Affiliations of authors: A. L. Feldman, H. R. Alexander, D. Lorang, C. E. Thiruvathukal, E. M. Turner, S. K. Libutti (Surgical Metabolism Section, Surgery Branch, Center for Cancer Research), S. M. Hewitt (Laboratory of Pathology, Center for Cancer Research), National Cancer Institute, Bethesda, MD.
Correspondence to: Steven K. Libutti, M.D., National Institutes of Health, Bldg. 10, Rm. 3C428, Bethesda, MD 20892 (e-mail: Steven_Libutti{at}nih. gov).
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ABSTRACT |
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INTRODUCTION |
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MATERIALS AND METHODS |
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The retroviral vector pCLNCX (5) and the vector pMD.G containing the G protein gene from vesicular stomatitis virus (VSV), as well as the cell line 293GP stably transfected with the retroviral gag and pol elements, were obtained from P. Robbins, National Cancer Institute (NCI), Bethesda, MD. The human elongation factor (EF) 1 promoter was isolated from the plasmid pAd.EF1 (Z. Guo, NCI) by BamHI/HindIII digestion (New England Biolabs Inc., Beverly, MA) and ligated in place of the downstream cytomegalovirus (CMV) promoter in BamHI/HindIII-digested pCLNCX to generate the vector pEF-null (Fig. 1
, a). Generation of the construct ss-mEndo, consisting of the 18-amino acid E3/19K signal sequence followed by the murine endostatin gene cloned from murine liver, has been described previously (6). The ss-mEndo construct was cloned into HindIII/ClaI-digested pEF-null to generate the vector pEF-Endo or into HindIII/ClaI-digested pCLNCX to generate the vector pCMV-Endo.
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Retroviral Transduction of NMuLi Cells
The cell line NMuLi, an epithelioid nonparenchymal cell line derived from the NAMRU mouse liver (7), was obtained from the American Type Culture Collection (Manassas, VA) and passaged in complete medium. We confirmed its ability to form malignant tumors in nude mice (8). For the retroviral transduction, cells were plated in six-well tissue culture plates at a density of 100 000 cells/well and allowed to adhere overnight. The medium then was replaced with 1 mL of fresh complete medium, 1 mL of retroviral supernatant, and 8 µg/mL of hexadimethrine bromide (Sigma Chemical Co., St. Louis, MO) to assist the uptake of viral particles. After incubation at 37 °C for 4 hours, the medium was replaced with 2 mL of fresh complete medium. The transduction was repeated the following day. Because the retroviral vector contains a selectable marker, the neomycin resistance gene, G418 (400 µg/mL; Life Technologies, Inc.), was added to the medium 24 hours after the second transduction. The surviving G418-resistant cells were amplified. To obtain individual clones, the endostatin-transduced cells (NEF-Endo) were plated in limiting dilution in complete medium containing G418 (400 µg/mL). The resultant clones were isolated by use of 8-mm diameter cloning cylinders (Specialty Media, Phillipsburg, NJ) and amplified.
To confirm endostatin expression, parental NMuLi cells, pEF-null-transduced cells (NEF-null), or NEF-Endo clones were plated in six-well plates at a density of 106 cells/well and allowed to adhere overnight. The medium was replaced with 1 mL/well of modified complete medium containing 5% FCS and incubated at 37 °C for 24 hours. Cell supernatants then were collected and passed through a 0.45-µm (pore size) filter. Endostatin concentrations in the supernatants were determined by competitive enzyme immunoassay (EIA) (Cytimmune Sciences, College Park, MD) according to the instructions of the manufacturer. Four endostatin-transduced clones with varying supernatant endostatin concentrations were selected for further study and designated NEF-Endo1 to 4, in decreasing order of endostatin production. The molecular weight of endostatin was determined from the culture supernatants by western blotting (NuPAGE; Novex, San Diego, CA) by use of 570 ng/mL of rabbit antimurine endostatin polyclonal immunogloblin G antibody (from Cytimmune Sciences). The EIA murine endostatin standard was used as a positive control.
Functional Assay of Retrovirally Derived Endostatin
To assess the functional activity of the retrovirally derived endostatin, we tested the culture supernatants in endothelial cell proliferation assays as described previously (6), with slight modifications. Briefly, bovine adrenal capillary endothelial cells (EJG; American Type Culture Collection) were plated in 200 µL of complete medium in collagen I-coated 96-well plates (Biocoat; Becton Dickinson Labware, Bedford, MA) at a density of 1000 cells/well and were incubated overnight at 37 °C. The medium then was aspirated and replaced with 100 µL of cell supernatant or modified complete medium containing 5% FCS. Basic fibroblast growth factor (R&D Systems, Inc., Minneapolis, MN) at a final concentration of 1 ng/mL was added to all wells as a stimulus of proliferation. After incubation at 37 °C for 72 hours, proliferation was analyzed by the WST-1 assay (Roche, Indianapolis, IN) according to the manufacturer's instructions. The inhibition of proliferation by each sample was calculated according to the formula: inhibition (%) = (mean ODmedia
ODsample/mean
ODmedia) x 100, where
OD represents the change in optical density at 450 nm (Multiskan MCC/340 plate reader; Titertek, Huntsville, AL) from before the addition of the WST-1 reagent to the completion of the 4 hours' incubation at 37 °C. Each sample was measured in six individual wells. The experiment was repeated to confirm results.
In Vitro Growth of Parental and Transduced NMuLi Cells
To place in perspective any differences noted in the in vivo growth of parental and transduced cell lines, we compared in vitro growth rates. Five identical 96-well tissue culture plates were prepared. On each plate, NMuLi, NEF-null, and NEF-Endo1 to 4 cells were each plated in eight identical wells at a density of 1000 cells/well in 100 µL of complete medium. WST-1 proliferation assays were performed daily on days 04, as described above. Cells were allowed to adhere for 3 hours before the day-0 assay was performed. Proliferation was expressed as the OD as defined above.
Tumor Formation by Retrovirally Transduced Cells
Animal experiments were conducted according to protocols approved by the National Institutes of Health Animal Care and Use Committee (Bethesda, MD). Eight-week-old female nude mice (Charles River Laboratories, Wilmington, DE) were given an injection of 5 x 105 parental NMuLi cells, NEF-null cells, or NEF-Endo1 to 4 cells. Subcutaneous injections were administered in the right flank in 100 µL of PBS. Tumors were measured in two dimensions by use of calipers at regular intervals, and tumor volumes were calculated according to the following formula: volume = width2 x length x 0.52, where 0.52 is a constant to calculate the volume of an ellipsoid. Intraperitoneal injections were administered in 2 mL of PBS. All animals were followed for survival. Each group consisted of eight mice.
Measurement of Endostatin Levels In Vivo
Mice were given an inoculation of NEF-null or NEF-Endo1 cells subcutaneously or intraperitoneally as described above. Serum samples obtained 45 days (subcutaneous model) or 21 days (intraperitoneal model) after tumor cell inoculation were analyzed for endostatin concentration by EIA. Serum samples also were obtained from non-tumor-bearing animals. Each group consisted of six mice.
To assess in vivo local endostatin expression, we harvested NEF-null or NEF-Endo1 tumors immediately after the mice were killed 38 days after subcutaneous injection as described above. Tumors were snap-frozen in liquid nitrogen and stored at -80 °C until ready for analysis. Tumors then were homogenized at room temperature in a protease inhibitor cocktail (Complete Mini; Roche) dissolved in 10 mM HEPES and 1 mM EDTA by use of a bead homogenizer (FastPrep; Savant Instruments, Holbrook, NY). Homogenates were centrifuged at 7500g for 5 minutes at 4 °C, and supernatants were analyzed for endostatin by EIA. Endostatin concentrations were normalized to total protein concentrations determined by use of a BCA protein assay kit (Pierce Chemical Co., Rockford, IL) and a linear bovine serum albumin standard curve, according to the manufacturer's instructions.
Immunohistochemistry and Histopathologic Evaluation
NEF-null or NEF-Endo1 tumors were harvested immediately after the mice were killed 38 days after subcutaneous injection, as described above. Tumors were snap-frozen in liquid nitrogen and stored at -80 °C. Frozen sections (7 µm) were cut on a cryostat and stained with hematoxylineosin (H&E) or antibodies specific for CD31 (PharMingen, San Diego, CA), proliferating cell nuclear antigen (PCNA) (Zymed Laboratories, San Francisco, CA), or caspase-3 (PharMingen). For immunostaining, sections were fixed in acetone for 10 minutes (CD31) or in 4% formalin for 1 hour (PCNA and caspase-3). After endogenous peroxidase activity was blocked by use of 3% hydrogen peroxide in methanol for 10 minutes, sections were incubated for 1 hour in a blocking solution containing 10% normal goat serum. Sections were incubated with primary antibody at 4 °C overnight (CD31 at 1 : 50 dilution or caspase-3 at a concentration of 1 µg/mL) or at room temperature for 1 hour (PCNA at 1 : 50 dilution). Slides were then washed three times in PBS, incubated in biotinylated species-specific appropriate secondary antibody for 1 hour, and exposed to avidinbiotinperoxidase complex (Vector Laboratories, Inc., Burlingame, CA). Sections were reacted with 0.06% 3,3'-diaminobenzidine (Sigma Chemical Co.) and counterstained with hematoxylin.
H&E and immunostained sections were analyzed by a pathologist (S. M. Hewitt), who was blinded to the identity of the groups. Only good-quality sections with uniform, well-demarcated staining and low background were analyzed. Microvascular density of each tumor was assessed by use of a scoring system (Table 1) based on the mean number of CD31-positive cells per high-power field (hpf, x600). Proliferative and apoptotic cells were analyzed by use of a similar scoring system (Table 1
) based on the number of cells per hpf staining positively for PCNA and caspase-3, respectively.
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Data are presented as means with 95% confidence intervals (CIs). Comparisons between groups were made by use of the MannWhitney U test or the KruskalWallis test, where appropriate. The endostatin dose dependence of endothelial cell inhibition was analyzed by use of the JonckheereTerpstra trend test. Two-tailed P values <.05 were considered to be statistically significant.
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RESULTS |
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Initial transduction of NMuLi cells with pCMV-Endo did not yield supernatant endostatin concentrations above baseline. We, therefore, replaced the downstream CMV promoter in pCLNCX with the EF1 promoter (Fig. 1
, a) based on favorable results reported with the use of the EF1
promoter in other viral gene delivery systems (9,10). Endostatin levels in supernatants from NEF-Endo1, 2, 3, and 4, NEF-null, and NMuLi cells were 223, 163, 42, 28, 20, and 4 ng/mL per 106 cells, respectively. The NEF-Endo clone supernatants produced specific 20-kd bands on western blot proportional in intensity to the endostatin concentration as measured by EIA (Fig. 1
, b).
In Vitro Function of Endostatin
To determine if the endostatin produced by the transduced clones was biologically active, we tested the supernatants in an endothelial cell proliferation assay. Compared with unconditioned medium, supernatants from the transduced clones NEF-Endo 1, 2, 3, and 4, and NEF-null inhibited endothelial cell proliferation by 20% (95% CI = 13% to 27%), 11% (95% CI = 3% to 19%), 9% (95% CI = 1% to 19%), 6% (95% CI = 0% to 12%), and 4% (95% CI = 8% to 17%), respectively (Fig. 1, c). The difference in inhibition between supernatants from NEF-Endo1 and NEF-null was statistically significant (P = .037). The dose-dependent inhibition of endothelial cell proliferation by endostatin also was statistically significant (P = .01).
In Vitro Growth of Parental and Transduced NMuLi Cells
We next compared the in vitro growth of the transduced cells with that of the parental NMuLi cells. All four NEF-Endo clones demonstrated similar patterns of in vitro growth to each other and to NEF-null cells (Fig. 1, d). Parental NMuLi cells grew more slowly in vitro than their transduced, G418-selected counterparts.
Subcutaneous Growth of Parental and Transduced NMuLi Cells
Mice given a subcutaneous injection of NMuLi or NEF-null cells developed rapidly growing tumors and were killed 63 days after injection when the tumor volumes were 2400 mm3 (95% CI = 1478 mm3 to 3300 mm3) and 2700 mm3 (95% CI = 2241 mm3 to 3144 mm3), respectively (Fig. 2, a). The mean tumor volumes were less than 30 mm3 in all four groups given an injection of NEF-Endo clones (P.<001 versus null-transduced tumors). Although the largest endostatin-transduced tumors were observed in the mice that were given an injection of the NEF-Endo4 clone, which produced the lowest amount of endostatin in vitro (NEF-Endo4; Fig. 2
, a, inset), the difference between the size of the endostatin-transduced tumors was not statistically significant (P = .52 at day 63). After 122 days of follow-up, mice given an injection of NEF-Endo clones 1, 3, and 4 had tumor volumes of 70 mm3 (95% CI = 0 mm3 to 202 mm3), 300 mm3 (95% CI = 123 mm3 to 419 mm3), and 90 mm3 (95% CI = 0 mm3 to 279 mm3), respectively. Mice given an injection of the NEF-Endo clone 2 had nonpalpable or barely palpable lesions.
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Mice given an intraperitoneal injection of parental NMuLi or NEF-null cells had median survival times of 58 days (95% CI = 49 days to 61 days) and 56 days (95% CI = 46 days to 92 days), respectively, and all mice were dead by day 123 (Fig. 2, b). At this time, only three (9%) of the 32 mice receiving intraperitoneal NEF-Endo clones had died. Autopsies of mice that died revealed massive peritoneal tumor deposits and ascites. Surviving mice were killed and had an autopsy, revealing only occasional, small peritoneal deposits.
In Vivo Endostatin Levels
Serum endostatin levels were similar in mice bearing subcutaneous NEF-Endo1 tumors (mean, 33.8 ng/mL; 95% CI = 18.5 ng/mL to 49.1 ng/mL) and NEF-null tumors (mean, 32.6 ng/mL; 95% CI = 15.8 ng/ml to 49.4 ng/mL), respectively (P = .87). Serum endostatin levels in mice bearing intraperitoneal NEF-Endo1 tumors (mean, 40.0 ng/mL; 95% CI = 7.8 ng/mL to 72.2 ng/mL) were slightly higher than those in mice bearing intraperitoneal NEF-null tumors (mean, 22.2 ng/mL; 95% CI = 18.3 ng/mL to 26.1 ng/mL), but this difference was not statistically significant (P = .42). Serum endostatin levels in non-tumor-bearing mice were 27.1 ng/mL (95% CI = 15.3 ng/mL to 38.9 ng/mL), similar to values we reported previously (11) and not statistically significantly different from any of the other groups tested.
To confirm endostatin production in the NEF-Endo tumors, we measured local endostatin levels in tumor lysates 38 days after subcutaneous injection. Tumor lysates from endostatin- and null-transduced tumor had 38.7 ng (95% CI = 20.7 ng to 56.6 ng) and 11.2 ng (95% CI = 8.8 ng to 13.7 ng) endostatin/mg total protein, respectively (P = .006).
Immunohistologic Characteristics of Transduced Tumors
Evaluation of H&E-stained tumor sections revealed small tumor nodules (mean diameter, 1.7 mm) derived from NEF-Endo1 and NEF-Endo2 cells (Table 1; Fig. 3
, a); tumors derived from NEF-null cells were larger (mean diameter, 4.9 mm) and had zones of central necrosis (Fig. 3
, b). Mean microvessel density scores for endostatin- and null-transduced tumors were 3 and 4, respectively (Table 1
; Fig. 3
, ce). Endostatin-transduced tumors did not have discernible vessels on H&E stains (Fig. 3
, a) but did contain CD31-positive endothelial cells as individual capillaries (Fig. 3
, c). By contrast, the null-transduced tumors contained easily identifiable vessels on H&E stains, away from zones of necrosis (Fig. 3
, b). The CD31 staining highlighted larger vessels as well as individual endothelial cells (Fig. 3
, d).
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DISCUSSION |
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Previously, we and others (6,15,16) have shown that adenoviral transfer of the endostatin gene to mice leads to high circulating endostatin levels that inhibit tumor growth. Several groups (1719) also have demonstrated that nonviral endostatin gene transfer can cause an antitumor effect in mice. Both of these approaches, however, are associated with limitations in their ability to be translated for the treatment of human patients: The use of adenoviral vectors is limited because of immunogenicity and toxicity, and nonviral vectors generally are associated with low levels of transgene expression. Because retroviruses have been used safely in human gene therapy trials (20,21), we investigated whether retroviral transfer of the endostatin gene to tumor cells could result in sufficient functional protein to inhibit tumor growth in vivo.
Because endostatin occurs naturally as a cleavage product of collagen XVIII, the coding sequence for endostatin lacks a secretion signal. We previously generated a construct in which the endostatin coding sequence is preceded by the adenoviral protein E3/19K signal sequence (6,22). We cloned this construct into the multiple cloning site of pCLNCX, a Moloney murine leukemia virus-derived retroviral expression plasmid (5), and generated pseudotyped retroviral particles containing the VSV glycoprotein G (23). NMuLi cells transduced with this construct were resistant to G418, but they did not generate high levels of endostatin in their cell supernatants. We hypothesized that this was a promoter-related phenomenon and replaced the downstream CMV promoter in the pCLNCX plasmid with the constitutively active human EF-1 promoter. Cells transduced with this modified construct secreted high endostatin levels, as measured in a competitive EIA.
To verify that the secreted protein measured in the EIA was, in fact, endostatin, we performed western blotting of aliquots from supernatants of the transduced cells. Supernatants from NEF-Endo clones produced bands of equal mobility to that of recombinant murine endostatin and of intensities proportional to the EIA-measured endostatin concentrations. When these supernatants were placed on bovine adrenal capillary endothelial cells, they inhibited endothelial cell proliferation in a dose-dependent fashion, confirming the activity of the secreted endostatin protein. The secreted endostatin was relatively endothelial cell specific, as has been reported previously (3), because the growth rates of the NEF-null cells and the NEF-Endo clones were similar, including NEF-Endo1 cells that secreted the highest level of endostatin.
By contrast with the in vitro growth, transduction of NMuLi cells with the endostatin gene profoundly inhibited their subcutaneous growth in mice. Consistent with the antiangiogenic function of endostatin, NEF-Endo cells were capable of forming tumors in vivo, although these tumors were very small and displayed fewer microvessels than their null-transduced counterparts. The NEF-Endo4 clone, which produced the least endostatin in vitro, initially produced larger tumors than the other NEF-Endo clones tested. However, the inhibition of in vivo tumor growth of all clones was not directly proportional to in vitro endostatin expression. In fact, the growth curves of the four NEF-Endo clones began to diverge after approximately 100 days, although tumors in all groups remained much smaller than tumors in the NEF-null mice, which exceeded 2 cm3. The eventual divergence of the growth curves among the NEF-Endo clones growth may reflect clonal variations unrelated to endostatin. Alternatively, local endostatin production at late time points in vivo may not correspond with in vitro expression by various clones.
Of interest, endostatin transduction of NMuLi cells also inhibited their ability to form aggressive lesions in the peritoneal cavity. Like tumors in other sites, the pathogenesis of tumors of the peritoneal cavity has been shown to be associated with angiogenesis (1,24,25). In addition, several inhibitors of angiogenesis have been reported to inhibit peritoneal tumor growth in rodent models, including protamine (26), TNP-470 (2729), and agents that inhibit the action of vascular endothelial growth factor (3032). Our data suggest that endostatin also can inhibit peritoneal tumor growth. Similar to the results seen with our subcutaneous model, the tumor lesions present in mice receiving intraperitoneal NEF-Endo cells were generally small. In mice with subcutaneous or intraperitoneal tumors, circulating endostatin levels were not statistically significantly increased in mice harboring endostatin-transduced tumors. Because of the minimal amount of tumor tissue in these animals and the baseline mean serum endostatin concentration of 27 ng/mL, small amounts of endostatin released into the circulation may not have been detectable by the assay employed. The assay was, however, sensitive enough to detect increased concentrations of endostatin in lysates of endostatin-transduced tumors, confirming that endostatin was being produced locally in these animals.
Our results indicate that retroviral transfer of the endostatin gene to eukaryotic cells does not inhibit their growth in vitro but generates a secreted, functional endostatin protein that inhibits endothelial cells in vitro and tumor formation at multiple sites in vivo. In addition, these data support previous evidence (2632) that inhibiting angiogenesis is a promising strategy for treating tumors of the peritoneal cavity. However, one limitation of this study is that ex vivo transduction of tumor cells is not a strategy applicable to clinical therapeutic use. Recently, retroviruses secreting another antiangiogenic molecule, interleukin 12 (IL-12), have been used in preclinical models of disseminated peritoneal cancer. Sanches et al. (33) transduced fibroblasts with the IL-12 gene ex vivo, then introduced them into mice with peritoneal ovarian carcinomatosis. Lechanteur et al. (34) used an intraperitoneal injection of retroviral packaging cells carrying the IL-12 gene as part of a combined approach to treating peritoneal colon carcinomatosis in rats. In each case, at least part of the tumor response was because of immune (i.e., nonantiangiogenic) mechanisms, and the immune effects of IL-12 have been associated with considerable toxicity in clinical and preclinical models. By contrast, endostatin appears to impart its antitumor effect solely by inhibiting angiogenesis, with minimal toxicity(3,35). Therefore, further studies using retroviral endostatin gene transfer for the treatment of cancer, including disseminated tumors of the peritoneal cavity, are warranted.
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NOTES |
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We thank S. Steinberg, National Cancer Institute (Bethesda, MD), for his help with the statistical analysis.
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Manuscript received December 15, 2000;
revised May 1, 2001; revised May 9, 2001;
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