c-Met, a receptor tyrosine kinase responsible for cellular migration, invasion, and proliferation, is overexpressed in human cancers. Although ligand-independent c-Met activation has been described, the majority of tumors are ligand dependent and rely on binding of hepatocyte growth factor (HGF) for receptor activation. Both receptor and ligand are attractive therapeutic targets; however, preclinical models are limited because murine HGF does not activate human c-Met. The goal of this study was to develop a xenograft model in which human HGF (hHGF) is produced in a controllable fashion in the mouse. Severe combined immunodeficient mice were treated with adenovirus encoding the hHGF transgene (Ad-hHGF) via tail vein injection, and transgene expression was determined by the presence of hHGF mRNA in mouse tissue and hHGF in serum. Ad-hHGF administration to severe combined immunodeficient mice resulted in hHGF production that was (a) dependent on quantity of virus delivered; (b) biologically active, resulting in liver hypertrophy; and (c) sustainable over 40 days. In this model, the ligand-dependent human tumor cell line SW1417 showed enhanced tumor growth, whereas the ligand-independent cell lines SW480 and GTL-16 showed no augmented tumor growth. This novel xenograft model is ideal for investigating c-Met/HGF–dependent human tumor progression and for evaluating c-Met targeted therapy. [Mol Cancer Ther 2007;6(4):1460–6]

The c-Met proto-oncogene encodes a transmembrane receptor tyrosine kinase that is responsible for cellular motility, invasion, and proliferation in embryogenesis (1, 2) as well as tumor progression and metastases (28). As with other receptor tyrosine kinases, binding of ligand hepatocyte growth factor (HGF) to c-Met results in receptor dimerization, autophosphorylation, and activation of downstream signaling cascades. In normal physiology and fetal development, HGF functions in a paracrine fashion. In human cancer, a variety of abnormal mechanisms of c-Met activation have been described, including autocrine stimulation [astrocytoma and select gastric cell lines (9, 10)] and ligand-independent activation related to gene rearrangement or mutation (2, 5, 8, 1117). However, the majority of human solid tumors seem to have receptor overexpression, without mutation, and are ligand dependent (8).

Based on the high prevalence of dysregulation noted in human tumors and its association with advanced disease, c-Met and HGF are being actively developed as therapeutic targets (3, 8). There are several methods for abrogating receptor tyrosine kinase activation: neutralization of ligand, direct inhibition of receptor, and blockade of mediators of downstream signaling pathways (8). Many of these approaches have been used to block c-Met, including HGF variants (NK1, NK2, and NK4; ref. 1820), antibodies directed against HGF (9, 21), antibodies directed against c-Met (22), and small-molecule c-Met kinase inhibitors (23).

A major obstacle in the development and implementation of therapeutic c-Met and HGF antagonists is the lack of preclinical models for testing potential agents. Xenograft models are inadequate when studying c-Met because murine HGF does not activate the human receptor (6). Multiple investigators have attempted to overcome this species specificity by using human tumors engineered to express both HGF and c-Met in an autocrine manner (6, 21, 2426) or by using human tumor transfected with constitutively activated c-Met (TPR-Met oncogene or activating mutations; refs. 15, 27). These model systems are limited, however, because the majority of human tumors do not contain gene rearrangements or activating mutations and show ligand-dependent activation in a paracrine fashion (28). Therefore, a model in which human HGF (hHGF) is present is necessary to examine the role of c-Met in human cancer.

Our goal was to develop a preclinical model for studying c-Met–dependent tumor progression, one that could easily be applied to any system and would not require breeding or maintenance of transgenic mice. We accomplished this by using an adenoviral vector containing a transgene for hHGF, which had previously been developed to study liver regeneration (28). By injecting the adenovirus into severe combined immunodeficient (SCID) mice, we developed a novel xenograft model in which hHGF is produced in controllable quantities and for a prolonged period. Using this model, we have shown that ligand-dependent tumors have augmented growth in mice producing hHGF. We believe that this model is ideal for investigating c-Met/HGF–dependent tumor progression and is well suited for preclinical evaluation of c-Met and HGF inhibitors.

Adenoviral Construct Propagation

A recombinant adenoviral vector encoding the hHGF transgene (Ad-hHGF) was kindly provided by Dr. J.M. Wilson and the Vector Core, Gene Therapy Program (Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA); this has been extensively studied in models of liver regeneration (28). A control adenoviral construct containing the alkaline phosphatase transgene was used in control experiments (29). These replication-deficient virus constructs were propagated as described previously (30). In summary, 293-H fibroblasts (American Type Culture Collection, Rockville, MD) were exposed to adenoviral constructs for 30 to 36 h, after which virus was extracted by freeze/thaw and then purified by a cesium chloride density gradient [2.5 mol/L cesium chloride overlaid 3.6 mol/L cesium chloride in 10 mmol/L Tris-HCl (pH 8.0)] followed by elution with a Sephadex column (Sigma, St. Louis, MO). Concentration was determined by UV spectrum at A260 nm, and virus was stored in a 10% glycerol solution using PBS at −80°C.

Generation of Mice That Produce hHGF

Five-week-old SCID mice (Taconic Farms, Germantown, NY) were injected with increasing concentrations of Ad-hHGF diluted in 100 μL of normal saline via tail vein using 28-gauge needles. Animals were sacrificed at sequential time points and underwent necropsy with organ harvest and blood collection. Serum was decanted following blood centrifugation at 14,000 rpm for 10 min and stored at −20°C for batch HGF determination. Liver, lung, and spleen were collected, weighed, and frozen for batch transgene expression by real-time reverse transcription-PCR.

Assessment of hHGF Transgene Expression in SCID Mice

hHGF transgene expression was measured in the mouse by detection of hHGF in serum, the presence of liver hypertrophy, and the presence of hHGF mRNA in tissue. Serum HGF measurement was determined by ELISA with Quantikine HGF Immunoassay (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. Liver hypertrophy indicates that a function protein is produced, as hHGF does activate mouse c-Met receptor (6). Liver hypertrophy was determined by normalizing liver mass to animal mass (liver/body weight = liver weight ratio). Transgene expression in liver, lung, and spleen was analyzed by quantitative real-time reverse transcription-PCR (31) with FAM dye-labeled Taqman MGM probe using the ABI PRISM 7700 (Applied Biosystems, Foster City, CA). Total RNA from mouse liver, spleen, and lung was isolated using RNeasy Mini kits (Qiagen, Santa Clarita, CA) following the manufacturer's instructions. For cDNA synthesis, ∼1 μg of total RNA was reverse transcribed with cDNA Transcription Reagents (Applied Biosystems) using random hexamers. hHGF and 18S rRNA sequence-specific primers and probes were obtained as Assay-on-Demand Gene Expression Products (Applied Biosystems). Real-time reverse transcription-PCR of cDNA specimens and standards was conducted in a total volume of 20 μL with 2× Taqman Master Mix (Applied Biosystems) according to the manufacturer's instructions. Thermal cycler variables included 2 min at 50°C, 10 min at 95°C, and 40 cycles involving denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. Relative standard curves were generated for hHGF and 18S rRNA using serial dilutions of cDNA obtained from high HGF-expressing liver samples as previously reported (31). All samples were analyzed in triplicate, and levels of mRNA expression are presented as the mean ± SE.

Cell Lines and Culture Conditions

The colon carcinoma cell lines SW1417 and SW480 were purchased from the American Type Culture Collection. SW1417 was derived from a Duke's C (American Joint Committee on Cancer stage III), grade III colon carcinoma. SW480 was derived from a Duke's B (American Joint Committee on Cancer stage II), grade II primary adenocarcinoma of the colon. The cells were cultured in Leibovitz's L-15 with 2 mmol/L l-glutamine and 10% FCS at 37°C. The GTL-16 gastric carcinoma cell line was supplied by Dr. Silvia Giordano (Institute for Cancer Research and Treatment, University of Torino School of Medicine, Candiolo, Italy) and stored in DMEM with high glucose and 4 mmol/L l-glutamine + 10% FCS at 37°C in 5% CO2. All human tumor cell lines were harvested and subcultured by brief treatment with 0.25% trypsin-0.02% EDTA and scraping.

Immunoblot Analysis of Ligand-Dependent SW1417 Cell Line Activation

Cells were plated in L-15 medium supplemented with 10% fetal bovine serum. At subconfluence, cells were serum starved for 18 h followed by treatment with or without HGF (100 ng/mL) for 10 min before lysis [1× radioimmunoprecipitation assay lysis buffer (Upstate, Lake Placid, NY), 1:100 Protease Inhibitor Cocktail Set I (Calbiochem, La Jolla, CA), and 1:100 Protease Inhibitor Cocktail Set II (Calbiochem)]. Cell lysate (25 μg/well) was loaded and separated by SDS-polyacrylamide electrophoresis [10% NuPAGE Novex Bis-Tris Gels (Invitrogen, Carlsbad, CA)]. The proteins were transferred to polyvinylidene difluoride membrane, and nonspecific binding was blocked with 5% dried milk in TBS for 1 h. The primary antibody used to probe the membrane was an unconjugated anti-c-Met (pYpYpY1230/1234/1235) phosphospecific antibody (Biosource, Camarillo, CA) diluted in TBS containing 5% dried milk. The secondary antibody was a peroxidase-conjugated donkey anti-rabbit IgG (Amersham, Piscataway, NJ) diluted in TBS-0.05% Tween 20 (v/v). Immunoreactive proteins were then visualized with an enhanced chemiluminescent detection system, ECL+ (Amersham).

Cellular Proliferation Assay

Cellular proliferation was measured using chemiluminescent bromodeoxyuridine ELISA assay (Roche Applied Biosciences, Indianapolis, IN) as directed by the manufacturer. In summary, SW1417, SW480, and GTL-16 cell lines were seeded in 96-well plates at various concentrations (5 × 103 to 1 × 104 per well), dependent on the particular cell line, in medium containing 10% fetal bovine serum for 12 to 24 h. Cells were then incubated with 0.5% fetal bovine serum and medium for 48 h before they were treated with increasing concentration of HGF (0–100 ng/mL) for 18 h in 2% FCS at 37°C. Solutions were removed, wells were incubated with 100 μmol/L bromodeoxyuridine labeling reagent for 2 h, and then cells were fixed and DNA was denatured with 100 μL FixDenat solution for 45 min. Next, 5% dry milk in 100 μL PBS was added to each well for 30 min to block nonspecific antibody binding. Cells were then washed once with PBS, exposed to anti-bromodeoxyuridine peroxidase–conjugated antibody (7.4 × 10−5 units/well) for 90 min, and then repeatedly washed with PBS. Immune complexes were detected colorimetrically by addition of 100 μL of substrate reaction at 630 to 450 nm. Each data point represents the mean ± SE of 10 wells.

Xenograft Tumor Growth in SCID Mice Expressing hHGF

SCID mice were injected with 1 × 1011 particles Ad-hHGF. Control mice were injected with no viral construct or the control construct Ad-AlkPhos via tail vein. At least 2 h after injection, the mice were shaven, and 1 × 107 human tumor cells suspended in 100 μL of saline were implanted s.c. in the right flank with a 28-gauge needle. Tumors were measured within the first week after injection and biweekly thereafter. Tumor nodules were measured in a cephalad-caudal and transverse fashion, and volume was estimated according to the formula V = w2 × l × 0.523, where w represents the shorter diameter and l represents the longer diameter of the tumor mass (32). Animals were euthanized when they showed signs of pain and suffering, tumor ulceration, or tumor growth >10% of body weight (usually representing 2 cm in diameter).

Statistics

Continuous data are presented as mean ± SE and analyzed with either Student's t test or ANOVA where appropriate. The Bonferroni correction was used for multiple comparisons. Correlations were measured with Pearson correlation coefficient. Significance was taken at the P < 0.05 level.

Generation of hHGF-Producing SCID Mice

I.v. injection of Ad-hHGF into SCID mice resulted in production of hHGF in a dose-dependent fashion as measured by serum ELISA (r = 0.950; P < 0.01; Fig. 1). The ELISA proved to be specific for hHGF, as control mice had undetectable levels of hHGF in the serum levels, whereas SCID mice treated with the Ad-hHGF transgene expressed levels ranging from 450 to 4,500 pg/mL (Fig. 1). To determine whether the expressed hHGF protein was biologically functional, liver mass was measured and normalized to total body weight (liver hypertrophy). I.v. administration of Ad-hHGF was associated with significant liver hypertrophy in a dose-dependent manner (r = 0.741; P < 0.01; Fig. 2). In time course experiments, transgene expression was shown to persist at least for 40 days with only 15% diminution in serum HGF levels (Fig. 3). Analysis of mRNA from liver, lung, and spleen indicates that transgene expression was only detected at significant levels in mouse liver (Table 1). In additional control experiments, flank tumors were resected at 20 days following implantation and treatment with Ad-hHGF. Analysis revealed that no tumors (n = 5) expressed the hHGF transgene, indicating that adenoviral infection was complete before xenograft implantation (data not shown).

Figure 1.

Serum hHGF expression levels in SCID mice 5 d following i.v. injection with Ad-hHGF. Quantity of injected Ad-hHGF into mice is associated with a dose-dependent increase in serum hHGF levels.

Figure 1.

Serum hHGF expression levels in SCID mice 5 d following i.v. injection with Ad-hHGF. Quantity of injected Ad-hHGF into mice is associated with a dose-dependent increase in serum hHGF levels.

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Figure 2.

Ad-hHGF produced is biologically active. Increased serum hHGF levels are associated with liver hypertrophy in a dose-dependent manner. Mice were analyzed 1 wk after i.v. Ad-hHGF injection.

Figure 2.

Ad-hHGF produced is biologically active. Increased serum hHGF levels are associated with liver hypertrophy in a dose-dependent manner. Mice were analyzed 1 wk after i.v. Ad-hHGF injection.

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Figure 3.

Transgene expression as measured by serum hHGF levels is sustained over time. Mice were injected i.v. with 2 × 1010 particles Ad-hHGF, and serum hHGF was measured over time. Points, mean of at least five animals per time point; bars, SE.

Figure 3.

Transgene expression as measured by serum hHGF levels is sustained over time. Mice were injected i.v. with 2 × 1010 particles Ad-hHGF, and serum hHGF was measured over time. Points, mean of at least five animals per time point; bars, SE.

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Table 1.

hHGF transgene expression as measured by reverse transcription-PCR in mice i.v. injected with or without Ad-hHGF

OrganNo Ad-hHGF*Ad-hHGF (1 × 1011 particles)P
Liver 0.1 ± 0.01 453 ± 53 0.001 
Lung 0.2 ± 0.01 3.2 ± 1.7 0.13 
Spleen 2.6 ± 0.08 2.2 ± 0.8 0.83 
OrganNo Ad-hHGF*Ad-hHGF (1 × 1011 particles)P
Liver 0.1 ± 0.01 453 ± 53 0.001 
Lung 0.2 ± 0.01 3.2 ± 1.7 0.13 
Spleen 2.6 ± 0.08 2.2 ± 0.8 0.83 

NOTE: Values represent mean ± SE.

*

Mice not injected with Ad-hHGF.

Mice injected with Ad-hHGF.

Tumor Cell Line Ligand Dependence

In vitro exposure of SW1417, SW480, and GTL-16 to increasing concentration of HGF showed that only the SW1417 cell line responded with augmented proliferation (Fig. 4). This is consistent with immunoblot analysis showing c-Met phosphorylation following exposure of SW1417 to HGF (Fig. 5). These data indicate that the SW1417 cell line is ligand dependent, whereas GTL-16, with high c-Met expression, is ligand independent. Previous reports concur with these findings and have shown that GTL-16 has c-Met amplification, which is associated with constitutive phosphorylation (23, 33). Further coculture of SW1417 with HGF has been shown to result in cellular migration and invasion (33). Not surprisingly, SW480, which has been shown to have low c-Met expression (31, 34), did not respond to HGF.

Figure 4.

Proliferation in cell lines following coculture with increasing concentrations of HGF. SW1417 shows increased proliferation following stimulation with increasing concentrations of HGF. GTL-16 and SW480 show no augmented proliferation in response to HGF. Points, mean of 10 wells; bars, SE. For the SW1417 cell line, P = 0.064 for 10 ng/mL HGF and P < 0.001 for 50 ng/mL compared with control with no HGF.

Figure 4.

Proliferation in cell lines following coculture with increasing concentrations of HGF. SW1417 shows increased proliferation following stimulation with increasing concentrations of HGF. GTL-16 and SW480 show no augmented proliferation in response to HGF. Points, mean of 10 wells; bars, SE. For the SW1417 cell line, P = 0.064 for 10 ng/mL HGF and P < 0.001 for 50 ng/mL compared with control with no HGF.

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Figure 5.

c-Met is phosphorylated in response to HGF stimulation in the human colorectal cancer cell line SW1417. Coculture of SW1417 cells with HGF after serum starvation resulted in c-Met phosphorylation as shown by immunoblot analysis.

Figure 5.

c-Met is phosphorylated in response to HGF stimulation in the human colorectal cancer cell line SW1417. Coculture of SW1417 cells with HGF after serum starvation resulted in c-Met phosphorylation as shown by immunoblot analysis.

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Enhanced Human Xenograft Growth in Mice Expressing hHGF

S.c. SW1417 tumor implants grew larger in SCID mice expressing hHGF compared with control (P < 0.01, t test; Fig. 6A). Significant differences in tumor size were noted at 14 days after implantation. As expected, the ligand-independent GTL-16 cell line showed comparable tumor growth in mice with and without hHGF (Fig. 6B). Due to the rapid growth of GTL-16 xenografts, tumor ulceration generally occurred at early time points, necessitating euthanasia. Similarly, SW480, which has low c-Met expression (33), did not show augmented growth when s.c. implanted in mice expressing hHGF (Fig. 6C).

Figure 6.

Growth of HGF-dependent s.c. tumor implants in SCID mice expressing hHGF is augmented in SCID mice treated with Ad-hHGF. Equal volume suspensions of tumor cell lines cells were s.c. implanted into the flanks of SCID mice that had been i.v. injected with or without 1 × 1011 particles of Ad-hHGF. In each panel, mean tumor volumes and SE are expressed. A, SW1417 colorectal cancer cell line (high c-Met expression and ligand dependent) shows augmented growth in hHGF-expressing mice with significance seen at 14 d. B, GTL-16 gastric cell line (high c-Met expression and ligand independent) shows no augmented growth in hHGF-expressing mice. Experiments with GTL-16 were terminated early due to rapid tumor growth and development of ulceration characteristic for this cell line. C, SW480 colorectal cancer cell line (low c-Met expression) shows no augmented growth in hHGF-expressing mice.

Figure 6.

Growth of HGF-dependent s.c. tumor implants in SCID mice expressing hHGF is augmented in SCID mice treated with Ad-hHGF. Equal volume suspensions of tumor cell lines cells were s.c. implanted into the flanks of SCID mice that had been i.v. injected with or without 1 × 1011 particles of Ad-hHGF. In each panel, mean tumor volumes and SE are expressed. A, SW1417 colorectal cancer cell line (high c-Met expression and ligand dependent) shows augmented growth in hHGF-expressing mice with significance seen at 14 d. B, GTL-16 gastric cell line (high c-Met expression and ligand independent) shows no augmented growth in hHGF-expressing mice. Experiments with GTL-16 were terminated early due to rapid tumor growth and development of ulceration characteristic for this cell line. C, SW480 colorectal cancer cell line (low c-Met expression) shows no augmented growth in hHGF-expressing mice.

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To control for possible nonspecific effects of adenoviral infection, repeat experiments were done with s.c. implants of SW1417 in SCID mice treated with no adenoviral construct, Ad-hHGF, or the control construct Ad-AlkPhos. Ad-AlkPhos produces the alkaline phosphatase transgene (29). Animals treated with no adenoviral construct or the control construct Ad-AlkPhos showed similar tumor growth, which was lower than that seen in mice treated with Ad-hHGF (Fig. 7). These data provide evidence that the augmented tumor growth seen in SW1417 is dependent on the hHGF transgene.

Figure 7.

S.c. implantation of SW1417 shows augmented growth in SCID mice treated with Ad-hHGF but not in control SCID mice treated with adenoviral construct (Ad-AlkPhos). Values represent percentage growth compared with SCID animals treated with no adenoviral construct.

Figure 7.

S.c. implantation of SW1417 shows augmented growth in SCID mice treated with Ad-hHGF but not in control SCID mice treated with adenoviral construct (Ad-AlkPhos). Values represent percentage growth compared with SCID animals treated with no adenoviral construct.

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There is growing evidence that the c-Met pathway is critical for tumor progression and metastasis (28). Although ligand-independent c-Met activation has been noted in select tumors related to mutation, gene rearrangement, or amplification, the majority of solid tumors seem to be responsive to ligand (8, 33). Therefore, to study c-Met tumor progression and metastases in human tumors, a system is necessary in which hHGF is present. We have successfully developed a novel xenograft model in which biologically active hHGF is produced in SCID mice. hHGF expression level is reliably controlled by the amount of i.v. Ad-hHGF injected. Transgene expression is sustainable, and a functional protein is produced as indicated by liver hypertrophy. Using this model system, we are able to show augmented growth of the c-Met–dependent, ligand-dependent human colorectal cancer cell line SW1417. As would be expected, the low c-Met–expressing cell line SW480 and ligand-independent cell line GTL-16 do not show enhanced growth in mice producing hHGF. We propose this as an ideal model for studying the HGF/c-Met activation pathway in human tumors and consider it potentially useful for preclinical studies of biological therapies.

Other model systems have been developed to overcome the species specificity currently limiting the study of c-Met–dependent tumor progression and metastasis. Mazzone et al. (20) used a lentivirus to deliver mutant and wild-type hHGF to tumor cells and directly to mice. They were able to show elevated serum HGF in the infected animals but did not identify sites of infection, dose dependence between viral infection and HGF production, or HGF-related liver hypertrophy.

Zhang et al. (35) recently produced a transgenic mouse by injecting hHGF cDNA driven by the mouse metallothionein-1 gene promoter into C3H/6 mouse embryos. In this system, hHGF serum levels are variable, ranging from 300 to 3,240 pg/mL, and HGF is produced in several organs, including the liver, lung, brain, and kidney. Our system provides an alternative xenograft model, which, in some respects, is more desirable. First, the cost of acquiring and maintaining transgenic mice, including development and specialized breeding/housing facilities, is not required in our model system. We would expect that any immunodeficient mouse could be used (36), although we have only used SCID mice. Second, the level of hHGF expressed is highly reproducible and manipulatable, responding to the quantity of virus introduced into the mouse. This allows the investigator to easily induce specific levels of HGF in the mouse, modeling human disease. For instance, generating elevated serum HGF levels may mimic certain advanced disease states, such as metastatic breast (37), prostate (38), or colorectal cancer (39, 40). Third, in our model, hHGF is predominately produced in the liver, which may be more physiologic than a transgenic model in which all tissues produce the ligand. Selective hHGF production by the liver may play a crucial role in the increased occurrence of gastrointestinal tumor metastases to the liver. One can postulate that tumor cells transported to the liver via the portal circulation would have a survival advantage related to the high local levels of HGF (41). Such hypotheses can be tested in our model system.

There are limitations to this model. First, levels of HGF diminish over time, although our studies indicated <15% reduction over 40 days when used in immunodeficient mice. However, the temporal change in serum levels of hHGF may be advantageous. For example, fluctuations in HGF have been measured clinically after resection of metastatic disease. Yoon et al. (40) noted that elevated HGF levels in patients with metastatic colorectal cancer initially increased following liver resection concomitant with liver regeneration and then either decreased to normal levels or remained elevated in a subset of patients with poor outcome. Our model can be used to further study the effects of fluctuation in serum HGF on tumor progression.

Another potential limitation to our model is the requirement of mice infection with an adenovirus. However, the effects of replication-deficient virus seem transient and limited. Phaneuf et al. (28) compared the effects of infection with adenovirus containing the hHGF transgene with an adenovirus containing the Escherichia coli gene for β-galactosidase (control). At currently used doses, no liver hypertrophy and no hHGF was produced in mice infected with the control adenovirus, indicating that the effect was specific for adenovirus containing the hHGF transgene. The authors did not report any long-term ill effects in animals infected with the replication-deficient virus. Similarly, we did not observe any ill effects in control animals followed for 6 weeks.

We also did experiments with another control adenoviral construct that produces alkaline phosphatase (Ad-AlkPhos). Augmented tumor growth was specific for the Ad-hHGF construct, and tumor growth was similar in SCID mice treated with no adenoviral construct or the control adenoviral construct.

In summary, we have developed a novel mouse model for study of c-Met–dependent human tumor progression and metastases, in which the quantity of ligand is easily controllable. Transgenic mice are not required, and this model overcomes the species specificity of the ligand-receptor interaction that has limited the study of human tumors in mice. It is ideal for preclinical evaluation of c-Met and HGF-directed therapy.

Grant support: American Society of Clinical Oncology Foundation Clinical Research Career Development Award (M.R. Weiser).

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

1
Birchmeier C, Gherardi E. Developmental roles of HGF/SF and its receptor, the c-Met tyrosine kinase.
Trends Cell Biol
1998
;
8
:
404
–10.
2
Trusolino L, Comoglio PM. Scatter-factor and semaphorin receptors: cell signalling for invasive growth.
Nat Rev Cancer
2002
;
2
:
289
–300.
3
Corso S, Comoglio PM, Giordano S. Cancer therapy: can the challenge be MET?
Trends Mol Med
2005
;
11
:
284
–92.
4
Gao CF, Vande Woude GF. HGF/SF-Met signaling in tumor progression.
Cell Res
2005
;
15
:
49
–51.
5
Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF. Met, metastasis, motility and more.
Nat Rev Mol Cell Biol
2003
;
4
:
915
–25.
6
Jeffers M, Rong S, Woude GF. Hepatocyte growth factor/scatter factor-Met signaling in tumorigenicity and invasion/metastasis.
J Mol Med
1996
;
74
:
505
–13.
7
Ma PC, Maulik G, Christensen J, Salgia R. c-Met: structure, functions and potential for therapeutic inhibition.
Cancer Metastasis Rev
2003
;
22
:
309
–25.
8
Christensen JG, Burrows J, Salgia R. c-Met as a target for human cancer and characterization of inhibitors for therapeutic intervention.
Cancer Lett
2005
;
225
:
1
–26.
9
Cao B, Su Y, Oskarsson M, et al. Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models.
Proc Natl Acad Sci U S A
2001
;
98
:
7443
–8.
10
Park WS, Oh RR, Kim YS, et al. Absence of mutations in the kinase domain of the Met gene and frequent expression of Met and HGF/SF protein in primary gastric carcinomas.
APMIS
2000
;
108
:
195
–200.
11
Jeffers M, Schmidt L, Nakaigawa N, et al. Activating mutations for the met tyrosine kinase receptor in human cancer.
Proc Natl Acad Sci U S A
1997
;
94
:
11445
–50.
12
Schmidt L, Junker K, Nakaigawa N, et al. Novel mutations of the MET proto-oncogene in papillary renal carcinomas.
Oncogene
1999
;
18
:
2343
–50.
13
Park WS, Dong SM, Kim SY, et al. Somatic mutations in the kinase domain of the Met/hepatocyte growth factor receptor gene in childhood hepatocellular carcinomas.
Cancer Res
1999
;
59
:
307
–10.
14
Lee JH, Han SU, Cho H, et al. A novel germ line juxtamembrane Met mutation in human gastric cancer.
Oncogene
2000
;
19
:
4947
–53.
15
Cooper CS, Park M, Blair DG, et al. Molecular cloning of a new transforming gene from a chemically transformed human cell line.
Nature
1984
;
311
:
29
–33.
16
Schmidt L, Duh FM, Chen F, et al. Germline and somatic mutations in the tyrosine kinase domain of the MET proto-oncogene in papillary renal carcinomas.
Nat Genet
1997
;
16
:
68
–73.
17
Wang R, Ferrell LD, Faouzi S, Maher JJ, Bishop JM. Activation of the Met receptor by cell attachment induces and sustains hepatocellular carcinomas in transgenic mice.
J Cell Biol
2001
;
153
:
1023
–34.
18
Parr C, Hiscox S, Nakamura T, Matsumoto K, Jiang WG. Nk4, a new HGF/SF variant, is an antagonist to the influence of HGF/SF on the motility and invasion of colon cancer cells.
Int J Cancer
2000
;
85
:
563
–70.
19
Jiang WG, Hiscox SE, Parr C, et al. Antagonistic effect of NK4, a novel hepatocyte growth factor variant, on in vitro angiogenesis of human vascular endothelial cells.
Clin Cancer Res
1999
;
5
:
3695
–703.
20
Mazzone M, Basilico C, Cavassa S, et al. An uncleavable form of pro-scatter factor suppresses tumor growth and dissemination in mice.
J Clin Invest
2004
;
114
:
1418
–32.
21
Burgess T, Coxon A, Meyer S, et al. Fully human monoclonal antibodies to hepatocyte growth factor with therapeutic potential against hepatocyte growth factor/c-Met-dependent human tumors.
Cancer Res
2006
;
66
:
1721
–9.
22
Zheng Z, Adams C, Moffatt B, Schwall R. A chimeric Fab antibody serves as an antagonist HGF/SF receptor c-met.
Proc Am Assoc Cancer Res
2004
;
44
:
5717
.
23
Christensen JG, Schreck R, Burrows J, et al. A selective small molecule inhibitor of c-Met kinase inhibits c-Met-dependent phenotypes in vitro and exhibits cytoreductive antitumor activity in vivo.
Cancer Res
2003
;
63
:
7345
–55.
24
Rong S, Oskarsson M, Faletto D, et al. Tumorigenesis induced by coexpression of human hepatocyte growth factor and the human met protooncogene leads to high levels of expression of the ligand and receptor.
Cell Growth Differ
1993
;
4
:
563
–9.
25
Rong S, Segal S, Anver M, Resau JH, Vande Woude GF. Invasiveness and metastasis of NIH 3T3 cells induced by Met-hepatocyte growth factor/scatter factor autocrine stimulation.
Proc Natl Acad Sci U S A
1994
;
91
:
4731
–5.
26
Jeffers M, Rong S, Anver M, Vande Woude GF. Autocrine hepatocyte growth factor/scatter factor-Met signaling induces transformation and the invasive/metastastic phenotype in C127 cells.
Oncogene
1996
;
13
:
853
–6.
27
Graveel CR, London CA, Vande Woude GF. A mouse model of activating Met mutations.
Cell Cycle
2005
;
4
:
518
–20.
28
Phaneuf D, Chen SJ, Wilson JM. Intravenous injection of an adenovirus encoding hepatocyte growth factor results in liver growth and has a protective effect against apoptosis.
Mol Med
2000
;
6
:
96
–103.
29
Dematteo RP, Chu G, Ahn M, et al. Immunologic barriers to hepatic adenoviral gene therapy for transplantation.
Transplantation
1997
;
63
:
315
–9.
30
Dematteo RP, Chu G, Ahn M, Chang E, Barker CF, Markmann JF. Long-lasting adenovirus transgene expression in mice through neonatal intrathymic tolerance induction without the use of immunosuppression.
J Virol
1997
;
71
:
5330
–5.
31
Kammula US, Kuntz EJ, Francone TD, et al. Molecular co-expression of the c-Met oncogene and hepatocyte growth factor in primary colon cancer predicts tumor stage and clinical outcome. Cancer Lett 2006. Epub ahead of print.
32
Jiang WG, Grimshaw D, Martin TA, et al. Reduction of stromal fibroblast-induced mammary tumor growth, by retroviral ribozyme transgenes to hepatocyte growth factor/scatter factor and its receptor, c-MET.
Clin Cancer Res
2003
;
9
:
4274
–81.
33
Francone T, Chen TC, Kunts E, Zeng Z, Paty PB, Weiser MR. Mechanisms of MET dysregulation and ligand-dependency in human colorectal cancer.
Ann Surg Oncol
2006
;
13
:
2
.
34
Zeng Z, Weiser MR, D'Alessio M, Grace A, Shia J, Paty PB. Immunoblot analysis of c-Met expression in human colorectal cancer: overexpression is associated with advanced stage cancer.
Clin Exp Metastasis
2004
;
21
:
409
–17.
35
Zhang YW, Su Y, Lanning N, et al. Enhanced growth of human met-expressing xenografts in a new strain of immunocompromised mice transgenic for human hepatocyte growth factor/scatter factor.
Oncogene
2005
;
24
:
101
–6.
36
Yang Y, Nunes FA, Berencsi K, Furth EE, Gonczol E, Wilson JM. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.
Proc Natl Acad Sci U S A
1994
;
91
:
4407
–11.
37
Toi M, Taniguchi T, Ueno T, et al. Significance of circulating hepatocyte growth factor level as a prognostic indicator in primary breast cancer.
Clin Cancer Res
1998
;
4
:
659
–64.
38
Naughton M, Picus J, Zhu X, Catalona WJ, Vollmer RT, Humphrey PA. Scatter factor-hepatocyte growth factor elevation in the serum of patients with prostate cancer.
J Urol
2001
;
165
:
1325
–8.
39
Fukuura T, Miki C, Inoue T, Matsumoto K, Suzuki H. Serum hepatocyte growth factor as an index of disease status of patients with colorectal carcinoma.
Br J Cancer
1998
;
78
:
454
–9.
40
Yoon SS, Kim SH, Gonen M, et al. Profile of plasma angiogenic factors before and after hepatectomy for colorectal cancer liver metastases.
Ann Surg Oncol
2006
;
13
:
353
–62.
41
Fidler IJ. The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited.
Nat Rev Cancer
2003
;
3
:
453
–8.