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From the Division of Vascular Biology, La Jolla Institute for Molecular Medicine, San Diego, California
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Abstract |
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We have previously demonstrated that endothelial cells can be stimulated to secrete the high molecular weight forms of FGF-2 in a regulated manner to levels capable of affecting cell behavior.13 The effects were twofold: stimulation of cell proliferation and inhibition of migration. The increase in proliferation was comparable to that promoted by 18-kd FGF-2, indicating that the stimulation of cell proliferation was independent of the additional amino terminal peptide. On the other hand, 24-kd FGF-2 inhibited migration of endothelial cells by 50% and mammary carcinoma MCF-7 cells by greater than 70%, even in the presence of unrelated mitogens such as vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF)-1 that promote cell migration. Overall, the 24-kd FGF-2 affects cell behavior differently than 18-kd FGF-2 and the ATE is responsible for this difference. Further studies from our laboratory using a series of truncated forms of 24-kd FGF-2 have demonstrated that the ability to induce cell proliferation is lost with the removal of 40 amino acids from the carboxy terminal end, while full ability to inhibit migration is retained. The smallest peptide generated which displayed maximal inhibition contained the ATE in addition to the first 31 amino acids of the 18-kd FGF-2 domain (ATE + 31).14
Migration is a fundamental cell behavior that is required for a number of physiological and pathological events such as angiogenesis,15 tumor development and metastasis,16,17 development,18 and leukocytic transmigration.19,20 Angiogenesis itself is involved in a variety of disease states, for example, tumor-dependent vascular formation and retinopathy.21,22 In the present study we used the murine dorsal skin-fold window chamber model23,24 to determine whether inhibition of migration by ATE + 31 could reduce angiogenesis and tumor growth in vivo. This model provides microscopic access to the vasculature of the skeletal skin muscle and subcutaneous connective tissue. It allows the implantation of multicellular aggregates made up of any of a variety of tumor cells within the chamber and enables monitoring of the tumor and its microcirculation over time in response to peptide treatment. Thus, we have the ability to measure changes in tumor characteristics at any point during the experiment and to generate a true picture of how a single tumor aggregate responds to the protein peptide over a prolonged period. Another advantage of this technique is that it overcomes the problems associated with endpoint analysis with multiple animals and the uncertainty of animal-to-animal variation. The results presented in this paper demonstrate that ATE + 31 is a potent inhibitor of both angiogenesis and tumor growth while, more importantly, failing to affect cellular events such as chemotaxis of inflammatory cells like neutrophils and eosinophils.
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Materials and Methods |
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To generate pure recombinant 24-kd FGF-2, full-length 24-kd FGF-2 cDNA was inserted in frame into a pPIC9K yeast expression vector (Invitrogen, San Diego, CA) between the SnaBI and Avrll restriction sites of the pPIC9K vector directly downstream of the DNA encoding the X-factor secretion signal region. The His4 mutant of P. pastoris GS 115, the methanol-utilization-positive phenotype (Mut+), was transformed by electroporation with the pPIC9K construct vector linearized with SacI. The His+ yeast transformants were grown on minimal dextrose medium (MD) plates and those carrying the kanT gene further selected for multiple integrated copies by replating on plates containing 4.0 mg/ml G418 antibiotic. The multicopy transformant was grown in 50 ml BMGY medium with glycerol as the sole carbon source until the cultures reached an OD600 = 2 to 6 (approximately 16 to 18 hours). The cells were collected by centrifugation at 1500 to 3000 x g for 5 minutes at room temperature and the cell pellet resuspended in BMGY medium to induce expression. The cells were grown for 4 days and 100% methanol was added every 24 hours to a final concentration of 2% to maintain protein expression. The medium was cleared of the yeast by filtration and mixed with 5 ml heparin-Sepharose for 2 hours at 4°C. The gel was washed with 50 ml buffer A [20 mmol/L Tris-HC1, 5 mmol/L EDTA, 2 mmol/L EGTA (pH 7.4)] and then with buffer A containing 0.5 mol/L NaCl. The recombinant 24-kd FGF was eluted with buffer A containing 3 mol/L NaCl. The eluate was dialyzed against 4 liters of 20 mmol/L Tris, 145 mmol/L NaCl (pH 7.4).
ATE + 31 was prepared by inserting SfiI restriction sites at the 5'end of the 24-kd FGF-2 cDNA and the 3' end of the sequence generating the required truncation of 24-kd FGF-2. The cDNA fragment was amplified by PCR and inserted into a modified pET15b vector carrying an N-terminal pelB signal sequence for periplasmic localization of the recombinant protein plus a C-terminal His-Tag sequence. The 5' primer was 5'-ATATATGGCCCAGC CGGCCATGGCACTGGGGGACCGCGGGCGCGG-3' (SfiI restriction site underlined) and the 3'-primer was 3'-ATATATGGCCCCCGAGGCCCCGCCTTGGGGTCCTTGAAGTGG-5'. The amplified fragments were cut with SfiI and ligated to the vector. The plasmids were used to transform BL-21-CodonPlus (DE-3)-RP competent cells. The cells were cultured in LB medium containing 0.4 mmol/L isopropyl-ß-D-thiogalacto pyranoside (IPTG) for 4 hours at 30°C. When the OD600 value of the culture reached 0.6 to 0.8, the cells were harvested and suspended in 50 mmol/L phosphate-buffered saline (PBS), 10% glycerol, 300 mmol/L NaCl, 5 mmol/L 2-mercaptoethanol; pH 8.0 (buffer B). The cells were lysed by freeze-thaw five times and the particulate matter removed by centrifugation and filtration through a 0.45-µm syringe filter. The filtrate was added to 5 ml of nickel-NTA agarose and the mixture shaken for 2 hours at 4°C. The resin was packed into a column and washed with buffer B containing 10 mmol/L imidazole, followed by the same buffer containing 50 mmol/L imidazole and the protein was finally eluted with buffer containing 400 mmol/L imidazole. The protein was dialyzed against 50 mmol/L sodium phosphate (pH 7.6) and analyzed by 10% SDS-PAGE followed by silver staining and Western blot analysis using antibodies generated against a 12-amino acid peptide found within the ATE domain of 24-kd FGF-2.13
Cell Culture and Migration Assay
MCF-7 breast carcinoma cells or Lewis lung carcinoma (LLC) cells were maintained in Dulbecco’s minimal essential media (DMEM) supplemented with 1 mmol/L sodium pyruvate, 10% fetal calf serum, and 1X penicillin/streptomycin at 37°C under 5% CO2 in humidified incubators. The cells were subcultured to densities of one-third to one-sixth that of confluent cultures.
For migration assays, MCF-7 cells were harvested with trypsin, counted, centrifuged, and resuspended at a cell density of 1 x 105 cells in 0.5 ml DMEM containing 0.5% bovine serum albumin (BSA). Cells were added to the upper well of a 6.5-mm Transwell microporous filter chamber (Fisher Scientific, Tustin, CA) containing an 8.0-µm pore size polycarbonate membrane, which separates the upper well from the lower well of the Transwell chamber. The filters were not coated with protein before use. The upper wells were placed into the lower wells containing 0.75 ml of DMEM with 0.5% BSA to which 10 ng/ml of IGF-1 (Sigma Chemical Co., St. Louis, MO) or VEGF (Sigma) was added as a chemoattractant. 24-kd FGF-2 or truncated forms of the protein was added to both wells of each chamber at the appropriate concentration. After 4 to 6 hours of incubation at 37°C in 5% CO2, non-migratory cells on the upper membrane surface were removed with a cotton swab and the cells that traversed and spread on the lower surface of the filter were fixed and stained with Diff-Quik (Dade-Behring, Deerfield, IL). The filter was mounted on a glass slide, and four phase-contrast photomicrographs/membrane were taken at a magnification of x100. The number of cells per field was counted from contact sheets and the results compared with control chambers, which had no 24-kd FGF-2 added.
Chemotaxis of eosinophils and neutrophils was performed using the same system except that the upper well of the Transwell chamber contained a polyethylene terephthalate filter of 3.0-µm pore size, which separated it from the lower chamber. The membrane was coated with Matrigel. For each determination, neutrophils and eosinophils labeled with (5(and-6)-(((4-chloro-methyl)benzoyl)amino)tetramethylrhodamine (CMTMR; Molecular Probes, Eugene, OR) were placed in the upper wells (105 labeled cells/well) of the chamber in the presence or absence of 1 x 10-7 M ATE + 31 or 10 µg/ml IB4. IB4, an antibody against ß2 integrin, was used as a positive control to demonstrate inhibition of migration. The lower well contained 1 mg/ml human serum albumin in DMEM with 1 x 10-7 M C5a. The chambers were incubated at 37°C for 2 hours. EDTA was added to a final concentration of 10 mmol/L to the bottom well, and the plates were allowed to stand for 10 minutes at room temperature before the inserts were removed from the wells. The cells, which had passed through the membrane and which were contained within a 1.1-mm2 central area on the bottom well, were counted using an inverted fluorescence microscope (Leitz Fluovert FS, Wetzlar, Germany).
To measure growth rates, MCF-7 cells (6 x 103) were plated in growth medium for 48 hours after which the medium was changed to assay medium containing phenol red-free modified Eagle’s medium (MEM) supplemented with 1 mmol/L sodium pyruvate and 0.3% lactalbumin hydrolysate with or without growth factors and the cultures were incubated for an additional 24 hours. Two hours before the termination of the experiment, 3H-thymidine was added. The cultures were washed two times with PBS followed by ice-cold methanol and 5% trichloroacetic acid (two times for 10 minutes each), and the DNA was finally extracted with 0.3 N NaOH. The number of counts per minute (cpm) incorporated was determined by liquid scintillation.
Preparation of Tumor Spheroids
Multicellular aggregates or tumor spheroids were prepared by suspending cells at a concentration of 5 x 106 cells/ml in the appropriate growth medium in a 50-ml flask and the suspension rocked on a gyratory shaker in a humidified incubator at 37°C in an atmosphere of 5% CO2 and 95% room air. Solid tumor spheroids are formed after 24 to 48 hours of shaking. Tumor spheroids with similar diameters (600 to 1000 µm) for implantation in the dorsal skin-fold chamber were selected by visualizing the spheroids under a microscope and individually picking the spheroids with a sterile pipette. This procedure insures single spheroids of similar size for implantation in the chamber. To facilitate visualization of the implanted tumors, spheroids were pre-labeled with CMTMR before transplantation into the skin-fold chamber.25
Dorsal Skin-Fold Chamber Model
To prepare the skin-fold chamber in mice, one layer of the dorsal skin was removed surgically and a pair of titanium frames were implanted into the dorsal skin-fold parallel to the dorsum creating a sandwich of the stretched layer of skin. The underlying thin layer of skin (M. cutaneous max.), subcutaneous tissue, and epidermis was covered with a coverslip enclosed in one of the frames and the animals allowed to recover from anesthesia.24-26 After an additional convalescence period of 2 to 3 days, the coverslip was removed and tumor spheroids were placed over the upper tissue layer of the chamber. The chamber was enclosed with the coverslip and the tumor spheroids were monitored over a 2-week period.
Evaluation of Tumor Development
The progress of tumor development and vascularization were followed by intravital microscopy. Unanesthetized mice containing the tumor spheroids were placed individually in a crouched position in a plexiglass tube and then placed on a microscope stage (Leitz Biomed) for observation of the tumor spheroids within the chamber. On the day of spheroid implantation, an overview video print was taken and the position of the implanted spheroids marked to facilitate future localization and for monitoring growth of the tumor. The CMTMR-labeled tumor spheroids and angiogenic blood vessels were observed by transillumination or stroboscopic epi-illumination using a video-triggered Xenon arc and a Leitz Ploemopak epi-illuminator. Tumor size was determined by measuring the diameter of the tumors at each time point and tumor volume was calculated according to the formula V = 2/3AH where A is the tumor area and H the thickness of the tumor. At the end of each experiment, the tissue containing the tumors was excised, fixed in 10% formalin, and prepared for sectioning by freezing in OCT. Ten-µm sections of the surrounding skin were also prepared and stained with hematoxylin-eosin (HE).
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Results |
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ATE + 31 was generated by deletion mutagenesis through the placement of a stop codon within the 24-kd FGF-2 cDNA after amino acid 86 (Figure 1A)
. Figure 1B
shows the effect of ATE + 31 on MCF-7 cell proliferation and migration compared to the full-length 24-kd FGF-2. At the optimum dose of 3.3 x 10-10 M, migration rates are reduced by 80% which is equal to the maximum inhibition by the full length 24-kd FGF-2.14
However, in contrast to the increase in proliferation induced by 24-kd FGF-2, ATE + 31 has no effect on the rate of cell growth when compared to untreated MCF-7 cells (Figure 1B)
14
. To determine whether ATE + 31 is effective in limiting tumor angiogenesis in vivo, MCF-7 cell spheriods (microtumors) were implanted into skin-fold chambers in mice and treated by superfusion with 20 ng ATE + 31 (or saline control) at 24 hours and every 2 days after. The infiltration of new blood vessels into the spheroid was observed by video microscopy. By day 5 the density of the neovasculature within the area of the spheroid exposed to ATE + 31 (Figure 2B)
was significantly less than that of the saline-treated spheroids (Figure 2A)
. The integrity of the growing vessels was also affected by the presence of the peptide (Figure 2, C and D)
. The vasculature in ATE-31-treated tumors contained fragmented vessels (arrowheads), an effect we attribute to the formation of poorly differentiated blood vessels (arrows, Figure 2D
). Differences in vascular density were further magnified by day 15. The vascular plexus in the untreated animals was extremely dense filling up most of the space within the spheroid (Figure 2E)
, whereas the treated animals, while showing a small increase in the number of blood vessels compared to that observed on day 5, had few blood vessels with large diameters such as those found in control animals (Figure 2F)
.
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. A dramatic decrease in tumor size was observed in the treated animals with the average size of the treated tumors 7.8% that of the control spheroids (1.3 mm2
vs. 16.8 mm2, n = 3) after 15 days (Figure 3, A versus B
). Blood vessels infiltrating the interior of the untreated tumor are visible in contrast to the treated tumor, where almost none are discernable (Figure 3, C and D
, respectively). Variation in final tumor size does occur and some animals were found to have no tumor at all by day 15 indicating that tumor maintenance is also compromised by the peptide, either by directly affecting tumor cell migration or through the inhibition of blood vessel formation.
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. Treatment with ATE + 31 at 20 ng as described earlier did not result in significant suppression of the tumor growth. However, when the concentration of ATE + 31 was increased to 100 ng, a dramatic effect on growth was observed. Over the first 7 days, untreated tumor spheroids grew in size ninefold (0.26 ± 0.05 mm3
to 2.5 ± 0.6 mm3), while the treated spheroids showed only a threefold increase (to 0.9 ± 0.3 mm3). After day 7, there was a further increase in the rate of tumor expansion in untreated animals with a final tumor volume 28 times larger than that of the original spheroid (8.5 ± 3.9 mm3) (Figure 4A)
. In contrast, significant suppression (81%) of tumor growth was observed in the ATE + 31-treated animals (1.5 ± 0.2 mm3) compared to the untreated tumor. Thus, ATE + 31 was most effective during the period of rapid tumor growth. This effect was also reflected in the vascularization of the tumor (Figure 4, B to E)
. At 6 days after implantation, a dense network of angiogenic blood vessels was seen in saline-treated control animals (Figure 4, B and D)
, while the area containing newly formed blood vessels in ATE + 31-treated animals was small, diffuse, and poorly developed (Figure 4, C and E)
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. After 48 hours, the spheroid in control animals spreads into a much larger and very diffuse structure without an inner core (Figure 5B)
and was found to increase in size by threefold (19.8 ± 2.4 vs. 6.9 ± 1.6 mm2, Figure 5D
). However, treatment with ATE + 31 mitigates this expansion and the spheroid body remains intact (Figure 5C)
with only a small halo of cells around it after 48 hours (6.1 ± 0.5 vs. 6.9 ± 1.6 mm2, Figure 5D
). Thus, suppression of tumor growth can occur even in the absence of a vascular system indicating that ATE + 31 is more than an anti-angiogenic molecule and functions via inhibition of tumor cell migration.
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While the inhibition of migration of endothelial and tumor cells is an advantage for suppressing tumor growth, its effect on other physiological processes such as leukocyte trafficking would be detrimental to the immune system. To determine whether leukocyte motility is affected by ATE + 31, in vitro chemotaxis experiments were performed with peripheral blood human eosinophils and polymorphonuclear neutrophils (Figure 6)
. In the presence of C5a,27
the number of migrating neutrophils increased from 6.5 ± 1.5% to 11.6 ± 2.0% and this did not change when ATE + 31 was added (12.0 ± 3.0%) (Figure 6A)
. IB4, a function blocking monoclonal antibody against CD18, reduced neutrophil migration to 3.8 ± 0.9%. Similar results were observed with eosinophils, where the number of migrating cells increased from 18.8 ± 1.0% to 28.4 ± 1.7% in the presence of C5a. While treatment with IB4 resulted in complete inhibition of chemotaxis (17.5 ± 1.5%), the presence of ATE + 31 failed to alter the C5a-induced migration of eosinophils (28.8 ± 3.0%) (Figure 6B)
.
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Discussion |
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In this study we analyzed the effect of ATE + 31 on LLC and MCF-7 tumor growth and angiogenesis using the dorsal skin-fold chamber model. This model allows us to monitor vascular development as well as the growth rate of a single well-defined microtumor over a period of time. We demonstrated that ATE + 31 is effective not only at suppressing tumor-induced angiogenesis, but tumor growth itself in the absence of a vascular network. When LLC cells were used, the final tumor volume after 10 days was less than 20% of saline-treated tumors (80% inhibition) while with MCF-7 cells the tumor was only 8% of saline-treated control tumors (>90% inhibition). These results support our hypothesis that inhibition of migration can lead to suppression of tumor growth involving very different types of cells in vivo. Equally important is the observation that suppression of tumor growth is not merely a consequence of inhibition of angiogenesis but is also due to an effect on the tumor cells themselves. This was indicated by the suppression of microtumor expansion even before the infiltration of blood vessels. Since we have shown that ATE + 31 does not affect proliferation,14
we interpret the differences in the sizes of the tumors and the diminished vascularization as a consequence of inhibition of migration. Suppressing tumor development through the inhibition of migration must address the possibility that other physiological processes that depend on cell motility would be compromised. One of the most crucial is the ability of blood cells to transmigrate into the vessel wall in response to inflammatory events. The results shown in Figure 6
demonstrate that ATE + 31 has no effect on the transmigration of either neutrophils or eosinophils in an in vitro chemotaxis assay. These experiments were performed under similar conditions as those using endothelial cells and MCF-7 cells so that the only variable was the cells themselves. Thus, despite the apparent widespread effect of ATE + 31 on endothelial cells and tumor cells, the mechanism by which it inhibits these cells is not applicable to leukocytes, a property that serves as an advantage for therapeutic use.
The mechanism through which ATE + 31 inhibits migration has not been elucidated although competition binding studies with the 24-kd FGF-2 and 18-kd FGF-2 indicate that it occurs independently of the FGF receptor14 and that the truncated protein does not contain a site that interferes with the binding of the full-length 24-kd FGF-2 to its receptor. ATE + 31 does bind MCF-7 cells in a specific and saturable manner suggesting that it has its own receptor/binding site distinct from the FGF receptor (unpublished observation). Since the inhibition of migration by ATE + 31 occurs in the presence of three unrelated growth factors, it is very likely that this protein affects the cells through a common point downstream of ligand-receptor interaction although neither extracellular signal-related kinase (ERK) nor AKT phosphorylation are affected by treatment with this protein.14 This still leaves a multitude of events that affect the mechanics of movement, eg, by altering cytoskeletal dynamics or adhesion. In fact, preliminary studies of the adhesive properties of treated versus untreated MCF-7 cells show an increase in adhesion to fibronectin, collagen, and laminin. For cells to migrate, they must adhere to and release from the substratum in a coordinated and polarized fashion. This process involves formation of filopodia and lamellipodia at the leading edge, formation of new adhesions in this region, and detachment of adhesions at the trailing edge of the cell.30,31 The inability to release these contacts at the required rate would compromise the motility of the cells leading to suppression of vascular formation or tumor growth. These studies demonstrate the pronounced effect that inhibition of migration can have on the growth of tumors and blood vessels.
In summary, this study demonstrates that ATE + 31 effectively inhibits both growth and neovascularization of various types of tumors potentially through the inhibition of migration of tumor cells. In contrast, ATE + 31 fails to affect the transmigration of neutrophils and eosinophils, indicating that this molecule specifically acts against tumor cells but not inflammatory leukocytes. These results suggest that ATE + 31 has the potential to function as an anti-tumor agent with multiple targets.
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Footnotes |
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Supported in part by funding from Breast Cancer Research Program grant 4JB-0164 and NIH 1RO1AI35796 (to P.S.) and NIH 1RO1CA81209 (to E.G.L.).
Accepted for publication December 9, 2003.
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References |
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-mediated apoptosis requires Ras and the activation of mitogen-activated protein kinase. J Biol Chem 1996, 271:14560-14566This article has been cited by other articles:
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  S. Chigurupati, T. Kulkarni, S. Thomas, and G. Shah Calcitonin Stimulates Multiple Stages of Angiogenesis by Directly Acting on Endothelial Cells Cancer Res., September 15, 2005; 65(18): 8519 - 8529. [Abstract] [Full Text] [PDF]
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) or 100 ng of ATE + 31 (
) every 48 hours for 10 days. At the days indicated the tumors were video recorded and tumor volume measured by offline analysis. Volume of treated tumors on day 10 was less than 20% of that of untreated tumors. Results represent the average ± SD of at least five tumors each from different mice. B–E: Angiogenic response of LLC tumors. Six days after implantation, vascular development in control (B and D) and ATE + 31-treated (C and E) tumors was analyzed by video microscopy at lower (B and C, x40) and higher (D and E, x100) magnification. The area of vasculature is indicative of the difference in size between the two tumors. The circle in C shows the area analyzed in E.
