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Experimental Therapeutics |
Departments of Cellular and Structural Biology [A. B., J. Y., L-Z. S.] and Surgery [S. N. M.], University of Texas Health Science Center, San Antonio, Texas 78229, and Departmento de Biologia Celular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, D.F., 04510, Mexico [F. L-C., J. L. M., V. M.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Betaglycan, also known as TGF-ß type III receptor, has two TGF-ß binding sites (7, 8, 9) and binds all three TGF-ß isoforms (TGF-ß1, TGF-ß2, and TGF-ß3) with high affinity (10) . A sBG consisting of its extracellular domain has been shown to bind and neutralize the activity of TGF-ß isoforms with high potency in vitro (7 , 11) . We have shown previously that ectopic expression of the sBG can suppress the malignant properties of human carcinoma cells by antagonizing the tumor-promoting activity of TGF-ß in vivo (12) . In the current study, we investigated the effectiveness of a recombinant sBG in suppressing malignant progression in a human breast carcinoma xenograft-bearing nude mouse model. We administered sBG by either p.t. or i.p. injection into mice with growing tumors and observed a significant reduction of the tumor growth rate, spontaneous metastasis, and tumor vascularization. Our study demonstrates the potential therapeutic utility of sBG as a TGF-ß antagonizing agent for the treatment of cancer.
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MATERIALS AND METHODS |
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Cell Lines.
Human breast cancer cell line MDA-MB-231 and mink lung epithelial cell line CCL64 were originally obtained from the American Type Culture Collection. To determine the effect of sBG administration on the early metastatic potential of the MDA-MB-231 cells, we stably transfected the enhanced GFP expression plasmid, pEGFP-N1 (Clontech Laboratories, Inc.), into MDA-MB-231 cells and obtained a pool of GFP-expressing cells called MDA-MB-231/GFP. The expression of GFP allowed us to detect micrometastatic colonies in the whole lungs under an inverted fluorescence microscope as others have reported (13)
. These cell lines were cultured in McCoy’s 5A medium supplemented with pyruvate, vitamins, amino acids, antibiotics, and 10% fetal bovine serum (14)
. HDMECs and the culture medium EGM-2MV were obtained from BioWhittaker (San Diego, CA). Working cultures were maintained at 37°C in a humidified incubator with 5% CO2.
Preparation of Recombinant sBG.
The procedures to generate, express, and purify the baculoviral recombinant human sBG were similar to those described earlier for the rat sBG (7
, 11)
. Briefly, the human BG cDNA (15)
was engineered to contain after Asp781 (the last residue of the predicted extracellular region) a hexa-histidine tail followed by a stop codon. The mutated cDNA was used to generate a high titer recombinant baculovirus, which was used to infect High Five cells (Invitrogen). After 2 days of infection, human sBG was purified from the conditioned medium using immobilized metal-ion affinity chromatography.
Affinity Labeling in Solution and Competition Assay.
TGF-ß2 affinity labeling in solution and TGF-ß competition assays were done as described previously (11)
. Briefly, 10 ng of human sBG and 100 pmol of 125I-labeled TGF-ß2 (in the absence or presence of the indicated concentrations of nonradioactive TGF-ß) were incubated for 3 h at 4°C in PBS supplemented with 0.05% (v/v) Triton X-100. Cross-linking was started by the addition of 0.1 mg/ml disuccinimidyl suberate (Pierce) and stopped after 15 min by the addition of Tris-Cl (pH 7.5) to a final concentration of 10 mM. The reaction mixture was immunoprecipitated with an antihuman sBG polyclonal antibody raised from rabbits following standard immunization protocols (16)
. The precipitated proteins were separated by SDS-PAGE and the 125I-labeled TGF-ß-complexed sBG was revealed by scanning in a PhosphorImager (Molecular Dynamics, Inc.). Quantitative densitometry of radiolabeled sBG was carried out using the ImageQuant software; data were analyzed using the Prism software.
Bioassays of TGF-ß1 Neutralizing Activity of Recombinant sBG.
Two bioassays were used to confirm that sBG can effectively antagonize the activity of TGF-ß1. In the first assay, sBG was used to neutralize the activity of TGF-ß1 in stimulating the promoter activity of the human PAI-1. We used a mink lung epithelial cell line that was stably transfected with a PAI-1 promoter-luciferase construct as described by Abe et al. (17)
. The cells were plated in a 96-well plate at 1000 cells/well and incubated for 3 days for them to reach the exponential growth phase. They were then treated with various concentrations of TGF-ß1 in the presence or absence of sBG at 5 µg/ml. After 16 h of incubation, cells were lysed, and the cell lysate was analyzed for luciferase activity. A second bioassay was used to test the ability of sBG in neutralizing TGF-ß1-mediated growth-inhibitory activity in the mink lung epithelial cells. The cells were plated in a 96-well plate at 2000 cells/well and incubated with 0.05 or 0.25 ng/ml of TGF-ß1 in the presence or absence of 5 µg/ml sBG. After 5 days of incubation, the relative cell number in each well was obtained with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (18)
and expressed as absorbance.
In Vivo Tumor Growth and Metastasis Studies.
The MDA-MB-231/GFP cells were harvested from exponential cultures and inoculated s.c. at 3 x 106 cells/inoculum in both sides of the inguinal mammary fat pad area of female athymic nude mice, 4 weeks of age. When the tumors grew with an average diameter of >4 mm after 
5 weeks, animals were ranked according to the tumor volume and divided into two groups such that the mean and median of tumor volume of the two groups were closely matched. In the first experiment, the mice in the experimental group were injected twice a week peritumorally (next to a tumor) with a rat recombinant sBG at a dosage of 50 µg/tumor in a total volume of 0.05 ml of PBS. The mice in the control (placebo) group received 50 µl of sterile PBS/tumor. In a second experiment, the experimental mice were injected every alternate day i.p. with a human sBG at a dosage of 100 µg/animal in a total volume of 0.1 ml of PBS. The control (placebo) mice received 0.1 ml of PBS. Each xenograft was monitored weekly by externally measuring tumors in two dimensions using a caliper. Xenograft volume (V) was determined by the following equation: V = (L x W2) x 0.5, where L is the length and W is the width of a xenograft.
At the termination of the growth studies, animals were sacrificed, and lungs were removed during autopsy to examine any spontaneous metastasis. The GFP-expressing green metastatic cancer cell colonies, if any, were identified and counted using a Nikon fluorescence microscope (TE-200) with a x20 objective (x200 magnification).
Measurement of sBG in the Tumor.
For the determination of sBG level in the tumors at the termination of sBG administration, tumors were extracted as described previously (19)
. Briefly, frozen tumor tissues were weighed, pulverized in liquid nitrogen with a pestle and a mortar, and extracted with an ice-cold extraction buffer containing10 mM Tris (pH 7.5), 0.1 M NaCl, 0.5% Triton X-100, 1.0 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride. For every 1 g of tissue, 6 ml of the extraction buffer were used. The extract was centrifuged at 10,000 x g for 15 min. sBG concentrations in the supernatant were determined by a two-antibody sandwich ELISA assay developed in our laboratory. An antihuman sBG polyclonal antibody was raised from rabbits by the injection of the purified recombinant human sBG following standard immunization protocols (16)
. The antibody was precoated (1:5000 dilution) to the bottom of a 96-well immunoplate (Dynatech laboratories, Inc.) as the capture antibody. The wells were washed with PBS containing 0.5% Tween 20 and blocked with 1% BSA. The purified human sBG and tumor extracts were added into the wells and incubated for 2 h at 37°C. The bound sBG was detected after subsequent incubation with an antihuman sBG goat antibody (R & D systems; 1:500 dilution), a polyclonal antigoat IgG conjugated with horseradish peroxidase (Pierce; 1:30,000 dilution), and a horseradish peroxidase substrate mixture containing H2O2 and o-phenylenediamine dihydrochloride. The final reaction was stopped with 6 N H2SO4, and the absorbance was determined with a microplate reader at 492 nm. The sBG concentration in the tumor extract was determined from a standard curve using known amounts of human sBG.
Assays for Tumor Vascularity.
To determine the effect of sBG treatment on tumor angiogenesis, we measured the vascularity of excised tumors at the termination of sBG treatment. Tumor tissues were fixed in 10% neutral buffered formalin (Fisher Scientific) overnight at 4°C and embedded in paraffin. Sections of 4 µm were cut from the embedded tissue and stained with H&E. Sections were examined by light microscopy under x400 magnification, and the number of blood vessels containing red cells from 10 high power fields was counted and averaged. CD31 immunostaining for mouse blood vessels was performed by incubating tumor sections with a rat antimouse CD-31 (PECAM-1) monoclonal antibody (PharMingen) at 5 µg/ml for 30 min at 37°C. Sections were then incubated with a biotin-labeled goat antirat IgG (Zymed; 1:200 dilution) for 30 min at room temperature, followed by ABC reagent kit (Vector Laboratories) for 30 min at room temperature. Color reaction was performed with 3,3'-diaminobenzidine (Vector Laboratories) and counterstained with hematoxylin. All sections were coded and observed by a pathologist who was blinded for the study protocol.
Hemoglobin Assay.
Hemoglobin levels in tumors were measured to corroborate the blood vessel density. Excised tumors were carefully cleaned for any external blood on their surface and then extracted as described above. Hemoglobin content in tumor extracts was measured using a hemoglobin assay kit (Sigma) following the manufacturer’s instruction. The hemoglobin content in systemic blood obtained through cardiac puncture at the termination of the experiment was also measured to obtain the blood volume/unit weight of tumor.
Endothelial Tube Assay.
Matrigel (Collaborative Biochemicals) was added to each well of a 24-well plate at 320 µl/well and allowed to polymerize at 37°C for 1 h. A suspension of 40,000 HDMECs in EGM-2MV (BioWhittaker) was added to a Matrigel-coated well. The cells were treated without or with different concentrations of the recombinant human sBG for 24 h at 37°C. The tubular web structures formed by HDMECs were observed with a Nikon T-200 inverted microscope under x40 magnification and captured with an Olympus MagnaFire digital camera. The web junctions defined as intersections formed by three or more tubules were counted in each microscopic field.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows that human sBG is expressed in the baculoviral system as a core protein devoid of glycosaminoglycans and binds TGF-ß2 with higher affinity than TGF-ß1, properties reported for its rat counterpart (Fig. 1B
; Ref. 11
). Although both human and rat sBG show a higher affinity for TGF-ß2 than for TGF-ß1, the rat sBG was shown to effectively neutralize the activity of TGF-ß1 in our previous study (11)
. To confirm its TGF-ß antagonistic activity, we determined the effect of human sBG treatment on TGF-ß1-induced activation of PAI-1 promoter. The mink lung epithelial cells stably transfected with a 800-bp PAI-1 promoter-luciferase construct (17)
were treated with various concentrations of TGF-ß1 in the presence or absence of 5 µg/ml of a human recombinant sBG. The activation of PAI-1 promoter activity reported with luciferase activity by TGF-ß1 was markedly attenuated in the presence of the sBG (Fig. 2A)
. Addition of the human or a rat recombinant sBG also significantly (P < 0.05) neutralized the growth-inhibitory activity of TGF-ß1 in the mink lung epithelial cells as shown in Fig. 2B
. These data represent the first description of the production of a recombinant human sBG and its TGF-ß antagonistic activity as reported previously for the rat sBG (11)
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4 mm in diameter, we started injecting a rat recombinant sBG next to each tumor at a dose of 50 µg/tumor. The injection of sBG twice per week resulted in a significant (P < 0.05) reduction of the mean tumor growth rate when compared with the placebo injection over a period of 3 weeks (Fig. 3A)
. This result prompted us to test whether systemic administration such as i.p. injection of sBG would yield similar results. Therefore, in a second experiment, when the average tumor diameter reached 4 mm, a recombinant human sBG was injected i.p. every alternate day at a dose of 100 µg/animal for 28 days. Similar to the p.t. injection, i.p. injection of sBG also generated a significant inhibition of tumor growth (Fig. 3B)
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60% (Fig. 3C)
. The average number of lung metastatic colonies/mouse were also reduced after p.t. (Fig. 3D)
and i.p. (Fig. 3E)
administration of sBG. These data indicate that recombinant sBG can inhibit the metastatic potential of MDA-MB-231 cells. Of note, sBG injection did not cause any weight loss or behavior change of the animals, suggesting that sBG did not induce any deleterious side effect.
To determine whether the systemic administration generated an appreciable level of sBG in tumors, tumor extracts were analyzed for sBG content after the termination of i.p. administration. We used a sandwich ELISA assay to determine the sBG level in tumor extracts from two placebo-treated mice and two sBG-treated mice. The tumor extracts from sBG-treated mice showed a significantly higher sBG level than those from placebo-treated mice (Fig. 4A)
. Previously, we have shown that ectopic expression of sBG in the MDA-MB-231 cells also inhibited tumor growth and metastasis (12)
. Because the inhibition by the ectopic expression of sBG appeared greater than by the i.p. injection of sBG, we measured sBG levels in the tumors formed by the control and sBG-transfected MDA-MB-231 clones (Cl.41 and Cl.44; Fig. 4B
). Apparently, the sBG transfection generated a much higher level of local sBG in the tumors than the i.p. sBG injection, suggesting that the sBG dosage for systemic administration may be further optimized to increase its tumor-suppressive activity. Nevertheless, our study, for the first time, demonstrates that the recombinant sBG can be administered systemically to inhibit carcinoma growth and metastasis using the MDA-MB-231 xenograft model.
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. Immunohistochemical staining of CD-31, an endothelial marker, also revealed a more extensive vasculature in the tumors from the control group than in the tumors from sBG-treated group (Fig. 5C)
. To correlate the vasculature with blood volume, we measured tumor hemoglobin content and calculated tumor blood volume. Consistent with the decrease in the number of microvessels, sBG treatment also significantly (P < 0.05) reduced tumor blood volume (Fig. 5B)
. A similar reduction of the tumor blood volume was also observed when a Texas Red-conjugated dextran was injected i.v., and its blood concentration was used to calculate tumor blood volume (data not shown). Thus, these data suggest that the inhibition of tumor growth and metastasis by sBG administration may be in part attributable to the inhibition of tumor angiogenesis.
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. Counting web junctions, the intersections formed by three or more tubules, in randomly selected microscopic fields revealed a statistically significant (P < 0.05) inhibition of web junction formation by sBG in a dose-dependent manner (Fig. 6B)
. This result suggests that sBG may inhibit angiogenesis by impairing the ability of endothelial cells to form blood vessels.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the current study, we have used a recombinant soluble form of betaglycan (sBG), produced in insect cells using the baculovirus expression system (11) , as a TGF-ß antagonist and evaluated its potential as a novel anticancer drug. We choose sBG over the soluble type II receptor because it has two TGF-ß binding sites/molecule (7 , 8) and binds all three TGF-ß isoforms with high affinity (11 , 24) . In contrast, the soluble type II receptor does not bind TGF-ß2 (25) . Because the MDA-MB-231 cell line produces both active TGF-ß1 and TGF-ß2 (14) , sBG should be a more effective antagonist in this model system. Administration of sBG via either p.t. or i.p. injection with a similar dosing regimen generated a significant inhibition of tumor growth and a marked reduction of metastatic incidence and colonies in the lung. The fact that i.p. administration of sBG inhibited the growth of a s.c. tumor and lung metastasis suggests that the administered sBG can be transferred to distant locations in mice. Indeed, our sandwich ELISA detected a significantly higher level of sBG in the tumors from the sBG-treated mice than from the control mice. Meanwhile, we also found that the tumor sBG level in the sBG-injected mice was much lower than that in the tumors produced by sBG-transfected MDA-MB-231 cells. Because the overexpression of sBG through transfection in our previous study (12) appears to be more tumor suppressive than the sBG administration in the current study, further studies are warranted to determine whether a higher tumor sBG level can be achieved by increasing sBG dosage in various tumor models. It will also be important to determine whether the administration of sBG can produce a synergistic effect with another cancer therapeutic strategy.
TGF-ß is believed to act as an autocrine as well as a paracrine factor to promote tumor progression. Although carcinoma cells are known to produce high levels of active TGF-ß isoforms, stromal cells from malignant breast tissue has also been shown to produce significantly more TGF-ß1 than those derived from normal breast (26) . Several mechanisms are believed to mediate the tumor-promoting activity of TGF-ß (3) . One of them is the stimulation of angiogenesis, which is the formation of new capillaries from preexisting vessels and is essential for the growth and metastasis of solid tumor (27) . TGF-ß has been shown to be angiogenic in vivo (28) . Overexpression of TGF-ß1 in Chinese hamster ovary cells significantly stimulated tumor growth and angiogenesis when they are inoculated into nude mice, and the effect could be attenuated by p.t. injection of a TGF-ß1 neutralizing antibody (29) . As such, we investigated whether the inhibition of tumor growth and metastasis by sBG administration is in part attributable to the inhibition of angiogenesis. Measurements of tumor blood vessel density and blood volume indicate that the systemic administration of sBG significantly inhibited tumor angiogenesis. Interestingly, we also observed a direct inhibitory effect of sBG on the capillary web structure formation by HDMECs, suggesting that sBG may inhibit angiogenesis by impairing the ability of endothelial cells to form new blood vessels.
In summary, our findings support the conclusion that sBG treatment can inhibit the malignant progression in the MDA-MB-231 xenograft model. This inhibition is apparently in part mediated by the impairment of tumor angiogenesis. Thus, the tumor-suppressive activity of sBG should be independent of the responsiveness of tumor cells to TGF-ß. Indeed, our recent study demonstrated that expression of sBG can inhibit in vivo growth of carcinoma cells that are insensitive to TGF-ß (30) . Future studies will need to address the pharmacokinetic and pharmacodynamic properties of sBG in various tumor models.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by NIH Grants CA79683 and CA75253 (to L-Z. S.) and by a Howard Hughes Medical Institute International Research Scholar Grant (to F. L-C.). 
2 To whom requests for reprints should be addressed, at Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, Mail Code 7762, San Antonio, TX 78229-3900. Phone: (210) 567-5746; Fax: (210) 567-3803; E-mail: sunl{at}uthscsa.edu
3 The abbreviations used are: TGF, transforming growth factor; sBG, soluble betaglycan; GFP, green fluorescent protein; HDMEC, human dermal microvascular endothelial cell; PAI-1, plasminogen activator inhibitor-1; p.t., peritumoral.
Received 4/23/02. Accepted 6/17/02.
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