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Expression of chemokine receptor CCR7 is associated with cervical lymph node metastasis of oral squamous cell carcinoma
Oral Oncology 45 (2009) 480–485
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Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Expression of chemokine receptor CCR7 is associated with cervical lymph node metastasis of oral squamous cell carcinoma Zheng Jun Shang a,b,*, Ke Liu b, Zhe Shao b a b
First Department of Oral and Maxillofacial Surgery, School of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, PR China Key Laboratory of Oral Biomedical Engineering, Ministry of Education of China, School of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, PR China
a r t i c l e
i n f o
Article history: Received 29 April 2008 Received in revised form 29 April 2008 Accepted 20 June 2008 Available online 26 August 2008 Keywords: Chemokine receptor Oral squamous cell carcinoma Cell migration Lymph node metastasis
s u m m a r y Tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. The present study was designed to examine the expression of chemokine receptor CCR7 in oral squamous cell carcinoma (OSCC), and to investigate the possible role of CCR7/CCL21 interaction in neck lymph node metastasis of OSCC. By using immunohistochemistry, RT-PCR and Western Blot, expression of CCR7 was examined in 85 cases of oral squamous cell carcinoma, and Tca8113 and ACC cell lines. CCL21-mediated cell migration was assayed in Matrigel-coated chemotaxis chamber. In vitro adhesion assay was shown for banding of tumor cell lines to submandibular lymph nodes with or without anti-CCR7 antibody treatment. Immunohistochemical staining showed 65.9% (56/85) of positive CCR7 expression in OSCC tissues. CCR7 expression was significantly higher in patients with lymph node metastasis compared with those without lymph node metastasis (P = 0.015) and was also associated with tumor size (P = 0.014), and clinical stage (P = 0.009). RT-PCR and Western Blot also confirmed positive CCR7 expression in oral squamous cell carcinoma and Tca8113 cell line, and negative CCR7 expression in normal oral mucosa and ACC cell line. CCL21 stimulation increased the ability of CCR7-positive Tca8113 cells passing through the Matrigel membrane. CCR7-positive Tca8113 cells also showed stronger adhesion to lymph nodes, which could be partly blocked by anti-CCR7 antibody incubation. These results indicated that the chemotactic CCR7/CCL21 interaction may be a possible mechanism for induction of directional lymph node metastasis by oral squamous cell carcinoma. Ó 2008 Elsevier Ltd. All rights reserved.
Introduction Oral squamous cell carcinomas (OSCCs) are characterized by a high degree of local invasion and a high rate of metastases to cervical lymph nodes. In particular, lymph node metastasis is an important prognostic factor for patients with oral carcinoma. Metastasis represents a highly organized, non-random, organ-specific, and multi-step processes.1 Although many molecules, such as integrins and matrix metalloproteinases (MMPs), play a key role in cancer cell invasion and metastasis,2–4 however, the precise factors and mechanisms affecting its preferred migration and invasion into cervical lymph nodes remain unknown. The chemokines comprise a family of small basic chemotactic proteins that mediate their effects by binding to G-protein-coupled receptors and play a key role in directing extravasation of inflammatory cells.5–7 A recent study reported that high levels of expres-
* Corresponding author. Address: Key Laboratory of Oral Biomedical Engineering, Ministry of Education of China, School of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, PR China. Tel.: +86 27 87646313; fax: +86 27 87873260. E-mail address: [email protected] (Z.J. Shang). 1368-8375/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2008.06.005
sion of chemokine receptors CXCR4 and CCR7 were found in breast cancer cells and the determination of the metastatic destination of tumor cells.8 It is well known that tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. Therefore, cancer cells might use chemokine-mediated mechanisms during the process of lymph node metastasis of cancer cells such as those regulating lymphocyte trafficking.9,10 In previous study, some authors examined the expression of CXCR4 in gastrointestinal carcinomas and oral cancers, and raised the possibility that CXCR4 could be involved in lymph node metastasis of these cancers.11,12 However, different chemokine receptors might determine distinct metastasis of cancer cells to specific organs. In fact, Murakami et al. found that chemokine receptors expressed by cancer cells may act to increase nodal metastasis (CCR7), increase adhesion to vascular endothelial cells (CXCR4) and promote distant metastasis, and/or allow malignant cells to evade host responses (CCR10), using a mouse model of melanoma.13 Here, we firstly reported the expression of CCR7 in OSCC, and its correlation with clinicopathological factors (especially nodal status) of the patient. We also investigated the migration of CCR7-expressing Tca8113 cells to Matrigel membrane under the
Z.J. Shang et al. / Oral Oncology 45 (2009) 480–485
chemoxis of its ligand CCL21, and the adhering of Tca8113 cells to submandibular lymph nodes with or without pretreatment with anti-CCR7 antibody. Materials and methods Cell lines and cell culture The TCa8113 cell line was established from a patient with human oral SCC of the tongue14, and ACC cell line from a patient with salivary adenoid cystic carcinoma.15 Both cell lines were purchased from China Center for Type Culture Collection (CCTCC). ACC and TCa8113 cells were maintained in DMEM supplemented with 10% (v/v) FCS, 100 g/ml streptomycin, and 100 U/ml penicillin in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Patients, specimen collection and preparation Our patient population consisted of 85 OSCC patients who received treatment at the Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, between 2004 and 2006. The diagnosis was pathologically conformed, and the classification of oral SCCs, including primary tumors (T), regional lymph nodes (N), distant metastasis (M) and stage grouping, was determined according to the International Union Against Cancer (UICC) rules for Head and Neck Cancer (Tumornode-metastasis, TNM classification, 1997). Patients undergoing preoperative chemotherapy and/or radiation therapy were not included. Local Ethical Committee approval was granted for the use of surgical trimming and informed consent was also obtained from the patients before surgery. About 10 cases of adjacent normal mucosa tissues were chosen as controls. All the specimens were collected immediately upon surgery, fixed in 10% formaldehyde solution, embedded in paraffin and used for pathological evaluation. Initially, hematoxylin and eosin (H&E) stained sections were examined by light microscopy to confirm histopathology. Five micrometer thick sections were cut and mounted on glass slides for the staining procedure. Immunohistochemical staining and evaluation Sections were deparaffinized in xylene for 10 min each and then were rehydrated through graded alcohols. To inhibit endogenous peroxide activity, sections were immersed in 100% methanol containing 0.3% hydrogen peroxide for 40 min. Then, they were put in a microwave oven in a jar filled with 10 mmol/L sodium citrate buffer (pH 6.0) for 10 min. Following this treatment, sections were allowed to cool at room temperature. Sections were incubated with normal goat serum (ZYMED, USA) at RT for 20 min and then incubated with a monoclonal anti-human CCR7 antibody (R&D Systems Inc., USA, MAB 197) at room temperature for 1 h. A negative control was performed in all cases by omitting the primary antibody. After the incubation period, sections were washed three times with PBS. Then, they were incubated with the linking reagent (biotinylated antiimmunoglobulin, ZYMED, USA) at room temperature for 1 h. After being washed three times with PBS, the sections were incubated with a complex of avidin DH and biotinlylated enzyme (ZYMED, USA) at room temperature for 30 min. Then, sections were washed three times with PBS again and incubated with a mixture of an equal volume of 0.02% hydrogen peroxide and diaminobenzidine tetrahydrochloride (Zhongshan Ltd., China) for 1 min in the dark. After chromogen development, sections were washed in water and counterstained with hematoxylin. The stained slides were investigated independently by two pathologists who had no knowledge of the clinical parameters and outcomes. For microscope analysis of the slides, they each se-
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lected five high-powered fields, whereby each field contained more than 200 tumor cells, and counted both positive and negative cancer cells. In total, at least 1000 tumor cells were counted. We calculated the average of 10 readings of positive cells as a percentage of all the cells to determine staining scores. We judged the expression of CCR7 according to histograms of the staining scores. Since the histogram of the CCR7 staining score showed two peaks, the bottom value of 30% between the two peaks was taken as the cut-off point. All SCC cases were graded as positive cases of CCR7 expression (CCR7-positive SCCs, over 30% CCR7 positive tumor cells) and negative cases (CCR7-negative SCCs, less than 30% CCR7 positive tumor cells) for the subsequent statistical analyses. Reverse Transcription-PCR Cells (TCa8113, ACC) grown in monolayers were harvested at early confluence. The preparation of cytoplasmic RNA and reverse transcription was performed as described previously. Then 1 ll of the products was subjected to PCR amplification using GeneAmp PCR System 9700 (ABI, USA) according to the manufacturer’s instructions. Semiquantitative RT-PCR was performed as described previously. PCR cycling conditions for CCR7 mRNA were: 95 °C for 5 min; then 30 cycles of 94 °C for 30 s, 60 °C for 1 min, 72 °C for 30 s; and finally extension at 72 °C for 7 min. The primer sequences of human CCR7 were designed as follows: forward 5-TCCTTCTCATCAGCAAGCTGTC-3 and reverse 5-GAGGCAGCCCAGGTCCTTGAAG3. Experiments were performed three times, and densitometric analysis of each set of data. PCR products were resolved by electrophoresis in 1–2% agarose gels and visualised by ethidium bromide staining. The densities of the CCR7 bands were divided by those of the b-actin bands. Protein extraction and Western Blot analysis Cell lysates were prepared by adding 200 AL ice-cold lysis buffer (0.5% Triton X-100, 50 mmol/L Tris (pH 7.2), 140 mmol/L NaCl, 10 mmol/L EDTA, 50 mmol/L NaF, 1 mmol/L Na3VO4) containing the protease inhibitor cocktail Complete Mini (Roche, Mannheim, Germany). Protein sample were mixed with an equal volume of 2sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer, boiled for 5 min, and then separated using 10% SDS–PAGE gels with 20 ug of protein loaded perlane. After electrophoresis, proteins were transferred to PVDF membranes by semi-dry electrophoretic transfer. The membranes were blocked in 5% dry milk, rinsed and then incubated with primary antibody of CCR7 overnight at 4 °C. The primary antibody was removed, membranes were washed four times, and followed by horseradish peroxidase-conjugated secondary Abs. Detection was then performed using an enhanced chemiluminescence kit and exposed to X-ray film. In vitro assay of cancer cells adhering to lymph nodes The adhesion assay was performed by the method of Stauder et a1.16,17 with some modifications. In brief, after treatment with or without anti-CCR7 antibody, 100 lL of 2 106 Tca8113 and ACC cells were suspended in HEPES-buffered RPMI 1640, and then incubated on 8 lL fresh-frozen sections of submandibular lymph nodes in a wax pen circle. These lymph nodes were taken from surgical or biopsy specimens pathologically determined to be normal. The sections were rotated on a shaker for 30 min at 4 °C during agitation. Medium and nonadherent cells were removed by rapping the edge of the slide against an absorbent towel. Subsequently, slides were fixed overnight in 1% paraformaldehyde in PBS, stained with hematoxylin, and counted under microscope (Olympus, Tokyo, Japan). Each value was expressed as a mean number of three different fields. Five experiments were done independently and the results were compared.
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In vitro migration assay In vitro cell migration was assayed in 24-well cell culture plates using inserts with 8 lm pore membranes (Costar, USA) according to the method mentioned by Katayama et al.18 In brief, membrane was precoated with 20 ll per insert of Matrigel (BD PharMingen, Bedford, MA) diluted 1:1 by RPMI 1640. TCa8113 and/or ACC cells (1 104) were resuspended in 500 ll migration buffer (DMEM/0.5% bovine serum albumin) per well and loaded into the upper compartment of the chamber. The lower compartment of the chamber was loaded with or without 50 ng/ml recombinant human CCL21 (R&D Systems) in 500 ll migration buffer. After incubation at 37 °C for 24 h, the cells on the lower surface of membrane were fixed in 70% ethanol, stained with hematoxylin, and counted under microscope (Olympus, Tokyo, Japan). Each value was expressed as a mean number of five different fields. Five experiments were done independently and the results were compared. Statistical analysis The association between CCR7 expression and clinicopathological factors was analyzed by the v2 test. The cancer cell numbers of adhesion and migration between different groups were analyzed by student t test. All statistical analysis was performed with SPSS 13.0 for windows software (SPSS, Inc., Chicago, IL, USA). For all tests, a P-value less than 0.05 was considered to be significant. Results Expression of CCR7 at mRNA and protein levels in primary OSCCs The distribution of CCR7 protein expression in oral SCC specimens was examined by immunohistochemical analysis. The cyto-
plasm and plasma membranes of tumor cells were positively stained ( Fig. 1A and B), whereas normal squamous epithelium had nearly negative staining in cytoplasm (Fig. 1C). Strong staining for CCR7 protein was observed in some infiltrating inflammatory cells in the specimens. 56 of 85 OSCC cases (65.9%) were positive for expression of CCR7 protein, and 1 of 10 (10%) adjacent normal mucosa were moderate positive for CCR7 staining. We also found the strong CCL21 protein expression observed in normal submandibular lymph node (Fig. 1D). To confirm these immunohistochemical findings, we analyzed protein extracts from oral cancer cell lines, adjacent normal mucosa and primary OSCC samples. Western Blotting showed positive expression of CCR7 in primary tissue and TCa8113 cell lines increased, and negative expression in the normal mucosa tissue and ACC cell lines were negative (Fig. 2). PCR products were cloned and then sequenced to be confirmed as identical to the expected fragments of cDNA in the CCR7 gene, as described in ‘‘Materials and Methods”. We found that CCR7 mRNA expression was detected in the primary tumors and TCa8113 cell lines, but not detected in ACC and adjacent normal mucosa (Fig. 3). Correlations between CCR7 expression and clinicopathological factors Table 1 summarized the relationship between the CCR7 expression and clinicopathological factors of the 85 OSCCs. The CCR7 expression was significantly correlated with cervical lymph node metastasis (P = 0.015). The CCR7-expressing cancers were characterized by significant frequent lymph node metastasis. There were also relatively obvious differences in tumor size (P = 0.014), and clinical stage (P = 0.009) between the two groups. However, there was no significant difference between CCR7 expression and age and gender. These findings demonstrated that the positive expression of CCR7 may play a critical role in cervical lymph node metastasis, and therefore influence the treatment outcomes.
Figure 1 Immunohistochemical staining of CCR7 in oral SCCs, normal tissue and lymph node. A and B showed strong expression of CCR7 in the cytoplasmic and plasma membrane of oral squamous cell carcinoma. C showed negative staining of CCR7 in normal oral mucosa. D showed positive CCL21 expression in a submandibular lymph node.
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Table 2 Comparison of adhering cells between Tca8113 and ACC cell lines treated or untreated with anti-CCR7 antibody
Figure 2 Western Blot analysis for CCR7 and b-actin protein expression in tumor tissue, cancer cell lines and normal oral mucosa. 1–5: OSCCs tissue, 6: normal mucosa tissue, 7: TCa8113 cell line, 8: ACC cell line.
Groups
No. of adhering cells (mean ± SE)
Tca8113 Tca8113 + CCR7 ACC ACC + CCR7
44.2 ± 5.05 21.6 ± 4.04 17.5 ± 2.16 11.3 ± 2.67
a b c
Figure 3 CCR7 and b-actin mRNA expression in tumor tissue, cancer cell lines and normal oral mucosa. M: Marker, 1–4: OSCCs tissue, 5: TCa8113 cell line, 6: ACC cell line, 7: normal mucosa tissue.
Table 1 CCR7 expression was associated with clinicopathological factors of OSCC patients Clinicopathological factors
No. of cases
CCR7 expression Positive
Negative
v2 test
P value
Age <40 P40
21 64
14 42
7 22
0.008
N.S.
Gender Male Female
53 32
31 25
22 7
3.422
N.S.
Tumor size T1/2 T3/4
49 36
27 29
22 7
5.982
0.014
Nodal metastasis No Yes
46 39
25 31
21 8
5.934
0.015
Clinical stage I + II III + IV
48 37
26 30
22 7
6.734
0.009
N.S., Not significant.
Anti-CCR7 antibody inhibited adhesion of cancer cells to lymph node The CCR7-positive Tca8113 cells showed stronger ability to adhere to lymph nodes as compared to CCR7-negative ACC cells. The adhesive capacity of CCR7-positive Tca8113 cells obviously decreased after incubation with anti-CCR7 antibody for 24 h (Table 2). In consideration of the abundance of CCL-21 in lymph nodes and the difference of CCR7 expression pattern in Tca8113 and ACC cells, these results might suggest that positive CCR7 expression plays an important role in mediating directional lymph node metastasis. CCL- 21-mediated migration of OSCC cells Both Tca8113 and ACC cells were tested for their ability to cross the reconstituted basement membrane barrier under the chemotaxis of CCL-21. Fig. 4A showed that Tca8113 cells migrated to CCL-21 in a dose-dependent manner (P < 0.01 for each CCL-21 concentration versus migration to control medium). Under the chemotaxis of 50 ng/ml CCL-21, Tca8113 cells showed stronger ability to
t test 3.494 4.858 1.806
df
P value
18 18 18
0.003a 0.000b 0.088c
Comparison between Tca8113 and Tca8113 + CCR7. Comparison between Tca8113 and ACC. Comparison between ACC and ACC + CCR7.
pass through the reconstituted basement membrane as compared to medium control. Although the ACC cells also migrated to 50 ng/ml CCL-21, there is significant difference in migrated tumor cell numbers between TCa8113 cells and ACC cells (Fig. 4B). Again, these findings supported our hypotheses that CCR7 may mainly mediate lymph node metastasis. Discussion The molecular basis of tumor cell migration and metastasis to lymph nodes in vivo is not fully understood, and is probably a result of the interaction between tumor cells and a supportive microenvironment.19 Malignant cells that have the capability to metastasize to a particular organ may have various properties supporting their tissue invasion or growth such as enhanced adherence to the microvascular cells of the organ, higher responsiveness to chemotactic signals released from the target organs and increased response to local soluble or tissue associated growth signals in the target organ.20–22 Recently, Muller et al.23 and Wang et al.24 suggested that CCR7 might play a role in directing SCC cells to the draining lymph nodes and contribute to the frequent presence of lymph node metastases in SCC patients. In this study, we investigated the mechanism of lymph node metastasis of oral SCC, focusing on the chemokine–receptor system. The findings obtained from the present series of experiments are as follows. First, 65.9% of oral SCC samples expressed CCR7. Second, the following clinicopathological features differed significantly between CCR7-positive and -negative cases: (a) lymph node metastasis; (b) tumor sizes; and (c) clinical stage. Third, CCL21, the ligand for CCR7, exhibited peak levels of expression in cervical lymph nodes. Forth, ACC cells, which prefers to metastasize to lung through hematogeneous tract and to invade the nerve, showed no expression of CCR7 at protein and mRNA levels. A natural mutation in mice designated plt that results in the loss of one of the forms of murine CCL2125 and targeted disruption of the CCR7 gene causes impaired homing of T cells to secondary lymphoid organs26, suggesting that the interaction between CCL21/ 6Ckine and CCR7 plays a critical role for lymphocytes to migrate to lymph nodes. Thus, our results suggested that CCR7/CCL21 interaction may mediate the homing of oral SCC cells to regional lymph nodes. A recent report by Muller et al.8 showed that chemokine receptors CXCR4 and CCR7 were highly expressed in breast cancer cells but not normal breast tissue and therefore provided evidence for preferential homing of breast cancer to lymph node. Murakami et al.13 also found that chemokine receptors expressed by cancer cells may act to increase nodal metastasis (CCR7), increase adhesion to vascular endothelial cells (CXCR4) and promote distant metastasis, and/or allow malignant cells to evade host responses (CCR10), using a mouse model of melanoma. Wiley et al .10 showed that expression of CCR7 enhanced metastasis of murine melanoma cells to draining lymph nodes in mouse models and that inhibition of CCL21/6Ckine blocked the metastasis to draining lymph nodes. Intravenous administration of CCR7-B16 or control B16 cells yielded comparable numbers of metastatic nodules in the lungs
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Figure 4 Migration assay of Tca8113 and ACC cell lines under the chemoxis of CCL21. A, the dose-dependent migration of Tca8113 cells under stimulation of CCL21. B, the difference of migrated cells between Tca8113 and ACC cell lines with or without stimulation of 50 ng/ml CCL21.
without forming visible metastasis in the lymph nodes, indicating that CCR7 expression specifically enhances the metastatic ability of B16 melanoma cells through a lymph-mediated but not a blood-borne pathway. These studies provided strong supporting evidence for our current findings. The interaction between CCR7 and CCL21/6Ckine may play crucial roles in the metastasis of cancer cells by direct effects on tumor cell migration and invasion. Recently, however, some authors found that the chemokine receptor CXCR4 was highly expressed in oral SCCs, and concluded that the interaction between CXCR4 and its ligand CXCL12/SDF-1 played an important role in lymph node metastasis of oral SCCs.12 We thought these results were not conflicted with that in the present study because CXCR4/SDF-1 interaction might result in tumor angiogenesis and/or chemotaxis or directed migration of tumor cells from their primary site via the circulation to preferential sites of metastasis. Migration and adhesion of tumor cells to target organ are crucial steps in tumor metastasis. Human cell-derived chemokines attracted and activated cancer cells with their receptors and the presence of this receptor, CCR7, and promoted cancer cell migration.27 In vitro assay analysis showed that Tca8113 cells migrated to CCL-21 in a dose-dependent manner. Under the chemotaxis of 50 ng/ml CCL-21, Tca8113 cells showed stronger ability to pass through the reconstituted basement membrane as compared to medium control. Although the ACC cells also migrated to 50 ng/ ml CCL-21, there is significant difference in migrated tumor cell numbers between TCa8113 cells and ACC cells. In CCR7-positive gastric cancer cell lines, Mashino et al. reported that the migration and invasive capabilities of cancer cells were increased by CCL21 stimulation, because of a transient increase in both intracellular F-actin and distinct pseudopodia formation.8 Furthermore, the chemokine signaling of CCL21 via CCR7 induced the directional migration and invasion of breast cancer cells through the basement membrane.9 This strongly supported the hypothesis that malignant cells may use CCR7 in lymph node metastasis dissemination. In our study, CCR7-expressing Tca8113 cells were easier to adhere to frozen section of CCL21-expressing cervical lymph nodes which could partly blocked after incubation with anti-CCR7 antibody for 24 h. CCR7-negative ACC cells, however, showed nearly no ability of adhering to CCL21-expressing cervical lymph nodes. Again, these data strongly suggested that the chemokine gradient affected the recruiting of the oral SCC cells to the lymph node. These results also indicate that the cervical lymph node might retain other chemotactic factors that influence the adhesion and lymph node metastasis of oral SCC cells. It can be considered that several factors, including inflammation of the tissues or degradation of proteins, affected the results, but every cervical lymph node
may have a different concentration of CCL21. We often encounter the skip lymph node metastasis in patients with oral SCC, which cannot be understood by the lymphatic stream, so-called sentinel lymph node metastasis. These results lead us to speculate that the lymph node with a high concentration of CCL21 might be involved in the sentinel lymph node metastasis. It has been previously reported that oral SCC cells easily intravasated into the circulation despite the establishment of distant metastasis.28 Such observations indicate that, for the establishment of distant metastasis in oral SCC, extravasation or ectopic growth potential may be more important, and microenvironmental factors, including adhesion molecules or growth factors, in these organs may not favor metastasis of the oral SCC cells. In conclusion, the chemotactic CCR7/CCL21 interaction may be a possible mechanism for induction by cancer cells of lymph node metastasis and tissue invasion. This hypothesis was supported by the findings that the expression of CCR7 was observed in 65.9% of OSCC tissues and was significantly correlated with the presence of lymph node metastasis, and the findings that increased migration under CCL21 stimulation and enhanced adhesion of Tca8113 to lymph nodes. These findings and those of previous studies suggest that CCR7 could be associated with directional lymph node metastasis of oral squamous cell carcinoma. Further, studies will needed to understand which signal pathway is involved in this process, and whether blocking of CCR7/CCL21 interaction by silencing CCR7 gene can inhibit the metastasis of OSCC to cervical lymph nodes or not.
Conflict of interest statement None declared. Acknowledgment This work was supported by Program for New Century Excellent Talents from the Ministry of Education of the People’s Republic of China. We also thank professor Xin Ming Chen for his kind help in evaluating the immunohistochemical staining. References 1. Nicolson GL. Paracrine and autocrine growth mechanisms in tumor metastasis to specific sites with particular emphasis on brain and lung metastasis. Cancer Metast Rev 1993;12:325–43. 2. Ramos DM, But M, Regezi J, et al. Expression of integrin beta 6 enhances invasive behavior in oral squamous cell carcinoma. Matrix Biol 2002;21: 297–307.
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Contents lists available at ScienceDirect
Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
Expression of chemokine receptor CCR7 is associated with cervical lymph node metastasis of oral squamous cell carcinoma Zheng Jun Shang a,b,*, Ke Liu b, Zhe Shao b a b
First Department of Oral and Maxillofacial Surgery, School of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, PR China Key Laboratory of Oral Biomedical Engineering, Ministry of Education of China, School of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, PR China
a r t i c l e
i n f o
Article history: Received 29 April 2008 Received in revised form 29 April 2008 Accepted 20 June 2008 Available online 26 August 2008 Keywords: Chemokine receptor Oral squamous cell carcinoma Cell migration Lymph node metastasis
s u m m a r y Tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. The present study was designed to examine the expression of chemokine receptor CCR7 in oral squamous cell carcinoma (OSCC), and to investigate the possible role of CCR7/CCL21 interaction in neck lymph node metastasis of OSCC. By using immunohistochemistry, RT-PCR and Western Blot, expression of CCR7 was examined in 85 cases of oral squamous cell carcinoma, and Tca8113 and ACC cell lines. CCL21-mediated cell migration was assayed in Matrigel-coated chemotaxis chamber. In vitro adhesion assay was shown for banding of tumor cell lines to submandibular lymph nodes with or without anti-CCR7 antibody treatment. Immunohistochemical staining showed 65.9% (56/85) of positive CCR7 expression in OSCC tissues. CCR7 expression was significantly higher in patients with lymph node metastasis compared with those without lymph node metastasis (P = 0.015) and was also associated with tumor size (P = 0.014), and clinical stage (P = 0.009). RT-PCR and Western Blot also confirmed positive CCR7 expression in oral squamous cell carcinoma and Tca8113 cell line, and negative CCR7 expression in normal oral mucosa and ACC cell line. CCL21 stimulation increased the ability of CCR7-positive Tca8113 cells passing through the Matrigel membrane. CCR7-positive Tca8113 cells also showed stronger adhesion to lymph nodes, which could be partly blocked by anti-CCR7 antibody incubation. These results indicated that the chemotactic CCR7/CCL21 interaction may be a possible mechanism for induction of directional lymph node metastasis by oral squamous cell carcinoma. Ó 2008 Elsevier Ltd. All rights reserved.
Introduction Oral squamous cell carcinomas (OSCCs) are characterized by a high degree of local invasion and a high rate of metastases to cervical lymph nodes. In particular, lymph node metastasis is an important prognostic factor for patients with oral carcinoma. Metastasis represents a highly organized, non-random, organ-specific, and multi-step processes.1 Although many molecules, such as integrins and matrix metalloproteinases (MMPs), play a key role in cancer cell invasion and metastasis,2–4 however, the precise factors and mechanisms affecting its preferred migration and invasion into cervical lymph nodes remain unknown. The chemokines comprise a family of small basic chemotactic proteins that mediate their effects by binding to G-protein-coupled receptors and play a key role in directing extravasation of inflammatory cells.5–7 A recent study reported that high levels of expres-
* Corresponding author. Address: Key Laboratory of Oral Biomedical Engineering, Ministry of Education of China, School of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, PR China. Tel.: +86 27 87646313; fax: +86 27 87873260. E-mail address: [email protected] (Z.J. Shang). 1368-8375/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2008.06.005
sion of chemokine receptors CXCR4 and CCR7 were found in breast cancer cells and the determination of the metastatic destination of tumor cells.8 It is well known that tumor cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. Therefore, cancer cells might use chemokine-mediated mechanisms during the process of lymph node metastasis of cancer cells such as those regulating lymphocyte trafficking.9,10 In previous study, some authors examined the expression of CXCR4 in gastrointestinal carcinomas and oral cancers, and raised the possibility that CXCR4 could be involved in lymph node metastasis of these cancers.11,12 However, different chemokine receptors might determine distinct metastasis of cancer cells to specific organs. In fact, Murakami et al. found that chemokine receptors expressed by cancer cells may act to increase nodal metastasis (CCR7), increase adhesion to vascular endothelial cells (CXCR4) and promote distant metastasis, and/or allow malignant cells to evade host responses (CCR10), using a mouse model of melanoma.13 Here, we firstly reported the expression of CCR7 in OSCC, and its correlation with clinicopathological factors (especially nodal status) of the patient. We also investigated the migration of CCR7-expressing Tca8113 cells to Matrigel membrane under the
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chemoxis of its ligand CCL21, and the adhering of Tca8113 cells to submandibular lymph nodes with or without pretreatment with anti-CCR7 antibody. Materials and methods Cell lines and cell culture The TCa8113 cell line was established from a patient with human oral SCC of the tongue14, and ACC cell line from a patient with salivary adenoid cystic carcinoma.15 Both cell lines were purchased from China Center for Type Culture Collection (CCTCC). ACC and TCa8113 cells were maintained in DMEM supplemented with 10% (v/v) FCS, 100 g/ml streptomycin, and 100 U/ml penicillin in a humidified atmosphere of 95% air and 5% CO2 at 37 °C. Patients, specimen collection and preparation Our patient population consisted of 85 OSCC patients who received treatment at the Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, between 2004 and 2006. The diagnosis was pathologically conformed, and the classification of oral SCCs, including primary tumors (T), regional lymph nodes (N), distant metastasis (M) and stage grouping, was determined according to the International Union Against Cancer (UICC) rules for Head and Neck Cancer (Tumornode-metastasis, TNM classification, 1997). Patients undergoing preoperative chemotherapy and/or radiation therapy were not included. Local Ethical Committee approval was granted for the use of surgical trimming and informed consent was also obtained from the patients before surgery. About 10 cases of adjacent normal mucosa tissues were chosen as controls. All the specimens were collected immediately upon surgery, fixed in 10% formaldehyde solution, embedded in paraffin and used for pathological evaluation. Initially, hematoxylin and eosin (H&E) stained sections were examined by light microscopy to confirm histopathology. Five micrometer thick sections were cut and mounted on glass slides for the staining procedure. Immunohistochemical staining and evaluation Sections were deparaffinized in xylene for 10 min each and then were rehydrated through graded alcohols. To inhibit endogenous peroxide activity, sections were immersed in 100% methanol containing 0.3% hydrogen peroxide for 40 min. Then, they were put in a microwave oven in a jar filled with 10 mmol/L sodium citrate buffer (pH 6.0) for 10 min. Following this treatment, sections were allowed to cool at room temperature. Sections were incubated with normal goat serum (ZYMED, USA) at RT for 20 min and then incubated with a monoclonal anti-human CCR7 antibody (R&D Systems Inc., USA, MAB 197) at room temperature for 1 h. A negative control was performed in all cases by omitting the primary antibody. After the incubation period, sections were washed three times with PBS. Then, they were incubated with the linking reagent (biotinylated antiimmunoglobulin, ZYMED, USA) at room temperature for 1 h. After being washed three times with PBS, the sections were incubated with a complex of avidin DH and biotinlylated enzyme (ZYMED, USA) at room temperature for 30 min. Then, sections were washed three times with PBS again and incubated with a mixture of an equal volume of 0.02% hydrogen peroxide and diaminobenzidine tetrahydrochloride (Zhongshan Ltd., China) for 1 min in the dark. After chromogen development, sections were washed in water and counterstained with hematoxylin. The stained slides were investigated independently by two pathologists who had no knowledge of the clinical parameters and outcomes. For microscope analysis of the slides, they each se-
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lected five high-powered fields, whereby each field contained more than 200 tumor cells, and counted both positive and negative cancer cells. In total, at least 1000 tumor cells were counted. We calculated the average of 10 readings of positive cells as a percentage of all the cells to determine staining scores. We judged the expression of CCR7 according to histograms of the staining scores. Since the histogram of the CCR7 staining score showed two peaks, the bottom value of 30% between the two peaks was taken as the cut-off point. All SCC cases were graded as positive cases of CCR7 expression (CCR7-positive SCCs, over 30% CCR7 positive tumor cells) and negative cases (CCR7-negative SCCs, less than 30% CCR7 positive tumor cells) for the subsequent statistical analyses. Reverse Transcription-PCR Cells (TCa8113, ACC) grown in monolayers were harvested at early confluence. The preparation of cytoplasmic RNA and reverse transcription was performed as described previously. Then 1 ll of the products was subjected to PCR amplification using GeneAmp PCR System 9700 (ABI, USA) according to the manufacturer’s instructions. Semiquantitative RT-PCR was performed as described previously. PCR cycling conditions for CCR7 mRNA were: 95 °C for 5 min; then 30 cycles of 94 °C for 30 s, 60 °C for 1 min, 72 °C for 30 s; and finally extension at 72 °C for 7 min. The primer sequences of human CCR7 were designed as follows: forward 5-TCCTTCTCATCAGCAAGCTGTC-3 and reverse 5-GAGGCAGCCCAGGTCCTTGAAG3. Experiments were performed three times, and densitometric analysis of each set of data. PCR products were resolved by electrophoresis in 1–2% agarose gels and visualised by ethidium bromide staining. The densities of the CCR7 bands were divided by those of the b-actin bands. Protein extraction and Western Blot analysis Cell lysates were prepared by adding 200 AL ice-cold lysis buffer (0.5% Triton X-100, 50 mmol/L Tris (pH 7.2), 140 mmol/L NaCl, 10 mmol/L EDTA, 50 mmol/L NaF, 1 mmol/L Na3VO4) containing the protease inhibitor cocktail Complete Mini (Roche, Mannheim, Germany). Protein sample were mixed with an equal volume of 2sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer, boiled for 5 min, and then separated using 10% SDS–PAGE gels with 20 ug of protein loaded perlane. After electrophoresis, proteins were transferred to PVDF membranes by semi-dry electrophoretic transfer. The membranes were blocked in 5% dry milk, rinsed and then incubated with primary antibody of CCR7 overnight at 4 °C. The primary antibody was removed, membranes were washed four times, and followed by horseradish peroxidase-conjugated secondary Abs. Detection was then performed using an enhanced chemiluminescence kit and exposed to X-ray film. In vitro assay of cancer cells adhering to lymph nodes The adhesion assay was performed by the method of Stauder et a1.16,17 with some modifications. In brief, after treatment with or without anti-CCR7 antibody, 100 lL of 2 106 Tca8113 and ACC cells were suspended in HEPES-buffered RPMI 1640, and then incubated on 8 lL fresh-frozen sections of submandibular lymph nodes in a wax pen circle. These lymph nodes were taken from surgical or biopsy specimens pathologically determined to be normal. The sections were rotated on a shaker for 30 min at 4 °C during agitation. Medium and nonadherent cells were removed by rapping the edge of the slide against an absorbent towel. Subsequently, slides were fixed overnight in 1% paraformaldehyde in PBS, stained with hematoxylin, and counted under microscope (Olympus, Tokyo, Japan). Each value was expressed as a mean number of three different fields. Five experiments were done independently and the results were compared.
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In vitro migration assay In vitro cell migration was assayed in 24-well cell culture plates using inserts with 8 lm pore membranes (Costar, USA) according to the method mentioned by Katayama et al.18 In brief, membrane was precoated with 20 ll per insert of Matrigel (BD PharMingen, Bedford, MA) diluted 1:1 by RPMI 1640. TCa8113 and/or ACC cells (1 104) were resuspended in 500 ll migration buffer (DMEM/0.5% bovine serum albumin) per well and loaded into the upper compartment of the chamber. The lower compartment of the chamber was loaded with or without 50 ng/ml recombinant human CCL21 (R&D Systems) in 500 ll migration buffer. After incubation at 37 °C for 24 h, the cells on the lower surface of membrane were fixed in 70% ethanol, stained with hematoxylin, and counted under microscope (Olympus, Tokyo, Japan). Each value was expressed as a mean number of five different fields. Five experiments were done independently and the results were compared. Statistical analysis The association between CCR7 expression and clinicopathological factors was analyzed by the v2 test. The cancer cell numbers of adhesion and migration between different groups were analyzed by student t test. All statistical analysis was performed with SPSS 13.0 for windows software (SPSS, Inc., Chicago, IL, USA). For all tests, a P-value less than 0.05 was considered to be significant. Results Expression of CCR7 at mRNA and protein levels in primary OSCCs The distribution of CCR7 protein expression in oral SCC specimens was examined by immunohistochemical analysis. The cyto-
plasm and plasma membranes of tumor cells were positively stained ( Fig. 1A and B), whereas normal squamous epithelium had nearly negative staining in cytoplasm (Fig. 1C). Strong staining for CCR7 protein was observed in some infiltrating inflammatory cells in the specimens. 56 of 85 OSCC cases (65.9%) were positive for expression of CCR7 protein, and 1 of 10 (10%) adjacent normal mucosa were moderate positive for CCR7 staining. We also found the strong CCL21 protein expression observed in normal submandibular lymph node (Fig. 1D). To confirm these immunohistochemical findings, we analyzed protein extracts from oral cancer cell lines, adjacent normal mucosa and primary OSCC samples. Western Blotting showed positive expression of CCR7 in primary tissue and TCa8113 cell lines increased, and negative expression in the normal mucosa tissue and ACC cell lines were negative (Fig. 2). PCR products were cloned and then sequenced to be confirmed as identical to the expected fragments of cDNA in the CCR7 gene, as described in ‘‘Materials and Methods”. We found that CCR7 mRNA expression was detected in the primary tumors and TCa8113 cell lines, but not detected in ACC and adjacent normal mucosa (Fig. 3). Correlations between CCR7 expression and clinicopathological factors Table 1 summarized the relationship between the CCR7 expression and clinicopathological factors of the 85 OSCCs. The CCR7 expression was significantly correlated with cervical lymph node metastasis (P = 0.015). The CCR7-expressing cancers were characterized by significant frequent lymph node metastasis. There were also relatively obvious differences in tumor size (P = 0.014), and clinical stage (P = 0.009) between the two groups. However, there was no significant difference between CCR7 expression and age and gender. These findings demonstrated that the positive expression of CCR7 may play a critical role in cervical lymph node metastasis, and therefore influence the treatment outcomes.
Figure 1 Immunohistochemical staining of CCR7 in oral SCCs, normal tissue and lymph node. A and B showed strong expression of CCR7 in the cytoplasmic and plasma membrane of oral squamous cell carcinoma. C showed negative staining of CCR7 in normal oral mucosa. D showed positive CCL21 expression in a submandibular lymph node.
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Table 2 Comparison of adhering cells between Tca8113 and ACC cell lines treated or untreated with anti-CCR7 antibody
Figure 2 Western Blot analysis for CCR7 and b-actin protein expression in tumor tissue, cancer cell lines and normal oral mucosa. 1–5: OSCCs tissue, 6: normal mucosa tissue, 7: TCa8113 cell line, 8: ACC cell line.
Groups
No. of adhering cells (mean ± SE)
Tca8113 Tca8113 + CCR7 ACC ACC + CCR7
44.2 ± 5.05 21.6 ± 4.04 17.5 ± 2.16 11.3 ± 2.67
a b c
Figure 3 CCR7 and b-actin mRNA expression in tumor tissue, cancer cell lines and normal oral mucosa. M: Marker, 1–4: OSCCs tissue, 5: TCa8113 cell line, 6: ACC cell line, 7: normal mucosa tissue.
Table 1 CCR7 expression was associated with clinicopathological factors of OSCC patients Clinicopathological factors
No. of cases
CCR7 expression Positive
Negative
v2 test
P value
Age <40 P40
21 64
14 42
7 22
0.008
N.S.
Gender Male Female
53 32
31 25
22 7
3.422
N.S.
Tumor size T1/2 T3/4
49 36
27 29
22 7
5.982
0.014
Nodal metastasis No Yes
46 39
25 31
21 8
5.934
0.015
Clinical stage I + II III + IV
48 37
26 30
22 7
6.734
0.009
N.S., Not significant.
Anti-CCR7 antibody inhibited adhesion of cancer cells to lymph node The CCR7-positive Tca8113 cells showed stronger ability to adhere to lymph nodes as compared to CCR7-negative ACC cells. The adhesive capacity of CCR7-positive Tca8113 cells obviously decreased after incubation with anti-CCR7 antibody for 24 h (Table 2). In consideration of the abundance of CCL-21 in lymph nodes and the difference of CCR7 expression pattern in Tca8113 and ACC cells, these results might suggest that positive CCR7 expression plays an important role in mediating directional lymph node metastasis. CCL- 21-mediated migration of OSCC cells Both Tca8113 and ACC cells were tested for their ability to cross the reconstituted basement membrane barrier under the chemotaxis of CCL-21. Fig. 4A showed that Tca8113 cells migrated to CCL-21 in a dose-dependent manner (P < 0.01 for each CCL-21 concentration versus migration to control medium). Under the chemotaxis of 50 ng/ml CCL-21, Tca8113 cells showed stronger ability to
t test 3.494 4.858 1.806
df
P value
18 18 18
0.003a 0.000b 0.088c
Comparison between Tca8113 and Tca8113 + CCR7. Comparison between Tca8113 and ACC. Comparison between ACC and ACC + CCR7.
pass through the reconstituted basement membrane as compared to medium control. Although the ACC cells also migrated to 50 ng/ml CCL-21, there is significant difference in migrated tumor cell numbers between TCa8113 cells and ACC cells (Fig. 4B). Again, these findings supported our hypotheses that CCR7 may mainly mediate lymph node metastasis. Discussion The molecular basis of tumor cell migration and metastasis to lymph nodes in vivo is not fully understood, and is probably a result of the interaction between tumor cells and a supportive microenvironment.19 Malignant cells that have the capability to metastasize to a particular organ may have various properties supporting their tissue invasion or growth such as enhanced adherence to the microvascular cells of the organ, higher responsiveness to chemotactic signals released from the target organs and increased response to local soluble or tissue associated growth signals in the target organ.20–22 Recently, Muller et al.23 and Wang et al.24 suggested that CCR7 might play a role in directing SCC cells to the draining lymph nodes and contribute to the frequent presence of lymph node metastases in SCC patients. In this study, we investigated the mechanism of lymph node metastasis of oral SCC, focusing on the chemokine–receptor system. The findings obtained from the present series of experiments are as follows. First, 65.9% of oral SCC samples expressed CCR7. Second, the following clinicopathological features differed significantly between CCR7-positive and -negative cases: (a) lymph node metastasis; (b) tumor sizes; and (c) clinical stage. Third, CCL21, the ligand for CCR7, exhibited peak levels of expression in cervical lymph nodes. Forth, ACC cells, which prefers to metastasize to lung through hematogeneous tract and to invade the nerve, showed no expression of CCR7 at protein and mRNA levels. A natural mutation in mice designated plt that results in the loss of one of the forms of murine CCL2125 and targeted disruption of the CCR7 gene causes impaired homing of T cells to secondary lymphoid organs26, suggesting that the interaction between CCL21/ 6Ckine and CCR7 plays a critical role for lymphocytes to migrate to lymph nodes. Thus, our results suggested that CCR7/CCL21 interaction may mediate the homing of oral SCC cells to regional lymph nodes. A recent report by Muller et al.8 showed that chemokine receptors CXCR4 and CCR7 were highly expressed in breast cancer cells but not normal breast tissue and therefore provided evidence for preferential homing of breast cancer to lymph node. Murakami et al.13 also found that chemokine receptors expressed by cancer cells may act to increase nodal metastasis (CCR7), increase adhesion to vascular endothelial cells (CXCR4) and promote distant metastasis, and/or allow malignant cells to evade host responses (CCR10), using a mouse model of melanoma. Wiley et al .10 showed that expression of CCR7 enhanced metastasis of murine melanoma cells to draining lymph nodes in mouse models and that inhibition of CCL21/6Ckine blocked the metastasis to draining lymph nodes. Intravenous administration of CCR7-B16 or control B16 cells yielded comparable numbers of metastatic nodules in the lungs
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Figure 4 Migration assay of Tca8113 and ACC cell lines under the chemoxis of CCL21. A, the dose-dependent migration of Tca8113 cells under stimulation of CCL21. B, the difference of migrated cells between Tca8113 and ACC cell lines with or without stimulation of 50 ng/ml CCL21.
without forming visible metastasis in the lymph nodes, indicating that CCR7 expression specifically enhances the metastatic ability of B16 melanoma cells through a lymph-mediated but not a blood-borne pathway. These studies provided strong supporting evidence for our current findings. The interaction between CCR7 and CCL21/6Ckine may play crucial roles in the metastasis of cancer cells by direct effects on tumor cell migration and invasion. Recently, however, some authors found that the chemokine receptor CXCR4 was highly expressed in oral SCCs, and concluded that the interaction between CXCR4 and its ligand CXCL12/SDF-1 played an important role in lymph node metastasis of oral SCCs.12 We thought these results were not conflicted with that in the present study because CXCR4/SDF-1 interaction might result in tumor angiogenesis and/or chemotaxis or directed migration of tumor cells from their primary site via the circulation to preferential sites of metastasis. Migration and adhesion of tumor cells to target organ are crucial steps in tumor metastasis. Human cell-derived chemokines attracted and activated cancer cells with their receptors and the presence of this receptor, CCR7, and promoted cancer cell migration.27 In vitro assay analysis showed that Tca8113 cells migrated to CCL-21 in a dose-dependent manner. Under the chemotaxis of 50 ng/ml CCL-21, Tca8113 cells showed stronger ability to pass through the reconstituted basement membrane as compared to medium control. Although the ACC cells also migrated to 50 ng/ ml CCL-21, there is significant difference in migrated tumor cell numbers between TCa8113 cells and ACC cells. In CCR7-positive gastric cancer cell lines, Mashino et al. reported that the migration and invasive capabilities of cancer cells were increased by CCL21 stimulation, because of a transient increase in both intracellular F-actin and distinct pseudopodia formation.8 Furthermore, the chemokine signaling of CCL21 via CCR7 induced the directional migration and invasion of breast cancer cells through the basement membrane.9 This strongly supported the hypothesis that malignant cells may use CCR7 in lymph node metastasis dissemination. In our study, CCR7-expressing Tca8113 cells were easier to adhere to frozen section of CCL21-expressing cervical lymph nodes which could partly blocked after incubation with anti-CCR7 antibody for 24 h. CCR7-negative ACC cells, however, showed nearly no ability of adhering to CCL21-expressing cervical lymph nodes. Again, these data strongly suggested that the chemokine gradient affected the recruiting of the oral SCC cells to the lymph node. These results also indicate that the cervical lymph node might retain other chemotactic factors that influence the adhesion and lymph node metastasis of oral SCC cells. It can be considered that several factors, including inflammation of the tissues or degradation of proteins, affected the results, but every cervical lymph node
may have a different concentration of CCL21. We often encounter the skip lymph node metastasis in patients with oral SCC, which cannot be understood by the lymphatic stream, so-called sentinel lymph node metastasis. These results lead us to speculate that the lymph node with a high concentration of CCL21 might be involved in the sentinel lymph node metastasis. It has been previously reported that oral SCC cells easily intravasated into the circulation despite the establishment of distant metastasis.28 Such observations indicate that, for the establishment of distant metastasis in oral SCC, extravasation or ectopic growth potential may be more important, and microenvironmental factors, including adhesion molecules or growth factors, in these organs may not favor metastasis of the oral SCC cells. In conclusion, the chemotactic CCR7/CCL21 interaction may be a possible mechanism for induction by cancer cells of lymph node metastasis and tissue invasion. This hypothesis was supported by the findings that the expression of CCR7 was observed in 65.9% of OSCC tissues and was significantly correlated with the presence of lymph node metastasis, and the findings that increased migration under CCL21 stimulation and enhanced adhesion of Tca8113 to lymph nodes. These findings and those of previous studies suggest that CCR7 could be associated with directional lymph node metastasis of oral squamous cell carcinoma. Further, studies will needed to understand which signal pathway is involved in this process, and whether blocking of CCR7/CCL21 interaction by silencing CCR7 gene can inhibit the metastasis of OSCC to cervical lymph nodes or not.
Conflict of interest statement None declared. Acknowledgment This work was supported by Program for New Century Excellent Talents from the Ministry of Education of the People’s Republic of China. We also thank professor Xin Ming Chen for his kind help in evaluating the immunohistochemical staining. References 1. Nicolson GL. Paracrine and autocrine growth mechanisms in tumor metastasis to specific sites with particular emphasis on brain and lung metastasis. Cancer Metast Rev 1993;12:325–43. 2. Ramos DM, But M, Regezi J, et al. Expression of integrin beta 6 enhances invasive behavior in oral squamous cell carcinoma. Matrix Biol 2002;21: 297–307.
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