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Clinical Cancer Research Vol. 11, 8789-8798, December 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Antitumor Vascular Strategy for Controlling Experimental Metastatic Spread of Human Small-Cell Lung Cancer Cells with ZD6474 in Natural Killer Cell–Depleted Severe Combined Immunodeficient Mice

Seiji Yano1, Hiroaki Muguruma1, Yuka Matsumori1, Hisatsugu Goto1, Emiko Nakataki1, Nobutaka Edakuni1, Hideki Tomimoto1, Soji Kakiuchi1, Akihiko Yamamoto1, Hisanori Uehara2, Anderson Ryan3 and Saburo Sone1

Authors' Affiliations: Departments of 1 Internal Medicine and Molecular Therapeutics and 2 Molecular and Environmental Pathology, University of Tokushima School of Medicine, Tokushima, Kuramoto-cho, Tokushima, Japan and 3 AstraZeneca, Macclesfield, United Kingdom

Requests for reprints: Seiji Yano, Department of Internal Medicine and Molecular Therapeutics, University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. Phone: 81-88-633-7127; Fax: 81-88-633-2134; E-mail: manae{at}clin.med.tokushima-u.ac.jp.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Background: Small-cell lung cancer is often characterized by rapid growth and metastatic spread. Because tumor growth and metastasis are angiogenesis dependent, there is great interest in therapeutic strategies that aim to inhibit tumor angiogenesis.

Methods: The effect of ZD6474, an orally available inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) and epidermal growth factor tyrosine kinases, was studied in experimental multiple-organ metastasis models with human small-cell lung cancer cell lines (SBC-3 or SBC-5) in natural killer cell–depleted severe combined immunodeficient mice.

Results: Intravenously inoculated SBC-5 cells produced experimental metastases in the liver, lung, and bone whereas SBC-3 cells produced the metastases in the liver, systemic lymph nodes, and kidneys. Daily oral treatment with ZD6474 (50 mg/kg), started on day 14 (after the establishment of micrometastases), significantly reduced the frequency of large (>3 mm) metastatic colonies (in the liver and lymph nodes) and osteolytic bone lesions. ZD6474 treatment did not significantly reduce the frequency of small (<2-3 mm) metastatic lesions found in the lung (SBC-5) or kidney (SBC-3), consistent with an antiangiogenic mechanism of action. Immunohistochemical analysis of SBC-5 metastatic deposits in the liver showed that ZD6474 treatment inhibited VEGFR-2 activation and induced apoptosis of tumor-associated endothelial cells, resulting in decreasing tumor microvessel density. ZD6474 treatment was also associated with a decrease in tumor cell proliferation and an increase in tumor cell apoptosis. The antitumor effects of ZD6474 were considered likely to be due to inhibition of VEGFR-2 tyrosine kinase because gefitinib, a small-molecule inhibitor of epidermal growth factor receptor tyrosine kinase, was inactive in these models.

Conclusions: These results suggest that ZD6474 may be of potential therapeutic value in inhibiting the growth of metastatic small-cell lung cancer in humans. Phase II trials with ZD6474 are currently ongoing in a range of solid tumors.

Lung cancer is the leading cause of malignancy-related deaths worldwide (1). Lung cancer is histologically subdivided into small-cell lung cancer and non–small-cell lung cancer. Small-cell lung cancer accounts for 15% to 20% of cases and presents aggressive clinical behavior characterized by rapid growth and metastatic spread to the systemic lymph nodes and distant organs. Because small-cell lung cancer frequently develops clinically undetectable micrometastasis early in its development, curative surgical resection does not provide long-term benefits. Although small-cell lung cancer is one of the most sensitive cancers to chemotherapy and radiotherapy, it usually recurs rapidly and becomes highly refractory to subsequent therapy (2). Therefore, novel therapeutic modalities to control growth of micro- and macrometastases, as well as primary tumor and recurrent diseases, are necessary for improving prognosis of this disease.

Recently, molecular targeted therapies, such as the inhibitors of epidermal growth factor receptor (EGFR), gefitinib and erlotinib, have been shown to be effective for the treatment of chemotherapy-refractory non–small-cell lung cancer (3). However, molecular targeted drugs have not yet been shown to be effective in small-cell lung cancer.

It has been established that angiogenesis is essential for the development and growth of solid tumors to provide oxygen and nutrients to the expanding tumor mass (4) and a number of reports have shown an inverse correlation between microvessel density in tumors and survival of the patients (5, 6). In particular, a recent study reported that surgically resected primary tumors of small-cell lung cancer were hypervascular, suggesting that angiogenesis may be a promising therapeutic target in small-cell lung cancer (7).

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and vascular permeability (8). VEGF overexpression has been correlated with tumor vascular density and is inversely correlated with survival of patients with various cancers, including non–small-cell lung cancer (9). In addition, VEGF induces vascular hyperpermeability that may be associated with malignant pleural effusions and ascites (10, 11). VEGF binds with high affinity to two tyrosine kinase receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), on endothelial cells inducing receptor dimerization, autophosphorylation, and signal transduction (12). Of these two receptors, VEGFR-2 is considered to be the most important mediator of VEGF-induced signaling responses (13). Several approaches to inhibiting VEGFR-2 signaling have been developed and evaluated in preclinical and clinical trials, including anti-VEGF neutralizing antibody, soluble VEGFR-1, and small-molecule inhibitors of VEGFR-2 tyrosine kinase (14). Of these approaches, an anti-VEGF neutralizing antibody, bevacizumab, has been shown to augment response rates of first-line chemotherapy and significantly prolong survival of colorectal cancer patients (15). In addition, bevacizumab enhanced the antitumor effects of first-line chemotherapy in patients with non–small-cell lung cancer although fatal hemoptysis occurred in some patients in a phase II clinical trial (16). The effects of bevacizumab support a role for VEGF-dependent signaling in non–small-cell lung cancer. This is further supported by results from a Japanese phase I study of ZD6474 in which four of nine patients with non–small-cell lung cancer had partial responses (17).

ZD6474 is a novel, orally active agent that inhibits VEGFR-2 and EGFR tyrosine kinases (18–20). Consistent with an antiangiogenic mechanism of action, ZD6474 inhibits VEGF signaling and angiogenesis in vivo and shows broad-spectrum antitumor activity in a range of histologically diverse tumor xenograft models (18). In spontaneous metastasis models, ZD6474 has been shown to reduce metastatic spread from the primary tumors to the lungs, possibly by inhibiting tumor growth at the primary site (18). It is, however, unknown whether ZD6474 has the potential to inhibit the growth of established micrometastases of small-cell lung cancer.

In the present study, we investigated whether ZD6474 could inhibit metastatic spread of human small-cell lung cancer cell lines in a natural killer (NK) cell–depleted severe combined immunodeficient (SCID) mouse model (21).


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. Human small-cell lung cancer cell lines, SBC-3 and SBC-5, were provided by Dr. K. Hiraki (Okayama University, Okayama, Japan; refs. 22, 23). Human lung adenocarcinoma PC-9 cells, used as a positive control for EGFR expression and susceptibility to gefitinib, were purchased from IBL Japan (Ibaraki, Japan). Human dermal microvessel endothelial cells (HMVEC) were purchased from KURABO (Osaka, Japan). Tumor cell cultures were maintained in MEM supplemented with 10% heat-inactivated fetal bovine serum and gentamicin at 37°C in a humidified atmosphere of 5% CO2 in air. HMVECs were maintained in HuMedia-MvG with growth supplements (KURABO) and used for in vitro assays at passages 2 to 5.

Reagents. ZD6474 and gefitinib (ZD1839) were supplied by AstraZeneca (Cheshire, United Kingdom). Recombinant human VEGF165 and recombinant human basic fibroblast growth factor (bFGF) were purchased from R&D Systems (Minneapolis, MN). An antimouse interleukin-2 receptor ß-chain monoclonal antibody (mAb), TM-ß1 (immunoglobulin G2b; ref. 24), was kindly supplied by Drs. M. Miyasaka and T. Tanaka (Osaka University, Osaka, Japan).

Expression of VEGF isoforms and VEGFRs. Expression of VEGF isoforms and VEGFRs was determined by reverse transcription-PCR. Total cellular RNA was extracted from the culture of cell lines by using the acid guanidinium thiocyanate/phenol-chloroform method (ISOGEN, Nippon Gene Co., Toyama, Japan) according to the protocol of the manufacturer. One microgram of total RNA was reverse transcribed. The PCR program used to amplify VEGF, VEGFRs, and glyceraldehyde-3-phosphate dehydrogenase consisted of a precycle of 5 minutes at 94°C, 45 seconds at 60°C, and 45 seconds at 72°C. After this cycle, the process was continued for 30 cycles of 1 minute at 94°C, 45 seconds at 65°C, 2 minutes at 72°C, concluding with 7 minutes at 72°C. The primers for VEGF, VEGFRs, and glyceraldehyde-3-phosphate dehydrogenase were as follows: VEGF sense (5'-TCCAGGAGTACCCTGATGAG-3') and antisense (5'-CTTTCCTGGTGAGAGATCTGG-3') immediately flanking the region of the VEGF open reading frame involved in the alternative splicing of several exons (25); VEGFR-1 sense (5'-ATTTGTGATTTTGGCCTTGC-3') and antisense (5'-CAGGCTCATGAACTTGAAAGC-3'); VEGFR-2 sense (5'-GTGACCAACATGGAGTCGTG-3') and antisense (5'-CCAGACATTCCATGCCACTT-3'; ref. 26); VEGFR-3 sense (5'-CCCACGCAGACATCAAGACG-3') and antisense (5'-TGCAGAACTCCACGATCACC-3'; ref. 27); and glyceraldehyde-3-phosphate dehydrogenase sense (5'-AGTCATCCACGAGCGATTTG-3') and antisense (5'-TGCTGCTTTTACAGCCTCCT-3'). Reverse transcription-PCR was done using a one-step RNA PCR kit (TAKARA, Tokyo, Japan). The bands were visualized by ethidium bromide staining.

Cell proliferation assay. Cell proliferation was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction method (28). Briefly, tumor cells (2 x 103 per well) plated in triplicate in 96-well plates were incubated in MEM containing 5% fetal bovine serum for 24 hours. HMVECs (5 x 103 per well) plated in triplicate in 96-well plates precoated with 1.5% gelatin were incubated in supplemented M131 medium for 24 hours. The cells were washed and incubated for 72 hours with ZD6474 or gefitinib in fresh MEM containing 5% fetal bovine serum in the presence or absence of VEGF or bFGF. Then, 50 µL of stock 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (2 mg/mL; Sigma, St. Louis, MO) were added to all wells and the cells were incubated for 2 hours at 37°C. The media containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution were removed and the dark blue crystals were dissolved by adding 100 µL of DMSO. Absorbance was measured with an MTP-120 microplate reader (Corona Electric, Ibaraki, Japan) at test and reference wavelengths of 550 and 630 nm, respectively.

In vivo metastasis models. To facilitate metastasis formation, SCID mice were pretreated with antimouse interleukin-2 receptor ß-chain antibody to deplete NK cells (21). Two days later, the mice were inoculated with SBC-3 or SBC-5 (1 x 106) cells into the tail vein. The mice were treated with ZD6474 at 10, 25, or 50 mg/kg/d or gefitinib at 50 mg/kg/d by oral gavage for the indicated periods. Five weeks (SBC-5) or 6 weeks (SBC-3) after tumor-cell inoculation, mice were anesthetized by i.p. injection of pentobarbital and X-ray photographs of mice were taken to determine bone metastasis (23). Mice were killed humanely under anesthesia and major organs were removed and weighed. The lungs were fixed in Bouin's solution for 24 hours. Metastatic lesions larger than 0.5 mm in diameter on the surface of the major organs were counted macroscopically. The numbers of osteolytic lesions on X-ray films were counted by two investigators independently (S.Y. and H.M.).

Immunohistochemical-immunofluorescent determination of endothelial cells, proliferating cells, apoptotic cells, and VEGF production. The major organs with metastases were cut into 5-mm fragments and placed into either buffered 10% formalin solution or optimum cutting temperature compound (Miles Laboratories, Elkhart, IN) and snap-frozen in liquid nitrogen for immunohistochemical analysis. Paraffin-embedded tissues (4 µm thick) were used to quantitate in vivo cell proliferation using mouse anti-human Ki-67 mAb (MIB1; 1:50 dilution; DAKO, Glostrup, Denmark), VEGF production using mouse anti-human VEGF mAb (1:100 dilution; PharMingen, San Diego, CA), and apoptosis using the terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) method. Antigen retrieval [microwave boiling for 10 minutes in 0.01 mol/L citrate buffer (pH 6.0)] was done for Ki-67 staining. TUNEL assay was done using the Apoptosis Detection System (Promega, Madison, WI) according to the instructions of the manufacturer.

Frozen tissue sections (8 µm thick) were used for identification of endothelial cells using rat anti-mouse CD31/platelet/endothelial cell adhesion molecule-1 mAb (1:100 dilution; PharMingen).

Double staining for CD31 with activated VEGFR-2, activated EGFR, proliferating cells, and apoptotic cells. For BrdUrd staining, the mice were injected i.p. with BrdUrd solution (200 µL; Zymed BrdUrd staining kit, Zymed Lab, South San Francisco, CA). Two hours later, the mice were killed and the major organs were collected and cut into 2- to 3-mm sections, which were placed into optimum cutting temperature compound to be snap-frozen on dry ice. The frozen tissue sections (8 µm thick) were fixed with cold acetone and washed with PBS. After blocking with 5% normal hoarse serum, the slides were incubated with rabbit anti–activated VEGFR-2/3 (Flk-1) mAb (1:1,000 dilution; Calbiochem, Cambridge, MA; ref. 29), rabbit anti–activated EGFR mAb [pY1173] (1:100 dilution; Biosource, Camarillo, CA; ref. 29), or mouse anti-BrdUrd mAb (Zymed BrdUrd staining kit) at 4°C overnight. After washing with PBS, the slides were stained with matched secondary antibodies conjugated with Alexa Fluor 594 (red) or Alexa Fluor 488 (green; 1:200 dilution; Molecular Probes, Eugene, OR).

For double staining for CD31 and TUNEL (30), the frozen tissue sections (8 µm thick) were fixed with cold acetone. The slides were washed with PBS and permeabilized with 20 µg/mL proteinase K. The samples were then equilibrated and DNA strand breaks were labeled with fluorescein-12-dUTP by adding nucleotide mix and terminal deoxynucleotidyl transferase enzyme using the Apoptosis Detection System (Promega). The reaction was stopped with saline sodium citrate. The samples were then stained with an anti-CD-31 mAb (PharMingen) and Texas red–conjugated secondary antibody (Vector Laboratories, Burlingame, CA). The localized green fluorescence and red fluorescence were detected by fluorescence microscope (30).

Quantification of immunochistochemistry and immunofluorescence. The five areas containing the highest numbers of staining within a section were selected for histologic quantitation under light microscopy or fluorescent microscopy with a 400-fold magnification. Results were evaluated by two authors independently (S.Y. and H.M.).

Statistical analysis. The statistical significance of difference in in vitro and in vivo data was analyzed by Student t test and Mann-Whitney U test, respectively.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Expression of VEGF and its receptors in human lung cancer cell lines in vitro. Neither SBC-3 nor SBC-5 cells expressed VEGFR-2 mRNA whereas PC-9 human non–small-cell lung adenocarcinoma cells expressed VEGFR-2 mRNA as determined by reverse transcription-PCR. SBC-3 cells, but not SBC-5 cells, expressed VEGFR-1 mRNA. Interestingly, both small-cell lung cancer cell lines expressed both VEGFR-3 and VEGF, primarily VEGF165 (Fig. 1). Because ZD6474 also inhibits EGFR tyrosine kinase activity, we also examined expression of EGFR on small-cell lung cancer cells by flow cytometry. EGFR expression was not detectable in SBC-3 or SBC-5 cells (data not shown). In parallel experiments, we explored cytokine secretion by small-cell lung cancer cells using ELISA. Whereas neither SBC-3 nor SBC-5 cells produced detectable levels of EGFR ligands, EGF, or transforming growth factor-{alpha} (detection limits were 0.7 and 2.27 pg/mL, respectively), they secreted 4.0 and 14.4 ng/mL VEGF protein, respectively.



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  		Fig. 1. Expression of VEGF isoforms and VEGFRs by small-cell lung cancer cell lines. Expression of VEGF isoforms and VEGFRs was determined by reverse transcription-PCR. Reverse transcription-PCR products were electrophoresed on a 1.2% agarose gel and stained with ethidium bromide. Both SBC-3 and SBC-5 cells expressed VEGF and the predominant isoform was VEGF165. Although neither SBC-3 nor SBC-5 cells expressed VEGFR-2 mRNA (human lung adenocarcinoma PC-9 expressed VEGFR-2), SBC-3 cells, but not SBC-5 cells, expressed VEGFR-1 mRNA. Both cell lines expressed VEGFR-3. Representative of three experiments with similar results.

 
Effects of ZD6474 on proliferation of human small-cell lung cancer and endothelial cell lines in vitro. As reported previously (31), human lung adeonocarcinoma PC-9 cells were highly sensitive to both ZD6474 and gefitinib (IC50 = 33 and 10 nmol/L, respectively; Fig. 2). Under the same experimental conditions, proliferation of human small-cell lung cancer SBC-3 or SBC-5 cells was not inhibited by ZD6474 or gefitinib at concentrations up to 1 µmol/L.



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  		Fig. 2. Effect of ZD6474 and gefitinib on proliferation of human lung cancer and endothelial cell lines. Tumor cells (2 x 103 per well) plated in triplicate in 96-well plates were incubated overnight in MEM containing 10% fetal bovine serum. Different doses of ZD6474 (A) or gefitinib (B) were then given. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye reduction was used after 72 hours to measure cell proliferation. Proliferation of small-cell lung cancer cells was not inhibited by ZD6474 or gefitinib even at a dose as high as 1 µmol/L, but proliferation of PC-9 non–small-cell lung cancer cells was inhibited in a dose-dependent manner. HMVECs (5 x 103 per well) plated in triplicate in 96-well plates precoated with 1.5% gelatin were incubated for 72 hours with different doses of ZD6474 (C) or gefitinib (D) in fresh MEM containing 5% fetal bovine serum in the presence or absence of VEGF (20 ng/mL) or bFGF (20 ng/mL). Then, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was done. ZD6474, but not gefitinib, abrogated the stimulation by VEGF in a dose-dependent manner. Representative of three experiments with similar results. Points, mean of triplicate cultures; bars, SD.

 
Proliferation of HMVECs was stimulated by addition of recombinant human VEGF165 or bFGF, but not EGF. The EGFR tyrosine kinase inhibitor gefitinib had no effect on proliferation of HMVECs irrespective of the presence of VEGF165 or bFGF. ZD6474 dose-dependently (IC50 = 55 nmol/L) abrogated cell growth stimulation by VEGF165 but not by bFGF, suggesting that ZD6474 is a potent and selective inhibitor of VEGF-stimulated endothelial cell proliferation.

Effect of ZD6474 on multiple-organ metastasis by small-cell lung cancer cells. We have previously shown that i.v. injection of SBC-5 cells into NK cell–depleted SCID mice produces experimental metastasis within 35 days of tumor cell injection (23). These metastatic lesions were located primarily in the liver where some were >3 mm in diameter. In addition, injection of SBC-5 cells produces lung metastases (<2 mm in diameter) and osteolytic bone metastases detectable by X-ray photography (Fig. 3; Table 1). To evaluate the effect of inhibition of VEGFR-2 tyrosine kinase activity (antiangiogenesis) on established micrometastases, oral dosing with ZD6474 commenced 14 days after tumor injection (when micrometastatic small-cell lung cancer lesions are present in the liver; refs. 22, 23) and continued until mice were sacrificed 35 days after tumor injection. Treatment with ZD6474 at 50 mg/kg/d significantly reduced the number of liver metastases (Fig. 3; Table 1) although lower doses (10 and 25 mg/kg/d) were not effective. Interestingly, in a dose-dependent manner, ZD6474 also reduced the number of SBC-5 liver metastases >3 mm in diameter and total liver weight. At the highest dose tested (50 mg/kg/d), ZD6474 also significantly reduced the number of SBC-5 osteolytic bone lesions but did not affect the number of lung metastases (all of lung metastases were <2 mm in diameter). In a parallel experiment, gefitinib (50 mg/kg/d) dosed on the same schedule did not affect the number or size of SBC-5 metastases, suggesting that the antimetastatic effects of ZD6474 in the liver and bone are not due to the inhibition of EGFR tyrosine kinase. On the basis of these results, we selected a dose of 50 mg/kg/d ZD6474 for further experiments.



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  		Fig. 3. Metastasis produced by SBC-5 cells treated with or without ZD6474 or gefitinib. SBC-5 cells (1 x 106) were injected i.v. into NK cell–depleted SCID mice. From 14 days after the inoculation, mice were treated with ZD6474 (50 mg/kg/d orally) or gefitinib (50 mg/kg/d orally). Five weeks after tumor-cell inoculation, metastatic burden was determined as described in Materials and Methods. SBC-5 cells produced large metastases in the liver and bone, some of which were >3 mm in diameter. The lung metastases were <2 mm in diameter. Treatment with ZD6474 inhibited production of large metastases in the liver and bone. Bar, 10 mm.

 

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  		Table 1. Therapeutic effect of oral treatment with ZD6474 on multiple organ metastasis by SBC-5 cells in NK cell–depleted SCID mice

 
We next examined the effect of ZD6474 on metastasis of another human small-cell lung cancer cell line, SBC-3. I.v. injection of SBC-3 cells into NK cell–depleted SCID mice produced metastatic lesions, measuring 1 to 5 mm in diameter, predominantly in the liver in addition to the kidney and systemic lymph nodes (Table 2), within 42 days of injection. Gefitinib (50 mg/kg, d14-d41) did not show any significant antitumor effects on SBC-3 metastatic growth in the liver, kidney, or lymph nodes (Table 2). ZD6474 treatment produced significant antitumor effects on SBC-3 liver metastases, reducing liver weight (P < 0.05), total number of liver metastases (P < 0.05), and most notably, the number of large (>3 mm in diameter) metastatic liver lesions (P < 0.01). ZD6474 treatment also reduced the number of SBC-3 lymph node metastases but did not affect kidney metastases in this tumor model (Table 2).


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  		Table 2. Therapeutic effect of oral treatment with ZD6474 on multiple organ metastasis by SBC-3 cells in NK cell–depleted SCID mice

 
Comparison of tumor vasculature among different organs. Because antimetastatic effect by ZD6474 was not the same among organs, we compared the metastatic lesions in terms of microvessel density, VEGFR-2 activation in endothelial cells, and VEGF expression by tumor cells. Because lung lesions of SBC-5 cells were too small for evaluation, we used metastatic lesions produced by SBC-3 cells in this set of experiments. There was no discernible difference among organs (the liver, kidney, and lymph node) on the intratumoral microvessel density (liver, 11.0 ± 1.9; kidney, 13.2 ± 1.0; lymph node, 10.4 ± 3.6) or VEGF production by metastatic SBC-3 cells (Fig. 4A). In addition, double staining for CD31 (red) and activated VEGFR-2 (green) revealed that VEGFR-2 in intratumoral endothelial cells (yellow) was focally activated and that there was no discernible difference among the organs (the number of double-positive cells was 2.0 ± 0.8, 2.8 ± 1.0, and 2.3 ± 0.5 per high-power field in the liver, kidney, and lymph node, respectively).




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  		Fig. 4. Histologic examinations of metastatic lesions. A, metastatic lesions produced by SBC-3 cells in the liver, kidney, and lymph node were stained for CD31 alone, CD31 (red), activated VEGFR-2 (green), and VEGF. There was no discernible difference among organs on these staining. B, the mice bearing SBC-5 cells were treated with or without ZD6474 (50 mg/kg/d) or gefitinib (50 mg/kg/d) from days 14 to 34 and killed on day 35. The liver metastases were analyzed by immunohistochemistry and immunofluorescence. Treatment with ZD6474 inhibited tumor vascularization (CD31) and tumor-cell proliferation (Ki-67) and enhanced total cell apoptosis (TUNEL). C, double immunofluorescence staining of the liver metastases produced by SBC-5 cells was done to evaluate the expression of activated (phosphorylated) VEGFR-2 and EGFR in tumor-associated endothelial cells and proliferating (BrdUrd) and apoptotic (TUNEL) endothelial cells. Endothelial cells in the liver metastasis did not express activated EGFR. Treatment with ZD6474, but not gefitinib, diminished activation of VEGFR-2 and proliferation of tumor-associated endothelial cells. In addition, ZD6474 enhanced apoptosis of tumor-associated endothelial cells. Bar, 100 µm. Arrows, double-positive cells.

 
Immunohistochemical and immunofluorescence staining to clarify the antimetastatic mechanisms of ZD6474. The liver lesions of SBC-5 cells treated with ZD6474 had decreased Ki-67-positive proliferating tumor cells, increased TUNEL-positive cells, and decreased microvessel density compared with the control or gefitinib-treated group (Figs. 4B and 5).



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  		Fig. 5. Quantitative immunohistochemical and immunofluorescence analyses of liver metastases produced by SBC-5 cells. A, vascularization determined by CD31 staining. B, proliferating tumor cells determined by Ki-67 staining. C, apoptotic cells determined by TUNEL staining. D, activated VEGFR-2-positive endothelial cells determined by double staining for CD31 and activated VEGFR-2. E, proliferating endothelial cells determined by double staining for CD31 and BrdUrd. F, apoptotic endothelial cells determined by double staining for CD31 and TUNEL. Treatment with ZD6474 (50 mg/kg) decreased vascularization, tumor cell proliferation, activated VEGFR-2-positive endothelial cells (EC), and proliferating endothelial cells and augmented apoptosis of total cells and tumor-associated endothelial cells in the liver metastases. Columns, mean number from five independent areas; bars, SD. HPF, high-power field (400x). *, P < 0.05, compared with control.

 
To assess the effect of ZD6474 on tumor-associated endothelial cells, we further did double staining for CD31/activated VEGFR-2, CD31/activated EGFR, CD31/BrdUrd, and CD31/TUNEL. VEGFR-2 of tumor-associated endothelial cells was focally activated in the control group and treatment with ZD6474, but not gefitinib, abrogated VEGFR-2 activation (Figs. 4C and 5). On the other hand, activated EGFR was rarely detected on tumor-associated endothelial cells irrespective of treatment with ZD6474 or gefitinib. The number of proliferating endothelial cells (double positive for CD31 and BrdUrd) was decreased in the tumors from mice treated with ZD6474, but not gefitinib. In contrast, the number of apoptotic endothelial cells (double positive for CD31 and TUNEL) was dramatically increased in the tumors from mice treated with ZD6474. These results suggest that ZD6474 induced apoptosis of tumor-associated endothelial cells presumably by inhibiting VEGFR-2 activation and thereby caused apoptosis of tumor cells.

Effect of timing of initiation of ZD6474 treatment on liver metastasis. In the final set of experiments, we evaluated the effect of timing of initiation of ZD6474 treatment on development and growth of SBC-5 metastases in the liver and bone 28 days after tumor cell injection. SBC-5 tumor–bearing mice were treated with or without 50 mg/kg/d gefitinib for 2 weeks started on day 14 (days 14-28) or 50 mg/kg/d ZD6474 for 3 weeks (days 7-28), 2 weeks (days 14-d28), or 1 week (days 21-28). Gefitinib treatment had no effect on metastases in the liver and bone. ZD6474 significantly inhibited both liver and bone metastases when treatment started on day 7 or 14 but it was less effective when treatment started on day 21 (Fig. 6).



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  		Fig. 6. Early treatment with ZD6474 is more effective for inhibition of metastatic growth by SBC-5 cells. SBC-5-bearing mice were treated with or without ZD6474 (50 mg/kg) or gefitinib (50 mg/kg) for indicated periods. A, weight of liver with metastases. B, bone metastases determined by X-ray photography. Solid lines, means. Dotted line, normal liver weight. *, P < 0.05, compared with the control. Treatment with ZD6474 starting at day 7 or day 14 significantly inhibited development of detectable liver and bone metastases.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Angiogenesis is considered essential for tumor growth and metastatic spread in a variety of cancers, including small-cell lung cancer (7); therefore, an antitumor vasculature treatment strategy seems to be ideal in small-cell lung cancer. Using a multiple-organ metastasis model with small-cell lung cancer cell lines in NK cell–depleted SCID mice, we showed that suppression of tumor angiogenesis resulted in growth inhibition of established experimental micrometastases of small-cell lung cancer cells. To inhibit tumor growth, we used ZD6474, an inhibitor of VEGFR-2 and EGFR tyrosine kinase activity. Although ZD6474 inhibited proliferation of small-cell lung cancer cells in vivo, it did not suppress in vitro proliferation of small-cell lung cancer cells at clinically relevant drug concentrations (<1 µmol/L; ref. 17), suggesting that growth inhibition of established micrometastases by ZD6474 is likely to be an effect on tumor-associated endothelial cells through blockade of VEGFR-2 signaling rather than direct growth inhibition of tumor cells through blockade of EGFR tyrosine kinase activity. This is further supported by the following lines of evidence: (a) ZD6474 potently and selectively inhibited VEGF-induced endothelial cell proliferation (Fig. 2); (b) ZD6474 (up to 1 µmol/L) did not significantly affect VEGF protein production by small-cell lung cancer (SBC-5 or SBC-3) cells in vitro (data not shown); (c) tumor-associated endothelial cells expressed activated VEGFR-2, but not activated EGFR, in our model with small-cell lung cancer cells (Fig. 4); (d) ZD6474 inhibited VEGFR-2 activation and proliferation and induced apoptosis of tumor-associated endothelial cells and thereby inhibited vascularization in the tumors (Fig. 4); and (e) ZD6474 inhibited the growth of metastatic lesions in a number of organ settings but was less effective in reducing the number of metastases (Tables 1 and 2).

ZD6474 is being developed as a potent and selective inhibitor of VEGFR-2 tyrosine kinase although it may also inhibit the tyrosine kinase activity of other receptors, particularly EGFR and RET, as well as VEGFR-3 (18, 32). For example, an in-frame deletion within the ATP-binding region encoded by exon 19 of the EGFR gene was recently reported to increase the sensitivity of non–small-cell lung cancer cells to ZD6474, as well as gefitinib (31, 33), showing the activity of ZD6474 to EGFR. Several reports showed the involvement of EGFR in tumor angiogenesis. EGFR could be expressed on tumor-associated endothelial cells if tumor cells produced EGFR ligands, such as EGF and transforming growth factor-{alpha} (34). In addition, blockade of EGFR decreased tumor cell production of proangiogenic molecules (such as VEGF; ref. 35) and inhibited angiogenesis (36). Nevertheless, neither of the human small-cell lung cancer cell lines (SBC-3 and SBC-5) used in this study expressed discernible levels of EGFR protein (data not shown) or produced ligands of EGFR (EGF and transforming growth factor-{alpha}, determined by ELISA; data not shown). Moreover, an inhibitor of EGFR (gefitinib) did not inhibit proliferation (in vitro) or metastasis formation (in vivo) of small-cell lung cancer cell lines. Tumor-associated endothelial cells hardly expressed activated EGFR in metastatic lesions produced by small-cell lung cancer cells (Fig. 4C). These results suggest that antimetastatic effect by ZD6474 was not mediated by EGFR inhibition, at least in this tumor model with small-cell lung cancer. Although involvement of VEGFR-3 is not completely ruled out in the present study, neither SBC-5 nor SBC-3 produced detectable levels of ligands for VEGFR-3 (VEGF-C and VEGF-D, determined by ELISA) irrespective of treatment with ZD6474 in vitro (data not shown).

The most serious problem in the management of small-cell lung cancer patients is metastatic spread to multiple organs, which is already established (microscopically or macroscopically) by the time of diagnosis. To investigate the molecular and biological mechanisms of metastasis and to develop novel therapeutic modalities against metastatic small-cell lung cancer, we established the multiple-organ metastasis models by i.v. dissemination of small-cell lung cancer cells (21, 23) as used in this study. Orthotopic tumor cell implantation and subsequent growth and metastasis may represent a more optimal preclinical animal model than i.v. inoculation but, to our knowledge, an orthotopic model with patient-like metastasis (multiple-organ metastases) is not available for small-cell lung cancer. In our model, i.v. inoculated small-cell lung cancer cells omit the initial steps of metastasis, such as the growth at the primary site and invasion into vessels. However, all the subsequent steps, such as the circulation in the blood, arrest at the secondary sites, extravasation, and the growth with vascularization as metastatic lesions, must be completed. In addition, our experimental metastasis model has several advantages as follows: (a) patient-like pattern of metastasis (liver, bone, lymph nodes, and lung); (b) production of metastasis is reproducible (all mice produce metastatic lesions); and (c) the number of metastatic lesions is consistent. Therefore, this model is suitable for evaluating therapeutic potential of anticancer modalities, especially against established micrometastases of small-cell lung cancer.

The outcome of metastasis is dependent on the properties of both tumor cells and host factors (37). Therefore, the host organ microenvironment (including endothelial cells) surrounding the tumor should always be considered when treating cancer. The characteristics of metastases in different organs may differ; for example, we have previously shown that the antimetastatic effects of both macrophage colony-stimulating factor (38) and matrix metalloproteinase inhibitors (39) are specific to particular organs although both macrophage colony-stimulating factor and matrix metalloproteinase inhibitors dramatically block the growth of s.c. inoculated tumor cells. For this reason, metastatic models should be used in preference to s.c. xenograft models for evaluating the therapeutic potential of new anticancer agents.

Treatment with ZD6474 suppressed enlargement of metastatic colonies in the liver, bone, and lymph nodes but did not markedly reduce the number of metastases in some organs, such as the lung and kidney (Tables 1 and 2). One possible explanation is that the expression of angiogenesis-related molecules is different among organs. However, this is not the case because there was no discernible difference among organs (the liver, kidney, and lymph node) on the intratumoral microvessel density, VEGFR-2 activation on tumor-associated endothelial cells, or VEGF production by metastatic SBC-3 cells (Fig. 4A). Although ZD6474 did not show a discernible effect on lung metastasis of SBC-5 cells, this is perhaps not unexpected because angiogenesis is not necessary for small tumors (<1-2 mm in diameter; ref. 40) and lung metastases produced by SBC-5 cells were <2 mm in diameter. Similar findings were observed in our previous study showing that treatment with a selective VEGFR-2 inhibitor (PTK787) did not reduce the number of lung metastasis by a non–small-cell lung cancer cell line (PC14PE6; ref. 41).

VEGF is reported to play a critical role in early tumor development rather than advanced phase cancer (42). Therefore, the timing of the initiation of antiangiogenic treatment may be important. We confirmed that micrometastases of small-cell lung cancer cells, at least in the liver, are produced 14 days after tumor-cell inoculation in our model (22). We found that treatment with ZD6474 was most effective when started early (from day 7 after tumor cell inoculation), suggesting that antiangiogenic treatment may be effective for controlling clinically undetectable sizes of micrometastases of small-cell lung cancer. Moreover, ZD6474 has been shown to augment the therapeutic efficacy of radiotherapy, antiandrogen therapy, and chemotherapy against a wide range of histologically diverse solid tumors (20, 43, 44). Therefore, it is possible that combined use of ZD6474 with other anticancer modalities may enhance antimetastatic effects in small-cell lung cancer.

In conclusion, we have shown that angiogenesis inhibition by the VEGFR-2 inhibitor (ZD6474) significantly inhibited growth of metastatic lesions in the experimental multiple-organ metastasis model with human small-cell lung cancer cell lines. These results suggest that antitumor vascular therapy with ZD6474 may be of potential value in controlling the metastatic spread of small-cell lung cancer in humans.


   Acknowledgments
 
We thank Dr. Masayuki Shono (University of Tokushima Graduate School) for technical assistance with immunohistochemical analysis.


   Footnotes
 
Grant support: Grant-in-Aid for Cancer Research from the Ministry of Education, Science, Sports, and Culture of Japan.

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.

Received 3/25/05; revised 9/ 8/05; accepted 9/30/05.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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