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Cell, Tumor, and Stem Cell Biology |
1 Department of Oncological Sciences, Mount Sinai School of Medicine and 2 ImClone Systems, New York, New York
Requests for reprints: Mihaela Skobe, Department of Oncological Sciences, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1130, New York, NY 10029. Phone: 212-659-5570; Fax: 212-987-2240; E-mail: mihaela.skobe{at}mssm.edu.
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Introduction |
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Angiogenesis is essential for the growth of most primary tumors and their metastases, and antiangiogenic therapy has been effective in suppressing tumorigenicity and metastases in experimental models of cancer. Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 play a central role in tumor angiogenesis, and agents that block VEGF signaling pathways have shown promising results in clinical trials (5). Recent studies showing the ability of tumors to induce lymphangiogenesis have identified also lymphatic vessels as a potential target for the antimetastatic therapy (6). Lymphatic vessel invasion by tumor cells, increased numbers of tumor-associated lymphatics and enlarged lymphatic vessels have been frequently correlated with lymph node metastases and poor patient prognosis (2, 7). Notably, a large number of studies showed a correlation between the expression of the lymphangiogenic factor VEGF-C in human tumors and lymph node metastases (2). VEGF-C plays a key role in lymphangiogenesis by activating the VEGFR-3 receptor tyrosine kinase on lymphatic endothelial cells (1, 8–10). Mature form of VEGF-C also binds and activates VEGFR-2 (11), but whether VEGFR-2 plays a direct role in lymphangiogenesis is less clear.
Several studies in animal tumor models have provided direct evidence for the causal role of VEGF-C in tumor lymphangiogenesis and metastasis. VEGF-C has been shown to induce tumor lymphangiogenesis and facilitate tumor spread to the regional lymph nodes in mouse models of breast (12–14), pancreatic (15), gastric (16), and colorectal cancer (17). Our previous studies in a mouse xenograft model of breast cancer showed increase of not only lymph node but also distant metastases upon VEGF-C overexpression (12). Here, we evaluated the VEGF-C/VEGFR-3 signaling pathway as a target for the antimetastatic therapy and have compared the effectiveness of antiangiogenesis versus antilymphangiogenesis approach for control of tumor metastases. Our results show that inhibition of VEGFR-3 signaling with the function-blocking antibody mF4-31C1 was more effective in suppressing metastases than inactivation of VEGFR-2, and that the combination therapy was more potent in suppressing metastases than single-antibody treatments. These results show a key role for VEGFR-3 activation in metastasis and validate the concept of targeting the lymphatic endothelium and lymphatic spread for metastasis control.
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Materials and Methods |
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Tail vein metastasis assay. MDA/pcDNA or MDA/VEGF-C cells were injected i.v. into the lateral tail vein of 8-week-old NCR female, athymic nu/nu mice (1 x 106/100 µL HBSS). Mice were sacrificed after 10 weeks, and lymph nodes, lungs, and liver were collected and processed for evaluation of metastases.
Blocking antibodies. Neutralizing rat monoclonal antibodies specific for mouse VEGFR-3 (mF4-31C1; ref. 19) and mouse VEGFR-2 (DC101; refs. 20, 21) were generated by ImClone Systems Inc. (New York, NY). The mF4-31C1 antibody has been shown to block VEGF-C–induced phosphorylation of mouse VEGFR-3 and VEGF-C–induced cell proliferation in vitro and to inhibit physiologic regeneration of lymphatic vessels in vivo (19).
Treatment modalities. Animals were randomly assigned to one of the four treatment groups (mF4-31C1, DC101, mF4-31C1+DC101, and control), 10 mice per group. In the prevention regimen, treatment was initiated at the time of orthotopic tumor cell injection. In the intervention regimen, treatment commenced when tumors and metastases were established, 4 weeks after the orthotopic tumor cell inoculation. Blocking antibodies were administered i.p. at 800 µg/mouse every second day, over the 6-week period. For the combination treatment, both antibodies were administered concurrently at 800 µg/mouse.
Quantification of metastases by flow cytometry. Tissue dissociation, sample preparation, and fluorescence-activated cell sorting (FACS) analysis were done essentially as described (22), with some modifications. Upon sacrifice, organs were collected and dissociated individually by mechanical disruption and incubation with 400 units/mL collagenase D (Roche, Indianapolis, IN) for 30 minutes at 37°C. Collagenase activity was stopped with 10 mmol/L EDTA, and samples were passed through a 70-µm cell strainer (BD Biosciences, Bedford, MA). Cell suspensions were centrifuged and incubated in ice-cold 0.17 mol/l NH4Cl for 10 minutes to lyse RBC. Finally, cells were washed with HBSS, stained with 3 µg/mL propidium iodide, and resuspended in FACS buffer (1%fetal bovine serum/PBS).
Samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Entire samples of each lymph node were analyzed. Total number of cells in lymph nodes typically ranged from 5 x 104 to 2 x 106. At least 2 x 106 cells were analyzed in each lung or liver sample. Background fluorescence was determined by using tissue from mice that were not implanted with tumor cells. Nonviable cells were excluded based on propidium iodide staining. Data acquisition and analysis were done with Cell Quest software (BD Biosciences). Tumor burden in organs was calculated as the percentage of fluorescent tumor cells detected in the total population of viable cells in the sample. As few as 1 fluorescent tumor cell in 1 x 105 host cells (0.001% total cells) could be accurately detected. Taking into consideration the minimal number of cells in lymph nodes and the sensitivity of detection, <1 fluorescent cell in 1 x 104 host cells (<0.01%) was considered negative.
Immunofluorescent staining. Cryosections (
7 µm) of tumors, lymph nodes, and lungs were stained as previously described (23), using antibodies against mouse CD31 and CD34 (1:50; BD Biosciences PharMingen, San Diego, CA), LYVE-1 (1:800; United Biomedical, Inc., Hauppauge, NY), VEGFR-3 (1:40; R&D Systems, Minneapolis, MN), VEGFR-2 (1:100; R&D Systems), Ki67 (1:1,000; Novocastra, Newcastle, United Kingdom), and corresponding secondary antibodies labeled with Alexa Fluor 488 or 594 (Molecular Probes, Eugene, OR). Cell nuclei were counterstained with 10 µg/mL Hoechst bisbenzimide (Sigma-Aldrich, St. Louis, MO). Specimens were examined with a Nikon E-600 microscope (Nikon, Melville, NY), and images were captured with a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI).
Quantification of tumor vasculature. Tumor sections were double-stained with antibodies to LYVE-1 and CD31, to visualize lymphatic (LYVE-1+) and blood vessels (LYVE-1–/CD31+). Computer-assisted morphometric analysis of digital images was done using the IPLab software (Scanalytics, Fairfax, VA). Five tumors were examined in each experimental group, and five areas of each tumor were evaluated at x20 magnification. Lymphatic and blood vasculatures were quantified in the tumor areas with the highest density of respective vessels ("hotspots"). Total vessel area was calculated per tumor, and data were expressed as the average vessel area of five tumors, or as the percentage of the tumor area occupied with the vasculature. The average vessel size was also determined for each tumor.
Statistical analyses. The data were analyzed using a mixed model ANOVA, to take into account correlation structure in the data (four lymph nodes in each mouse). Because the outcome variables of percent GFP+ cells and the area of the vasculature both exhibited right-skewed distributions, these variables were log-transformed before analysis. The least-squares means for each treatment group were back-transformed to the original scale to provide the geometric means. The Ps for the differences between the means of treatment group versus control were calculated using Dunnett's adjustment for multiple comparisons of treatment means versus a single control. The Tukey-Kramer–adjusted Ps were used for the comparisons between the different treatments. All statistical analyses were done using SAS statistics software (SAS Institute, Inc., Cary, NC).
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Results |
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35 mm3 within 3 weeks after tumor cell injection.
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In the intervention treatment regimen, mF4-31C1 antibody lowered tumor burden in the lymph nodes by 47% (Fig. 1B). The number of mice with lymph node metastases was comparable between the mF4-31C1–treated and control group, but the number of lymph nodes with metastases in each mouse was lower in the anti-VEGFR-3–treated group (Table 1).
Systemic treatment with the anti-VEGFR-3 blocking antibody inhibits lung metastases. Blocking antibody to VEGFR-3 drastically reduced tumor burden in the lungs in the prevention and less prominently in the intervention treatment regimen (Fig. 1C and D). In the prevention regimen, blocking VEGFR-3 decreased tumor burden in the lungs by 87% compared with the control (% GFP+ tumor cells: 0.016 ± 0.01 versus 0.119 ± 0.12, respectively; P < 0.05). The effect of VEGF-C on promoting lung metastases was abolished with the anti-VEGFR-3 antibody treatment. In the intervention regimen, the inhibition was much less pronounced (%GFP+ tumor cells: mF4-31C1 treated 0.05 ± 0.07 versus control 0.07 ± 0.1; P > 0.05).
Blocking VEGFR-2 is less effective in halting metastases than blocking VEGFR-3. Inhibition of VEGFR-2 signaling had only minor effect on the incidence of metastases to lymph nodes in the prevention regimen and no effect in the intervention treatment regimen (Table 1). However, anti-VEGFR-2 blocking antibody DC101 decreased overall tumor burden in the lymph nodes by 64% in the prevention regimen (control, 0.035 ± 0.0836 versus DC101, 0.0128 ± 0.019; P > 0.05), although the effect was not statistically significant. In the intervention regimen, blocking VEGFR-2 also did not show a significant effect (control, 0.833 ± 1.51 versus DC101, 0.633 ± 0.88; P > 0.05; Fig. 1A and B). Because the number of lymph nodes with metastases was comparable between the control and anti-VEGFR-2–treated mice, reduced tumor burden in lymph nodes of DC101-treated mice reflects primarily the lower number of tumor cells in lymph nodes with metastases. Indeed, the average number of metastatic cells in lymph nodes was decreased by 59% with the DC101 treatment (control, 0.058 ± 0.1 versus DC101, 0.024 ± 0.02; P < 0.05).
Blocking VEGFR-2 also proved less effective in reducing lung metastases. In the prevention regimen, DC101 antibody decreased lung tumor burden, by 42% (%GFP+ tumor cells: control, 0.119 ± 0.12 versus DC101, 0.069 ± 0.082; P > 0.05) compared with 87% decrease of lung tumor burden with the mF4-31C1 antibody (Fig. 1C). In the intervention regimen, the extent of lung metastases was comparable between control and DC101-treated groups (%GFP+ tumor cells: 0.07 ± 0.102 versus 0.1 ± 0.084, respectively; P > 0.05; Fig. 1D).
Blocking of either VEGFR-3 or VEGFR-2 inhibits tumor lymphangiogenesis. Our previous studies showed that overexpression of VEGF-C in MDA-MB-435/GFP cells resulted in increased intratumoral lymphangiogenesis and enlargement of peritumoral lymphatic vessels (12). Thus, we investigated whether the different effects of VEGFR-3 and VEGFR-2 blocking on tumor metastasis can be attributed to distinct roles of these two receptors in tumor lymphangiogenesis. Lymphatic vessels were visualized by immunostaining with the antibody for LYVE-1, a specific marker of lymphatic endothelium (24, 25). Blocking VEGFR-3 activation resulted in the complete normalization of peritumoral lymphatic vasculature (Fig. 2). In contrast to the hyperplastic lymphatic vessels observed around the tumors of control animals, lymphatic vessels surrounding tumors of mice treated with mF4-31C1 were small, compressed, and indistinguishable from the lymphatic vessels in normal skin. Moreover, anti-VEGFR-3–treated mice displayed complete inhibition of intratumoral lymphangiogenesis compared with control-treated mice (Fig. 3); intratumoral lymphatic vessel densities were decreased by 95% in the prevention and in the intervention treatment regimen (P < 0.001). Notably, blocking VEGFR-2 also resulted in the normalization of peritumoral lymphatic vasculature as well as in the significant reduction of intratumoral lymphangiogenesis (Fig. 2). DC101 antibody decreased intratumoral lymphatic vessel densities by 81% (P < 0.001) in both treatment regimens (Fig. 3).
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Phenotypic characteristics of metastases. To gain insight into the mechanism by which inactivation of VEGFR-2 and VEGFR-3 signaling suppresses metastases, we evaluated lymph node and lung metastases by histology and examined metastatic nodules for proliferation and angiogenesis. At the time points examined (i.e., 6 weeks for the prevention and 10 weeks for the intervention experiments), we have not observed any macrometastases on the surface of the organs examined. Micrometastases, which were detected in the lymph node sections by GFP fluorescence, were localized predominantly in the subcapsular zone. Typical appearance of metastases in the lymph nodes is shown in Supplementary Fig. S1. The size of lymph node metastases varied, from only few cells per section to different size nodules ranging from 100 to 400 µm in diameter (Supplementary Fig. S1A-H). For comparison, the size of lymph nodes enlarged by the presence of a tumor ranged from 1 to 3 mm in diameter. Most commonly, the metastatic nodules were composed entirely of viable cells, as indicated by the uniform expression of GFP (Supplementary Fig. S1A and B, E-H). In larger metastases, necrosis was apparent in the center of the nodule, and tumor cell proliferation was restricted to the outer cell layers (Supplementary Fig. S1C and D; Fig. 6A and B). There were no obvious differences in the appearance of lymph node metastases among the different antibody treatments. Typical histology of a large lymph node metastasis is shown in Fig. 6A. Unexpectedly, we found that all lymph node metastases, including the largest nodules in the nontreated group, were avascular, as shown by the CD31 and CD34 immunostaining (Fig. 6C and D). Tumor cell proliferation in the lymph node was apparently high, as shown by Ki67 labeling (Fig. 6B). Similarly, even the largest lung metastases were not vascularized yet clearly showed proliferative activity (Fig. 6E-G). Upon different antibody treatments, both the size and the number of the metastatic lesions were decreased (Supplementary Fig. S2A-D). Although the appearance and the size of the metastatic lesions were rather heterogeneous within the same group of specimens (Supplementary Fig. S2E and F), the phenotype of metastases was not notably different between the control and the treated groups.
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Combination of DC101 and mF4-31C1 antibodies decreased tumor angiogenesis by 79% (compared with control; P < 0.0001), which was slightly more potent than with the each antibody alone (69% and 71% inhibition with mF4-31C1 and DC101 antibody, respectively; P < 0.01). Lymphangiogenesis was not inhibited more than with the single-antibody treatments. Finally, the combined treatment was not more effective in inhibiting tumor growth than blocking VEGFR-2 alone (Fig. 5E).
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Discussion |
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Our previous studies have shown that MDA-MB-435 breast cancer cells producing high levels of VEGF-C induce tumor lymphangiogenesis, and that the VEGF-C–mediated activation of tumor-associated lymphatic vessels facilitates metastases in a mouse xenograft model (12). By using this tumor model, we show here that the systemic treatment with the function-blocking antibody mF4-31C1, which selectively inhibits murine VEGFR-3 signaling (19), potently reduced tumor cell dissemination to the lymph nodes, indicating that the activation of VEGFR-3 signaling is critical for lymphogenous spread. Notably, the blockade of VEGFR-3 signaling also reduced lymph node metastases when the antibody was administered after the metastases were established, indicating for the first time a potential use of lymphatic vessel targeting for halting metastases after the tumor dissemination has occurred and not only as a prevention strategy.
The soluble form of VEGFR-3 has been reported to prevent formation of lymph node metastases in several experimental tumor models. Adenoviral administration of soluble VEGFR-3-Ig fusion protein reduced incidence of lymph node metastases from lung, melanoma, and prostate tumors implanted s.c. in immunodeficient mice (26, 27). In another study, VEGFR-3-Ig overexpressed in breast cancer cells suppressed formation of lymph node metastases in immunocompetent rats (14). Most recently, small interfering RNA (siRNA)–mediated VEGF-C gene silencing has been shown to inhibit tumor lymphangiogenesis and metastasis (28). Because soluble VEGFR-3 and the VEGF-C siRNA approach interfere with the activation of both VEGFR-2 and VEGFR-3, these studies could not assess the specific role of each receptor in metastasis. It has been reported that the monoclonal antibody AFL4 specifically blocks murine VEGFR-3 signaling and inhibits corneal lymphangiogenesis (29). In experimental tumor models, AFL4 has been found to inhibit angiogenesis and tumor growth (30) and to suppress lymph node metastases (31). In contrast, Pytowski et al. reported that AFL4 binds to VEGFR-3 but does not block VEGFR-3 signaling and have suggested that the observed effects of AFL4 may be mediated by nonantagonist mechanisms, such as the steric hindrance of VEGFR-3 dimerization or antibody-induced reduction in surface receptor expression (19). In our own studies, we have not observed any effects of the AFL4 antibody on tumor lymphangiogenesis or metastasis.3 In view of the above, the results presented in this report provide the first direct evidence for the pivotal role of VEGFR-3 signaling in lymphogenous metastasis.
Importance of VEGFR-3 signaling in metastases to lymph nodes is further underscored by our findings that blocking VEGFR-2 was less effective in halting metastases then blocking VEGFR-3, although VEGFR-2 blockade more potently inhibited angiogenesis and primary tumor growth. Because blocking of VEGFR-2 mainly affected angiogenesis and tumor growth, we hypothesized that inactivation of VEGFR-2 suppresses metastases by restraining metastatic growth, whereas VEGFR-3 inactivation mainly inhibits tumor spread. To test this hypothesis, we analyzed lymph node and lung metastases for angiogenesis and proliferation. We found, to our surprise, that despite prominent proliferation, metastases were not vascularized in any of the control and treatment groups, indicating that the growth of metastases was not dependent on angiogenesis at the secondary site within the duration of the experiment. This could explain the limited efficacy of the antiangiogenic therapy with the DC101 antibody for metastasis control in this experimental model. It is conceivable that the metastatic cells were not highly sensitive to the antiangiogenesis treatment because the metastatic lesions were too small to depend on angiogenesis for growth. It has also been suggested in the literature that the vascular dependence of malignant cells may be heterogeneous, particularly, that in the late stages of tumor progression vascular demand may be lowered (32–34). Furthermore, in well-vascularized organs, such as lungs, the dependence on angiogenesis may be diminished because of the extensive preexisting vascular network which can be co-opted by the metastatic cells (35, 36). Hence, the observed suppression of metastases with the DC101 antibody could be due to the significant decrease in the size of a primary tumor and decreased lymphangiogenesis, which may limit the rate of tumor cell escape from the tumor.
Effects of VEGFR-3 inhibition on metastases could be explained, at least in part, by inhibition of lymphangiogenesis. We and others have shown that increased lymphangiogenesis and recruitment of lymphatic vessels into the tumor increase the propensity of tumors to metastasize (12–15, 17, 37). Accordingly, decreasing the number of lymphatic vessels in the vicinity of tumor cells may restrain tumor spread by restricting tumor cell access to the lymphatic vasculature. Because VEGFR-2 blockade also inhibited tumor lymphangiogenesis but without decreasing the incidence of lymph node metastases, the reduction of lymphatic vessel densities alone can not explain the suppression of metastases by VEGFR-3 inactivation, raising a question about the mechanism by which VEGF-C and VEGFR-3 activation promote tumor dissemination. We have proposed previously that activation of lymphatic endothelium by VEGF-C may facilitate tumor cell entry into the lymphatics by promoting molecular interactions between tumor cells and lymphatic endothelium (12, 38, 39). Activation of lymphatic endothelial cells by VEGF-C and other VEGFR-3 ligands may facilitate the escape of cancer cells from the primary tumor by promoting release of chemokines that may attract tumor cells into the lymphatics or by altering adhesive properties of lymphatic endothelium to support tumor invasion.
In support of this concept, high levels of VEGF-C expression have been associated with lymphatic vessel invasion and lymph node metastases in the multitude of human tumors, whereas tumor metastases have not always been associated with increased lymphangiogenesis (2). In the present study, we have observed fewer lymphatic vessels infiltrated with tumor cells in tumors treated with the anti-VEGFR-3 antibodies compared with anti-VEGFR-2–treated tumors. Furthermore, our in vitro studies showed that lymphatic endothelial cells potently promote tumor cell chemotaxis.4 Taken together, these results suggest that in addition to lymphangiogenesis, activation of lymphatic vessels via the VEGFR-3 pathway may facilitate tumor metastases by promoting entry of tumor cells into the peripheral lymphatics.
To gain insight into the mechanism by which VEGF-C/VEGFR-3 signaling promotes distant metastases, the ability of control and VEGF-C–overexpressing tumor cells to form colonies in target organs was assessed in the experimental metastasis assay. In this assay, which models later stages of the metastatic process, there was no difference in the colonization of lungs or liver between control and VEGF-C–expressing cells. These findings show that VEGF-C does not provide an advantage for tumor cell survival in the bloodstream, arrest, extravasation, or growth at the secondary site and further support the concept that VEGF-C increases formation of distant metastases via the lymphatic pathway. Nevertheless, a possibility remains that VEGF-C may also facilitate tumor cell invasion of the blood vasculature at the primary tumor site. Interestingly, when injected directly into the blood circulation, tumor cells also entered lymph nodes. In this case, tumor burden in the lymph nodes was not increased by VEGF-C, reinforcing the conclusion that VEGF-C does not increase growth of metastases in lymph nodes but facilitates tumor cell arrival from the primary tumor.
In summary, we show that selective inhibition of the VEGF-C/VEGFR-3 signaling pathway is an effective strategy for control of regional and distant metastases. It has been suggested that targeting the late steps of the metastatic process, such as growth of metastases at the secondary site, may be the most effective antimetastatic strategy (4, 40). The present study validates the concept of targeting the lymphatic endothelium, and thereby very early stages of the metastatic process, for metastasis control. Our results, showing the combination treatment with mF4-31C1 and DC101 antibodies to be more potent in suppressing metastases than either treatment alone are encouraging and suggest a potential promise for strategies which combine targeting both tumor dissemination and growth for the treatment of metastatic disease.
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Acknowledgments |
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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.
We thank Drs. Peter Bohlen and Dan Hicklin (ImClone Systems, New York, NY) for helpful discussions and for generously providing the DC101 antibody and Dr. James Godbold for his expert assistance with the statistical analyses.
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Footnotes |
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Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
3 N. Roberts and M. Skobe, unpublished data. 
4 S.K. Das and M. Skobe, unpublished data.
Received 5/26/05. Revised 11/11/05. Accepted 12/28/05.
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