Digestive and Liver Disease
Volume 43, Issue 1 , Pages 40-47, January 2011

The CCL21/CCR7 pathway plays a key role in human colon cancer metastasis through regulation of matrix metalloproteinase-9

  • Jiang Li

      Affiliations

    • Department of Laparoscopic Surgery Unit, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, PR China
    • ,
  • Renhu Sun

      Affiliations

    • Department of Endoscopy Center, the Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, Nanjing 210029, Jiangsu Province, PR China
    • Corresponding Author InformationCorresponding author.
    • ,
  • Kaixiong Tao

      Affiliations

    • Department of Laparoscopic Surgery Unit, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, PR China
    • ,
  • Guobin Wang

      Affiliations

    • Department of Laparoscopic Surgery Unit, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, PR China
    • Corresponding Author InformationCorresponding author at: Department of Laparoscopic Surgery Unit, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Road, Wuhan 430022, PR China. Tel.: +86 27 85726301.

Received 3 December 2009; accepted 20 May 2010. published online 08 July 2010.

Article Outline

Abstract 

Purpose

CC chemokine receptor 7 (CCR7) and matrix metalloproteinase-9 (MMP-9) have been associated with lymph node metastasis in human colon cancer. Studies have suggested a potential link between CCR7 and MMP-9 in cancer; however, the molecular mechanism by which C–C ligand 21/CCR7 promotes tumour dissemination in human colon cancer is not well understood. Thus, we aimed to determine whether MMP-9 is regulated by the C–C ligand 21/CCR7 in human colon cancer.

Method

RNA interference technology was employed to detect effect of CCR7 deficiency on the expression of MMP-9 in SW480 human colon cancer cells. We also evaluated the ability of CCR7 short hairpin RNA to inhibit MMP-9 production and tumour invasion in a xenografted mouse model by using whole-body fluorescence imaging and gelatin zymography.

Result

We found that CCR7 short hairpin RNA significantly inhibited C–C ligand 21/CCR7-induced up-regulation of MMP-9 in SW480 cells. Furthermore, knockdown of CCR7 significantly limited the production of MMP-9 and colon cancer metastasis in a xenografted mouse model. Mice that received SW480/control cells had progressively enlarging tumours and more lymphatic metastases, and these animals did not survive as long as mice that received SW480/CCR7− cells.

Conclusion

MMP-9 and CCR7 may be useful targets for the treatment of lymphatic metastasis in colon cancer.

Abbreviations: MMP-9, matrix metalloproteinase-9, CCR7, CC chemokine receptor 7, ECM, extracellular matrix, CCL21, C–C ligand 21

Keywords: Matrix metalloproteinase-9, CC chemokine receptor 7, Whole-body fluorescence imaging, Colon cancer metastasis

 

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1. Introduction 

Colon cancer is the third most prevalent cancer worldwide, and in recent years, its incidence has increased in developing nations. The main cause of colon cancer-related deaths is the lymphatic dissemination of malignant cells from the primary site [1], [2]. Recent clinical pathological studies have indicated that the spread of malignant cells to regional lymph nodes is an early event in colon cancer metastasis [3], [4]. The chemokine receptor family of G-protein-coupled receptors (GPCRs) plays a key role in this process [5], [6], [7].

CC chemokine receptor 7 (CCR7), a member of the GPCR family, is expressed on memory T cells, B cells and mature dendritic cells. Previous studies have demonstrated a positive correlation between CCR7 expression and lymphatic metastasis and poor prognosis in colorectal cancer [8], [9]. Stimulation of CCR7 by its ligand CCL21 induces the migration and invasion of CCR7+ malignant cells into the lymph nodes in a manner that is similar to the process of lymphocyte homing [10], [11]. Data from animal models support CCR7 as a potential target for the treatment of lymphatic metastasis in colon cancer [12].

Another important contributor to colon cancer metastasis is matrix metalloproteinase-9 (MMP-9). MMP-9 cleaves specific factors present in the extracellular matrix (ECM), such as collagen IV in basement membranes, and induces ECM remodelling [13]. Early clinical evidence demonstrating a correlation between colon cancer progression and the expression level or activation status of MMP-9 suggests the involvement of MMP-9 in colon cancer progression. A high ratio of MMP-9 expression was associated with overall survival in patients with colon cancer. Patients with increased plasma levels of MMP-9 did not survive as long as patients with normal plasma levels in Dukes’ stage IV colon cancer [14], [15]. Furthermore, MMP-9 secreted by colon cancer cells has been found to promote metastasis [16], and over-expression of MMP-9 in cancerous tissues correlates with lymph node metastasis in colon cancer [17]. Together, these findings clearly demonstrate that MMP-9 is critical in colon cancer metastasis.

Interestingly, the CCL21/CCR7 pathway has been shown to regulate MMP-9, and MMP-9 has been demonstrated to play an important role in CCL21-induced cell invasion in B-cell chronic lymphocytic leukaemia [18], [19]. These previous findings suggest a potential link between the CCL21/CCR7 pathway and MMP-9 in cancer metastasis; however, an association between CCL21/CCR7 and MMP-9 in colon cancer has not been demonstrated. Furthermore, the mechanism by which the CCL21/CCR7 pathway promotes lymph node metastasis is not well understood, and novel mechanisms of activation of MMP-9 continue to be elucidated. Therefore, we aimed to determine whether MMP-9 was regulated by the CCL21/CCR7 pathway in the human colon cancer cell line SW480 in vitro and in vivo. To address this question, we inhibited CCR7 expression using short hairpin RNA (shRNA) [20], [21]. Using whole-body fluorescence imaging and gelatin zymography, we demonstrate that knockdown of CCR7 significantly inhibits colon cancer metastasis and the production of MMP-9 in a xenografted animal model.

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2. Materials and methods 

2.1. Cell line and treatment 

The human colon cancer cell line SW480 (China Center for Type Culture Collection, Wuhan, China) was cultured in RPMI-1640 medium supplemented with 10% foetal bovine serum (Gibco, USA) and 1% streptomycin–penicillin (Gibco, USA). The cells were treated with CCL21 (PeproTECH, UK).

2.2. Construction of short hairpin interfering RNA expression vectors 

Based on the sequence of the CCR7 cDNA in GenBank (accession no: NM_001838.2), three 19-bp nucleotide fragments were selected as targets. The homology of these sequences was confirmed in the BLAST database. We used the PGC-silencer-CRM30 vector to construct CCR7-shRNA expression vectors in which expression of CCR7-shRNA and green fluorescent protein (GFP) was driven by the CMV promoter. The vectors contained human miR-30 framework [22]. The target and primer sequences were as follows: CCR7-1, 5′-GGACGTGCGGAACTTTAAA-3′ (216–234) (sense: 5′-CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGCaGGACGTGCGGAACTTTAAAgAGTGAAGCCACAGATGTAcTTTAAAGTTCCGCACGTCCtTTGCCTACTGCCTCGCAATTG-3′); CCR7-2, 5′-GCTCATCTTTGCCATCTAC-3′ (453–471) (sense: 5′-CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGCaGCTCATCTTTGCCATCTACaAGTGAAGCCACAGATGTAtGTAGATGGCAAAGATGAGCtTTGCCTACTGCCTCGCAATTG-3′); CCR7-3, 5′-CCCTTTCTTGTACGCCTTC-3′ (1029–1047) (sense: 5′-CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGCaCCCTTTCTTGTACGCCTTCaAGTGAAGCCACAGATGTAtGAAGGCGTACAAGAAAGGGtTTGCCTACTGCCTCGCAATTG-3′); negative control, 5′-TTCTCCGAACGTGTCACGT-3′ (sense: 5′-CTCGAGAAGGTATATTGCTGTTGACAGTGAGCGCaTTCTCCGAACGTGTCACGTaAGTGAAGCCACAGATGTAtACGTGACACGTTCGGAGAAtTTGCCTACTGCCTCGCAATTG-3′). Each oligonucleotide sequence was annealed at 94°C for 5minutes and inserted into the XhoI and MfeI restriction endonuclease sites in the PGC-silencer-CRM30 vector. The vectors were then sequenced by Invitrogen Corporation (Shanghai, China).

2.3. RNA extraction and real-time PCR 

SW480 human colon cancer cells were transfected with CCR7-shRNA or control vectors. After treatment for 24h, total RNA was extracted using TRIzol reagent (Invitrogen, USA) to evaluate the effect of the CCR7-shRNA vectors. The RNA was quantified and assessed for purity by UV spectrophotometry. Two micrograms of RNA was used for reverse transcription. The CCR7 and β-actin genes were amplified with SYBR Green using a FTC-2000 Real-time PCR Detection System (Feng Ling Bio-engineering Co., Ltd, Shanghai, China). The primer sequences for CCR7 were 5′-AACCAATGAAAAGCGTGCTG-3′ (forward) and 5′-CGAACAAAGTGTAGTCCACTG-3′ (reverse) [23]. The primer sequences for β-actin were 5′-GAAACTACCTTCAACTCCATC-3′ (forward) and 5′-CGAGGCCAGGATGGAGCCGCC-3′ (reverse). Cycling conditions were as follows: 30 cycles of denaturation at 94°C for 30s, annealing at 58°C for 30s and extension at 72°C for 30s. Each experiment was carried out in triplicate using β-actin as an internal standard.

2.4. Stable transfection of the human colon cancer cell line SW480 

Based on PCR results, SW480 human colon cancer cells were transfected with 4μg CCR7-3 shRNA vector or control vector with 10μl Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions in 6-well culture plates. After treatment for 24h, the transfected cells were treated with 800μg/ml G418 (Sigma, USA). Two weeks later, monoclonal GFP+ cells were selected under fluorescence microscopy according to the manufacturer's instructions for cell cloning by serial dilution in 96-well culture plates. The monoclonal cells were maintained in RPMI-1640 medium supplemented with 15% foetal bovine serum and 400μg/ml G418. After 1 month, stably transfected cells were obtained. The cells transfected with the CCR7-3 shRNA vector and control vector, referred to as SW480/CCR7− and SW480/control cells, respectively, were used in subsequent experiments.

2.5. Flow cytometric analysis 

Single-cell suspensions of the stably transfected cells were prepared. The cells were then analysed by flow cytometry using parental SW480 cells as a negative control. Single colour flow cytometric analysis for assessment of positive GFP expression was performed on a BD FACScalibur flow cytometer. To rule out potential cytotoxic effects of the CCR7-shRNA, necrosis was detected by flow cytometric analysis using an Annexin-V/PI assay (Jingmei Biological Technology Co., Ltd, Shenzhen, China). Briefly, the stably transfected cells were collected. After adjusting the cell number to 1×105/ml in each group, 5μl FITC-labelled Annexin-V and 10μl propidium iodide were added to each 200μl cell suspension. Samples were incubated for 10min and analysed.

2.6. Proliferation assay 

The parental cells and stably transfected cells were seeded into 96-well culture plates (5×103/well) and allowed to adhere overnight. The cells were then treated with 0ng/ml or 100ng/ml CCL21 for 24h. Proliferation was assessed using a CCK-8 assay (Dojindo, Japan). The proliferation stimulation or inhibition index was calculated as follows: [(the absorbance of treated cells/the absorbance of untreated control cells)−1]×100%. Each experiment was performed in triplicate.

2.7. Protein extraction and immunoblotting 

The cells stably transfected with shRNA or control vectors were harvested and lysed. Proteins (25μg) from the cell lysates were separated by 12% SDS-PAGE, transferred to nitrocellulose membranes (Millipore, USA) and blotted with primary antibodies (rabbit anti-CCR7, 1:1000, Abcam, USA; mouse anti-β-actin, 1:3000, Santa Cruz, CA, USA) at 4°C overnight. Membranes were then washed and probed with secondary antibodies (for CCR7, goat anti-rabbit-HRP, 1:5000, Santa Cruz, CA, USA; for β-actin, goat anti-mouse-HRP, 1:5000, Santa Cruz, CA, USA) for 1h at room temperature. Bands were visualized using ECL (Pierce, USA) and quantified by densitometry. We calculated the CCR7/β-actin expression ratio to evaluate CCR7 protein expression. Each experiment was performed in triplicate.

2.8. Invasion assay 

Invasion assays were performed in 24-well cell culture plates containing inserts with 8μm pores (Millipore, USA) according to the manufacturer's instruction. The inserts were coated with Matrigel (50μl/cm2; R&D Systems, USA). The cells were suspended at 5×105/ml and added 100μl to the inserts, which were transferred to wells containing the medium with or without CCL21. After incubation at 37°C for 24h, cells on the lower surface of the inserts were stained and counted under a light microscope in six different fields. The experiment was performed in triplicate and the results were expressed as the mean number of invaded cells/well.

2.9. In vivo xenograft animal model 

Nude BALB/c mice were purchased from the Academy of Preventive Medicine (Wuhan, China). The mice were housed and maintained under specific pathogen-free conditions in the Animal Center of Tongji Medical College (Wuhan, China) in accordance with the regulations and standards published by the National Institutes of Health. The mice were used according to institutional guidelines when they were 4–6 weeks of age. The SW480/CCR7− and SW480/control cells were harvested and suspended in PBS, and the viability of cells in each single-cell suspension was determined by trypan blue exclusion. BALB/c nude mice were divided into two groups of 32 mice. One million SW480/CCR7− cells (0.1ml) were injected into the neck region of each mouse in the shRNA vector group, and an equal number of SW480/control cells were injected into the neck region of each mouse in the negative control group. The xenografts were measured in two dimensions with a caliper twice a week. The volume was calculated as follows: volume=(width2×length)/2, where width=shorter diameter and length=longer diameter. Sixteen mice per group were killed 8 weeks after the cell transfer. Xenografts and regional lymph nodes were collected. The remaining 16 mice in each group were monitored for survival studies.

2.10. Gelatin zymography 

Supernatants from 1×106 stably transfected SW480 cells treated with medium containing 100ng/ml CCL21 or medium alone for 24h were harvested and stored at −70°C. A 0.05-g cancerous tissue fragment was cut from each xenograft and extracted with 1ml PBS buffer. These cancerous tissue samples were separated by centrifugation at 12,000×g for 10min, and aliquots of the supernatants were collected and stored at −70°C. A 20-μl sample of each supernatant was separated by electrophoresis on a 10% acrylamide gel containing 0.1% gelatin. After renaturation with 2.5% Triton X-100, the gels were incubated overnight at 37°C in a buffer containing 50mM Tris, pH 7.6, 150mM NaCl, 5mM CaCl2, 1μM ZnCl2, and 0.02% Brij-35. The gels were stained with 0.5% Coomassie blue in 40% methanol and 10% acetic acid for 2h at room temperature and then destained with a gradient of methanol ranging from 40% to 10% containing 10% acetic acid to visualize the bands. The experiment was performed in triplicate and the results were quantified by densitometry.

2.11. Enzyme-linked immunosorbent assay 

The cells and tissues were treated and collected as described above. Concentrations of MMP-9 in the supernatants of SW480 cells and in the xenografted cancerous tissues were quantified using an MMP-9 ELISA kit (R&D Systems, USA). The limit of detection was 15 pg/ml. The absorbance was measured at 450nm using a micro-plate reader (Bio-Rad, USA). Each sample was analysed in triplicate.

2.12. Whole-body fluorescence imaging 

The development of fluorescent xenografts in subcutaneous regional sites in the neck was detected using a whole-body fluorescence imaging system (Image Station 2000MM, Kodak, USA).

2.13. Immunohistochemistry 

Paraffin-embedded xenografted tissues and regional lymph nodes were immunohistochemically stained for CCR7 using a streptavidin–biotin complex method. The tissues were deparaffinized and treated with 0.3% H2O2 to inhibit endogenous peroxidase. After microwave antigen retrieval, tissues were incubated with 10% normal goat serum (SABC immunohistochemical kit; Wuhan Boster Biological Technology Co., Ltd, Wuhan, China) for 30min. The tissues were incubated with 1:100 rabbit anti-human CCR7 antibody (Abcam, USA), 1:100 rabbit anti-human MMP-9 (Santa Cruz, CA, USA) and 1:100 rabbit anti-human proliferating cell nuclear antigen (PCNA, Santa Cruz, CA, USA) at 4°C overnight. PBS was used as a negative control. The tissues were then incubated with biotin-conjugated secondary antibodies, streptavidin–biotin complex and diaminobenzidine (DAB kit, Wuhan Boster Biological Technology Co., Ltd, Wuhan, China). Immunostaining for CCR7 was performed to evaluate the efficiency of CCR7 silencing by CCR7-shRNA in vivo. Immunostaining for MMP-9 was performed to evaluate the effect of CCL21/CCR7 on MMP-9 expression. Immunostaining for PCNA was performed to evaluate the presence of cancer cells in the regional lymph nodes.

2.14. Statistical analysis 

Statistical analysis was performed with SPSS 13.0. Independent t-tests were used to evaluate the significance of differences between means in two groups. One-way ANOVA analyses were used to compare three groups. Survival rates were determined by Kaplan–Meier curves, and P values were determined by the log-rank test. Groups for which P<0.05 were considered to be significantly different.

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3. Results 

3.1. Detection of shRNA vector efficiency 

To evaluate the function of the CCL21/CCR7 pathway in colon cancer, we generated three shRNA vectors targeting CCR7. Real-time PCR analysis showed that transient transfection with shRNA vectors suppressed CCR7 mRNA levels (Fig. 1A). Of the vectors tested, the CCR7-3 shRNA vector inhibited CCR7 mRNA expression the most efficiently (P=0.000). Thus, we generated stable cell lines expressing the CCR7-3 shRNA vector or a control vector (SW480/CCR7− and SW480/control, respectively) by selecting transfected cells in G418. Stable cell lines were confirmed by flow cytometric analysis, which demonstrated that 87.4% of SW480/CCR7− cells and 86.2% of SW480/control cells expressed GFP (Fig. 1B). Immunoblotting demonstrated that CCR7 protein levels in SW480/CCR7− cells were lower than those in SW480/control cells (P=0.000; Fig. 1C).

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  • Fig. 1. 

    (A) Decreased CCR7 mRNA expression in SW480 cells 24h after transfection with CCR7-shRNA vectors. CCR7 mRNA levels were normalized to β-actin mRNA levels. (B) Left, flow cytometric analysis of the frequency of GFP+ cells among parental SW480 cells and stably transfected SW480/CCR7− cells. Right, flow cytometric analysis of the frequency of GFP+ cells among parental SW480 cells and stably transfected SW480/control cells. (C) Immunoblotting analysis of inhibition of CCR7 protein expression by CCR7-shRNA vectors in stably transfected cell lines (lanes 1 and 2: parental SW480 cells; lanes 3 and 4: SW480/control cells; lanes 5 and 6: SW480/CCR7− cells). Upper panel, quantification of immunoblots.

3.2. Effect of CCR7-shRNA on proliferation of SW480 cells 

The potential cytotoxicity of CCR7-shRNA was evaluated by CCK-8 assay and flow cytometric analysis. The SW480/CCR7− and SW480/control vectors inhibited the growth of SW480 cells by 1.97% (P>0.05) and 2.00% (P>0.05), respectively, over a period of 24h, and 0.07% (P>0.05) of SW480/control cells and 0.21% (P>0.05) of SW480/CCR7− cells underwent necrosis. These results indicated that shRNA cytotoxicity had only a minor effect on cell growth. Treatment with CCL21 enhanced the proliferation of SW480/CCR7− and SW480/control cells by 1.62% (P>0.05) and 0.72% (P>0.05), respectively.

3.3. Effect of CCL21/CCR7 on MMP-9 up-regulation and invasion of colon cancer cells in vitro 

To investigate the effects of CCL21 on SW480/CCR7− and SW480/control cells, we performed invasion assays. The SW480/control cells exhibited significant responses to 100ng/ml CCL21 for 24h, whereas SW480/CCR7− did not. Upon treatment with 100ng/ml CCL21, the number of SW480/control cells that invaded through a reconstituted basement membrane was higher than that of SW480/CCR7− cells (113.5±3.9 and 42.8±3.9, respectively, P=0.000) (Fig. 2A–C). To study the mechanism by which the CCL21/CCR7 pathway promotes tumour invasion, we performed ELISAs and gelatin zymography to determine whether MMP-9 was up-regulated by CCL21/CCR7. CCL21 significantly increased MMP-9 secretion and activation in SW480/control cells. The concentration of secreted MMP-9 increased from 126.45±2.27 pg/ml to 260.02±1.57 pg/ml (P=0.000) when SW480/control cells were treated with CCL21. Furthermore, the relative activity of MMP-9 in culture supernatants increased from 0.048±0.001 to 0.089±0.002 (P=0.000) when SW480/control cells were treated with CCL21. Importantly, CCR7 gene silencing significantly inhibited CCL21-induced MMP-9 up-regulation (Fig. 3A and C).

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  • Fig. 2. 

    (A) Invasion assay in SW480/control cells treated with 100ng/ml CCL21. (B) Invasion assay in SW480/CCR7− cells treated with 100ng/ml CCL21. (C) Quantification of invasion assays.

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  • Fig. 3. 

    (A) Concentration of MMP-9 in CCL21-treated or untreated SW480/control and SW480/CCR7− cell supernatants as measured by ELISA. (B) Concentration of MMP-9 in SW480/control and SW480/CCR7− xenografted tissues as measured by ELISA. (C) MMP-9 activity in CCL-21 treated or untreated SW480/control and SW480/CCR7− cell supernatants as measured by gelatin zymography (lane 1: untreated SW480/control cells; lane 2: SW480/control cells treated with 100ng/ml CCL21; lane 3: untreated SW480/CCR7− cells; lane 4: SW480/CCR7− cells treated with 100ng/ml CCL21). Lower panel, quantification of gelatin zymography. (D) MMP-9 activity in SW480/control and SW480/CCR7− xenografted tissues as measured by gelatin zymography (lane 1: the SW480/control group; lane 2: the SW480/CCR7− group). Lower panel, quantification of gelatin zymography.

3.4. Effects of the CCL21/CCR7 pathway on MMP-9 up-regulation during colon cancer metastasis in vivo 

To discern the effects of the CCL21/CCR7 pathway in vivo, SW480/CCR7− or SW480/control cells were injected into the subcutaneous region of the neck of nude mice (n=32 per group). After 8 weeks, the tumour volume in mice that received SW480/control cells was 474.69±51.31mm3; however, tumour volume was significantly lower in mice that received SW480/CCR7− cells (P=0.000). To further verify that the CCL21/CCR7 pathway regulates MMP-9 production in vivo, half of the mice in each group (n=16) were killed for evaluation of MMP-9 production and regional lymph node metastasis. The xenografted tissues were examined by immunohistochemistry, ELISA and gelatin zymography. MMP-9 was expressed in xenografts from 11 of 16 mice that received SW480/CCR7− cells and 12 of 16 mice that received SW480/control cells (P>0.05); however, the average expression levels of MMP-9 in xenografts from mice that received SW480/CCR7− cells were significantly lower than those in xenografts from mice that received SW480/control cells. The relative activity of MMP-9 in xenografts from mice that received SW480/CCR7− cells was also down-regulated (P=0.000; Fig. 3B and D). All 16 animals in the SW480/control group developed regional lymph node metastases. In contrast, only four animals in the SW480/CCR7− group developed regional lymph node metastases. The lymph nodes that contained cancer cells were evaluated by immunohistochemical analysis of PCNA expression. The total number of PCNA+ lymph nodes from mice that received SW480/control cells was 24, whereas that of mice that received SW480/CCR7− cells was four (P=0.000). The expression of CCR7 in xenografted mice was also evaluated by immunohistochemistry. All mice that received SW480/control cells and two mice that received SW480/CCR7− cells expressed CCR7 in cancer cells (P=0.000), indicating that CCR7 expression was blocked by shRNA (Fig. 4). CCR7 gene silencing also caused a significant decrease in tumour volume (Fig. 5A). Kaplan–Meier analysis showed that the animals in the SW480/control group did not survive as long as those in the SW480/CCR7− group (P=0.000; Fig. 5B).

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  • Fig. 4. 

    (A) Immunohistochemical staining of CCR7 in xenografts from mice that received SW480/CCR7− cells (SABC; original magnification, 400×). (B) H&E staining of xenografts from mice that received SW480/CCR7− cells (original magnification, 400×). (C) Immunohistochemical staining of CCR7 in xenografts from mice that received SW480/control cells (SABC; original magnification, 400×). (D) H&E staining of xenografts from mice that received SW480/control cells (original magnification, 400×). (E) Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in regional lymph nodes (SABC; original magnification, 400×). (F) H&E staining of regional lymph nodes (SABC; original magnification, 400×). Immunostaining showed that CCR7 was located in the cell membrane and PCNA was located in the cell nucleus.

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  • Fig. 5. 

    (A) Growth curve of xenografted tumours in mice that received SW480/control cells or SW480/CCR7− cells. (B) Kaplan–Meier analysis showed that the SW480/control group did not survive as long as the SW480/CCR7− group (group 1=SW480/control group; group 2=SW480/CCR7− group).

3.5. Whole-body fluorescence imaging 

Whole-body fluorescence imaging of nude mice 4 weeks after injection of SW480/CCR7− or SW480/control cells demonstrated the development of xenografts in primary sites. The xenografts in mice that received SW480/CCR7− cells were smaller in size than those in mice that received SW480/control cells (Fig. 6C and D).

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  • Fig. 6. 

    (A) Fluorescence microscopy of SW480/control cells (Nikon, original magnification, 200×). (B) Fluorescence microscopy of SW480/CCR7− cells (Nikon, original magnification, 200×). (C) Whole-body fluorescence image of a mouse that received SW480/control cells (Image Station 2000MM, original magnification, green fluorescence light). (D) Whole-body fluorescence image of a mouse that received SW480/CCR7− cells (Image Station 2000MM, original magnification, green fluorescence light).

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4. Discussion 

In our study, CCR7 protein levels in SW480 colon cancer cells expressing an shRNA construct specific for CCR7 were lower than those in control SW480 cells. The SW480/control cells exhibited significant invasion responses to CCL21, whereas the SW480/CCR7− cells did not. These results confirmed the silencing of CCR7 expression in stably transfected colon cancer cell lines. Using these stable cell lines, we found that CCL21 significantly increased MMP-9 secretion and activation as determined by ELISA and gelatin zymography, and CCR7 gene silencing inhibited CCL21-induced MMP-9 up-regulation. These results suggest that the CCL21/CCR7 pathway might regulate MMP-9 production in human colon cancer. To further establish that the CCL21/CCR7 pathway was responsible for MMP-9 up-regulation, in vivo experiments were performed. Using whole-body fluorescence imaging technology and gelatin zymography, we demonstrated that silencing of CCR7 significantly limited the metastasis of colon cancer and the production of MMP-9 in a xenografted animal model. Mice that received SW480/control cells had progressively enlarging tumours and more lymphatic metastases than mice that received SW480/CCR7− cells, and these animals did not survive as long as mice that received SW480/CCR7− cells.

A growing body of clinical and experimental evidence has demonstrated that MMP-9 and CCL21/CCR7 play key roles in cancer progression and metastasis [24], [25], [26], [27]. Indeed, colon cancer cells secrete MMP-9, which promotes ECM degradation and tumour cell seeding. MMP-9 cleaves specific factors in the ECM, such as collagen IV in basement membranes, creating a physical and biochemical pathway for tumour invasion. Actin cytoskeleton remodelling associated with membrane protrusion formation induces polarized MMP-9 release [28], [29]. Furthermore, the CCL21/CCR7 pathway has been linked to cytoskeleton remodelling because CCR7 signalling induces actin polymerization in various cancer cell types [30], [31], [32]; however, the molecular mechanism of colon cancer cell metastasis to lymph nodes has not been fully elucidated. Our results therefore establish a novel role for the CCL21/CCR7 pathway as an MMP-9 regulator in human colon cancer.

We have described a previously unknown function for the chemokine receptor CCR7 in promoting MMP-9 production specifically in colon cancer cells. The chemokine ligand CCL21 might influence cancer cells to secrete MMP-9, which is considered one of the most critical steps in the metastatic process. These findings might have important implications for the development of novel therapies for metastatic tumours. In the animal model, we showed for the first time that CCR7 expression was correlated with regulation of MMP-9 and lymph node metastasis. A reasonable explanation for the ability of shRNA-CCR7 to inhibit regional lymph node metastasis is that malignant cells are unable to attach to the regional lymph nodes due to the low level of MMP-9 present as a result of decreased CCR7 expression. Thus, CCL21 in the regional lymph nodes may not be able to attract and induce these tumour cells, which express inadequate levels of CCR7 protein on their cell surface, to secrete enough MMP-9. This argument is consistent with the suggested roles of these pathways in lymph-angiogenesis and lymph node metastasis [33]; however, CCR7-shRNA may only block CCR7 activity but not completely suppress metastasis because cancer metastasis is mediated by complex molecular mechanisms.

Colon cancer is the third most common neoplasm in developed nations. Presurgical and postsurgical adjuvant chemotherapy has improved outcome in patients with metastatic spread [34], [35]; however, advanced metastasis remains untreatable [35]. The heterogeneity and complexity of malignant transformation makes it difficult to identify common molecular mechanisms governing the metastatic progression of cancer and to develop effective treatments for malignant cancers. Thus, targeting both MMP-9 and CCR7 may represent a novel therapeutic strategy for colon cancer that disrupts fundamental processes.

We also demonstrated for the first time that the silencing of CCR7 influenced tumour growth in vivo, suggesting that MMP-9 production promotes angiogenesis and supports tumour progression by activating ECM-embedded vascular endothelial growth factor [36]. Our future studies will focus on the mechanisms of this novel function for the CCL21/CCR7 pathway.

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Conflict of interest statement 

The authors declare that there is no conflict of interest.

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Acknowledgment 

The work was fully supported by grants from the National Natural Science Foundation of China (30772128).

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PII: S1590-8658(10)00195-7

doi:10.1016/j.dld.2010.05.013

Digestive and Liver Disease
Volume 43, Issue 1 , Pages 40-47, January 2011

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