Affiliation of authors: Dermatology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD.
Correspondence to: Sam T. Hwang, M.D., Ph.D., National Institutes of Health, Bldg. 10, Rm. 12N246, 10 Center Dr., MSC 1908, Bethesda, MD 208921908 (e-mail: hwangs{at}mail.nih.gov).
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ABSTRACT |
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INTRODUCTION |
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Our knowledge of the molecular determinants of metastasis is clearly limited, although a small number of gene products have been reported to suppress (5) or to increase (6) metastasis. For example, stimulation of intratumor lymphangiogenesis with growth factors such as vascular endothelial growth factor-C (7) markedly stimulates lymph node metastasis. Most of these studies (8,9) focused on dissemination of cells through blood vessels to organs, such as lungs or liver, although lymphatic vessels are clinically important routes for metastatic melanoma and breast cancer cells.
The migration of cancer cells to regional lymph nodes via lymphatic vessels resembles the normal migration of antigen-presenting cells such as dendritic cells during the course of inflammation. Antigen-bearing dendritic cells from tissues enter afferent lymphatic vessels and migrate to draining lymph nodes. Dendritic cell migration has been studied extensively. Although several proteins (i.e., E-cadherin, P-glycoprotein, and 6-integrin) are potentially important to the migration of dendritic cells to draining lymph nodes [reviewed in (10)], the role of CC chemokine receptor-7 (CCR7) has been established conclusively. When dendritic cells are activated by a variety of inflammatory stimuli (such as lipopolysaccharide) or cytokines (such as tumor necrosis factor-
), the cell surface expression of CCR7 is increased and the cells become responsive to CCL21 (also termed secondary lymphoid tissue chemokine and 6Ckine/exodus-2) (11). CCL21 is a chemokine that is produced constitutively by lymphatic endothelial cells in the skin (12) and other organs (13), and CCL21 induces a CCR7-mediated Ca2+ flux. Targeted deletion of the CCR7 gene (14), spontaneous mutations that prevent the expression of CCL21 (15), and inhibition of CCL21 with neutralizing antibodies (12) show that interference with either CCR7 or CCL21 prevents the migration of dendritic cells from peripheral tissues to regional lymph nodes.
Of interest, CCR7 is expressed by human adult T-cell leukemia cells with lymph node involvement (16) and by some human breast and melanoma cell lines (17). Thus, CCR7 may play a role in lymph node metastasis.
Herein we investigate whether functional expression of CCR7 enhances murine melanoma cells to metastasize to regional lymph nodes at a higher rate than control melanoma cells and explore the mechanism used.
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MATERIALS AND METHODS |
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B16/F1 melanoma cells (18), derived from C57BL/6 mice, were provided by the cell repository of the National Cancer Institute (NCI)-Frederick Cancer Research and Development Center (Frederick, MD). The cells were grown in Dulbecco's modified Eagle medium (MEM) (Life Technologies, Inc. [GIBCO BRL], Rockville, MD) with 10% heat-inactivated fetal calf serum, 10 mM MEM nonessential amino acids, 100 mM sodium pyruvate, and 200 mM L-glutamine. Recombinant chemokines were purchased from Peprotech (Rocky Hill, NJ). Female C57BL/6 mice (610 weeks old) from the NCI-Frederick Cancer Research and Development Center Animal Facility were used in all in vivo experiments, which were approved by the NCI Animal Care and Use Committee.
Retroviral Transduction of B16/F1 Melanoma Cells
Murine CCR7 complementary DNA (cDNA) (from A. Iwasaki, Yale University School of Medicine, New Haven, CT) was subcloned into the vector pLNCX2 (Clontech Laboratories, Inc., Palo Alto, CA) immediately downstream of the human cytomegalovirus immediate early promoter. GP-293 packaging cells (Clontech Laboratories, Inc.) were cotransfected with pLNCX2-CCR7 (or pLNCX2 alone) and pVSV-G (Clontech Laboratories, Inc.), a plasmid encoding the viral envelope glycoprotein (VSV-G) of vesicular stomatitis virus, by use of Effectene transfection reagent (Qiagen, Valencia, CA). Supernatants from transfected GP-293 were incubated with 50% confluent B16/F1 cells in the presence of Polybrene (8 µg/mL, final concentration; Sigma Chemical Co., St. Louis, MO). Transduced B16/F1 cells were propagated in medium containing G418 (Life Technologies, Inc.) at 400 µg/mL.
The retroviral vector MSCV-IRES-GFP (19) was made by inserting the internal ribosomal entry site (IRES)-green fluorescent protein (GFP) sequence into the retroviral vector MSCV 2.2 and used to transfer the gene for CXC chemokine receptor 5 (CXCR5) into B16 cells. CXCR5 cDNA (20) was inserted upstream of the IRES-GFP sequence to create the bicistronic retroviral vector MIG-CXCR5 encoding both CXCR5 and GFP genes. Transduction of B16/F1 cells was carried out as above with greater than 95% efficiency, as estimated by GFP fluorescence in the transduced cells. This CXCR5 retroviral expression vector can mediate chemotaxis in transduced bone marrow-derived dendritic cells in response to CXCL13 (a chemokine localized to B-cell follicles in secondary lymphoid organs) in vitro (Wu M, Hwang ST: unpublished results).
Quantitative Real-Time Reverse TranscriptionCoupled Polymerase Chain Reaction (RTPCR) Assay for Lymph Node Metastases
Melanoma lines were removed from plates with trypsin/EDTA and placed in Hanks' balanced salt solution (Life Technologies, Inc.). B16 cells (400 000 cells in 20 µL) were injected into the left hind footpads of C56BL/6 female mice in groups of five mice per cell line. One week after injection, the mice were killed and the left popliteal lymph nodes were removed. These lymph nodes were pooled, and total RNA was extracted by use of Qiagen Rneasy (Qiagen). After first-strand cDNA synthesis from deoxyribonuclease-treated RNA, real-time quantitative PCR was performed with an ABI PRISMTM 7700 DNA Sequence Detector and SYBRTM Green to quantitate the PCR product (PE Biosytems, Foster City, CA). Threshold cycle (CT) values were assigned to various products according to the cycle number at which a fixed, low fluorescence intensity was achieved. CT = CT(target) CT(glyceraldehyde-3-phosphate dehydrogenase [GAPD]), where
CT is the difference between the CT value of the target gene and the CT value of the reference gene (GAPD). The tyrosinase-related protein-1 (TRP) gene was chosen as the melanocyte/melanoma-specific marker (i.e., target gene) to identify melanoma cells that had metastasized to a lymph node (21,22). The fold increase of TRP mRNA in lymph nodes from CCR7-B16- or CXCR5-B16-injected mice relative to those from pLNCX2-B16-injected mice was calculated according to the expression, 2
CT, where
CT is the difference between
CT (chemokine receptor-B16) and
CT (pLNCX2-B16). The following murine primer sequences were used in the real-time PCR amplifications, all of which generated products 6588 base pairs long: TRP, F5`-GAAAATATGACCCTGCTGTTCGA-3` and R5`TTGTCCTCCCGTTCCATTCA-3`; GAPD, F5`-CGTGTTCCTACCCCCAATGT-3` and R5`-TGTCATCATACTTGGCAGGTTTCT3-'; CCR7, F5`-GGACACGCTGAGATGCTCACT-3` and R5`-CCATCTGGGCCACTTGGAT-3`; CCR4, F5`-AACAGAGCAGTGCGCATGAT-3` and R5`-CGTTGTACGGCGTCCAGAA-3`; and CCR5, F5`-GCCATGCAGGCAACAGAGA-3` and R5`-TCTCCAACAAAGGCATAGATGACA-3` (where F = forward and R = reverse). Dilution studies demonstrated that the TRP signal could be detected from cDNA derived from as few as 50 melanoma cells mixed with 5 x 106 murine lymph node cells (5% of the sample was used for each PCR assay).
Statistical Methods
Statistical tests were performed with the exact Wilcoxon rank sum test or 2 test as indicated, and both tests were two-sided.
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RESULTS |
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To determine whether CCR7 alone was sufficient to direct B16 melanoma cells to regional lymph nodes, we transduced B16 melanoma cells with retroviral vectors carrying CCR7 (CCR7-B16 cells), vector alone (pLNCX2-B16 cells), or CXCR5 (CXCR5-B16 cells). CXCR5 was chosen as a control because it is believed to participate in the recruitment of various immune cells to B-cell areas of the lymph node (23) but has no known role in directing dendritic cells to the afferent lymphatic vessels in the skin.
Real-time, quantitative RTPCR showed that CCR7-B16 cells expressed approximately 1000-fold more CCR7 than pLNCX2-B16 cells; levels of other chemokine receptors tested were essentially equivalent (Fig. 1, A). CCL21 stimulated a calcium flux in CCR7-B16 melanoma cells (Fig. 1, D
), demonstrating the functional expression of CCR7 in CCR7-B16 cells but not in pLNCX2-B16 melanoma cells (Fig. 1, B
). To rule out a selective growth advantage of CCR7-B16 cells, we cultured both cell lines in the presence or absence of CCL21 and found essentially no difference in growth (Fig. 1, C
). Thus, we have stably transduced CCR7 into B16 melanoma cells without affecting expression of several other chemokine receptors, demonstrated a functional response to the CCR7 ligand by CCR7-B16 cells, and showed essentially no in vitro growth differences between CCR7-B16 and pLNCX2-B16 cells.
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B16 melanoma cells have a low spontaneous rate of metastasis from tissue to local draining lymph nodes (see Fig. 3). To assay migration of B16 melanoma lines from the periphery to draining lymph nodes, we injected CCR7-B16, pLNCX2-B16, and CXCR5-B16 melanoma cells into the footpad of C57BL/6 mice. One week later, when tumors in the footpad were not grossly visible, we collected popliteal lymph nodes (Fig. 2, AC
) and isolated RNA for quantitative, real-time RTPCR. Although visible lymph node metastases were occasionally observed for CCR7-B16-injected cells at that time (Fig. 2, B
), they were never observed for pLNCX2-B16 (Fig. 2, A
) or CXCR5-B16 cells (Fig. 2, C
). We assessed expression of TRP messenger RNA (mRNA), a melanoma/melanocyte-specific marker, by quantitative, real-time RTPCR. This assay is a sensitive, quantitative measure of melanoma metastasis that has been used clinically to stage human melanoma (21). In five experiments, lymph nodes collected from mice given an injection of CCR7-B16 cells showed 701-fold (range, 200- to 1400-fold; 95% confidence interval [CI] = 64- to 1336-fold) more TRP mRNA than lymph nodes from mice given an injection of pLNCX2-B16 cells (Fig. 2, G
). In two experiments, lymph nodes from CXCR5-B16-injected mice showed only a minimal threefold increase (95% CI = 9- to 16-fold) of TRP mRNA over that of the vector control. Comparison of the groups injected with CCR7-B16 and CXCR5-B16 cells provided evidence of a trend toward increased metastasis in the CCR7-B16 group, despite the lack of statistical significance (P = .095 by a two-tailed exact Wilcoxon rank sum test) because of a limited number of observations with CXCR5-B16-injected animals. CXCR5 was chosen as negative control because its ligand, CXCL13, is expressed within the lymph node within B-cell zones (24). However, unlike CCR7, CXCR5 apparently has no role in the entry of dendritic cells into lymphatic vessels. In pLNCX2-B16 mice, the levels of TRP mRNA in draining lymph nodes and in contralateral popliteal lymph nodes were comparable, indicating that very few of the control B16 melanoma cells migrated to lymph nodes soon after injection (data not shown).
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Two weeks after tumor cell injection into footpads, B16 tumors increased rapidly in size. Three weeks after injection, when these tumors were 57 mm in diameter, the animals were killed, and the draining lymph nodes were removed for visual inspection. The tumor volumes in CCR7-B16- and pLNCX2-B16-injected animals were similar (data not shown). However, in four experiments, large metastases (multiple in some lymph nodes) were found in lymph nodes of CCR7-B16-injected mice (Fig. 3, A). In four experiments, 11 (58%) of 19 (range, 25%100%) lymph nodes from 19 CCR7-B16-injected mice had obvious metastases, but only one (5%) of 19 lymph nodes from 19 pLNCX2-B16-injected mice had a metastasis that was relatively small (Fig. 3, A
) (P<.001,
2 test).
Because retroviral transduction may lead to heterogeneous expression of the gene of interest, we repeatedly subcultured B16 melanoma cells from one of the metastatic lymph nodes shown in Fig. 3, A. If CCR7 expression contributed to the lymph node migration of CCR7-B16 cells, metastatic cells within the lymph node should express CCR7. Indeed, even after seven passages, CCL21 induced a Ca2+ flux in these B16 melanoma cells (Fig. 3, B
), indicating the presence of active CCR7. Thus, CCR7 expression enhanced the metastatic potential of B16 melanoma cells at both early and late times and was functionally maintained during the metastatic process.
Effect of Anti-CCL21 on CCR7-B16 Metastasis to Draining Lymph Nodes
To demonstrate that the metastasis of CCR7-B16 cells to draining lymph nodes was dependent on the CCR7/CCL21 pathway used by dendritic cells to migrate to draining lymph nodes (12,32), we repeatedly injected neutralizing antimurine CCL21 antibodies (12) or a control goat immunoglobulin G (IgG) into the mouse footpads containing CCR7-B16 cell implants. At the end of 10 days, metastases were detected in three of five lymph nodes from five IgG-treated mice, but none were detected in the lymph nodes from the five mice treated with anti-CCL21 antibodies (Fig. 4). Thus, inhibition of CCL21 blocks the metastasis of CCR7-B16 cells to draining lymph nodes.
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DISCUSSION |
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A number of genes are likely to be involved in metastasis, including genes that suppress metastasis (5,25) or genes that enhance metastasis, such as RhoC (a small guanosine triphosphatase) (6) or vascular endothelial growth factor-C (7). Transcription factors, such as AP-2 (26) and CREB (27), may also be involved in the regulation of metastasis.
The role of chemokine receptors, however, in metastasis has been explored only recently. Chemokine receptors have been identified in human breast carcinoma lines that respond chemotactically to various chemokines in vitro (28). Furthermore, interleukin 8 has been reported to induce haptotactic migration of A2056 human melanoma lines (29). In a study of adult T-cell leukemia/lymphoma (ATLL) patients with and without lymph node involvement, Hasegawa et al. (16) showed that ATLL cells from patients with lymph node involvement had increased expression of CCR7 and enhanced functional responses to CCL21, suggesting that the expression of CCR7 may alter the trafficking patterns of ATLL cells by recruiting these cells via CCL21-rich high endothelial venules within lymph nodes. Finally, Müller et al. (17) showed functional expression of CCR7 and CXCR4 in breast cancer cells and of these receptors and also CCR10 (30) in several melanoma cell lines. Although the functional role of CCR7 was not explored, striking inhibition of breast cancer metastasis was obtained with a function-blocking anti-CXCR4 antibody but not with a nonbinding isotype control antibody, raising the possibility that CXCR4 may be involved in the metastasis of breast cancer cells (17).
Given that the rate of genetic mutations in cancer cells is much higher than that in normal cells, perhaps genetic or transcriptional changes lead to enhanced expression of CCR7, as demonstrated by Müller et al. (17). Based on our understanding of dendritic cell migration, it is conceptually clear how CCR7 expression may work to recruit tumor cells to (and perhaps into) lymphatic channels that constitutively express CCL21 (13). After passive transport of tumor cells to the draining lymph nodes, CCR7 may help to retain malignant cells in the lymph nodes, where the T-cell paracortical regions are rich in the CCR7 ligands CCL21 and CCL19 (EBI1-ligand chemokine/MIP-3). In summary, our data demonstrate that the expression of a single chemokine receptor gene (CCR7) by murine melanoma cells increases metastases to lymph nodes, raising the possibility that cancer cells may use normal mechanisms of lymph node homing for metastatic dissemination.
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NOTES |
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H. E. Wiley is in the Howard Hughes Medical Institute-National Institutes of Health Research Scholars Program.
We thank Dr. Mark Udey (National Cancer Institute [NCI]) for helpful comments, Dr. Seth Steinberg (NCI) for statistical advice, and Drs. Akiko Iwasaki (Yale University School of Medicine) and David Chaplin (University of Alabama, Birmingham) for providing CCR7 and CXCR5 complementary DNA, respectively.
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Manuscript received May 2, 2001; revised August 16, 2001; accepted August 27, 2001.
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