbreviations used are: TSP-2, thrombospondin-2; LLC, Lewis lung carcinoma; VEGF, vascular endothelial growth factor; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; TGF, transforming growth factor.
5 J. P. Vacanti, unpublished observations.
Received 8/ 3/01. Accepted 1/28/02.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) at 5 days (arrow) after intradermal implantation of B16F10 malignant melanoma cells significantly inhibited tumor growth, as compared with control cell grafts (
). Values represent means (n = 10 tumors per group); bars, ± SE. *, P < 0.05.
