SEARCH

SEARCH BY CITATION

Keywords:

  • chemokine;
  • bladder cancer;
  • metastasis;
  • migration;
  • invasion

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Transitional cell carcinoma of the urinary bladder remains life threatening due to the high occurrence of metastases. Emerging evidence suggests that chemokines and their receptors play a critical role in tumor metastases. In our study, we performed a systematic analysis of the mRNA and protein expression levels of all 18 chemokine receptors in normal urothelium and bladder cancer. CXCR4 was the only chemokine receptor whose mRNA expression level was upregulated in bladder cancer cell lines as well as in invasive and locally advanced bladder cancer tissue samples (pT2–pT4). In contrast, superficial bladder tumors (pTa and pT1) displayed low CXCR4 expression levels and normal urothelial cells were negative for CXCR4. Immunohistochemistry of a bladder cancer tissue microarray (TMA) confirmed that a subgroup of invasive bladder cancers revealed a high CXCR4 protein expression, while superficial bladder tumors showed low immunoreactivity. To investigate the functional significance of CXCR4 expression, we performed migration and invasion assays. Exposure of CXCR4-positive bladder cancer cells to CXCL12 in a Boyden chamber type assay provoked a significant increase in migration as well as invasion across a Matrigel barrier. Enhanced migration and invasion were inhibited by a CXCR4-specific blocking antibody. In contrast, normal urothelial cells did not respond to CXCL12 and lacked chemotactic migration. In conclusion, bladder cancer cells express CXCR4 progressively with advanced tumorigenesis and this receptor interacts with CXCL12 to mediate tumor chemotaxis and invasion through connective tissue. These properties identify CXCR4 as a potential target for the attenuation of bladder cancer metastases. © 2004 Wiley-Liss, Inc.

Transitional cell carcinoma of the bladder is the most frequent tumor of the urinary tract. Despite advances in early detection and therapy, bladder cancer remains life threatening due to the high occurrence of metastases. Radical cystectomy is the preferred treatment for muscle-invasive bladder cancer in the United States, Japan and some countries of Europe. However, at least 50% of patients with locally advanced disease are expected to develop systemic progression within 3 years.1 Patients suffering from metastatic bladder cancer are commonly treated with systemic chemotherapy. Analyzing survival rates from standard chemotherapy schedules such as M-VAC (methotrexate, vinblastine, adriamycin and cisplatin), results are more or less discouraging. In the Loehrer trial, only 5 of 133 (3.7%) of the patients randomized to M-VAC were alive at the 6-year follow-up time. These results indicate that patients with systemic metastases continue to be incurable by chemotherapy.2, 3 Despite the fact that it is the metastases rather than the primary tumors that cause most cancer deaths, there has been little progress in identifying drugs to specifically block metastases.

Metastasis is not a simple phenomenon, but a highly organized process composed of a series of molecular events. Several molecular families have been identified to play pivotal roles in cancer metastases. These include adhesion proteins such as integrins as well as enzymes like the metalloproteinases (MMPs), which support tumor invasion in connective tissue.4, 5 Emerging evidence suggests that a third family, the chemokines and their receptors, are involved in organ-specific metastasis. Chemokines are a superfamily of small, secreted proteins (8–10 kDa) that function to set up immune and inflammatory reactions. Chemokines also affect angiogenesis and collagen production, as well as B-cell lymphopoiesis and bone marrow myelopoiesis.6, 7 The interaction of these soluble chemokines with their specific, transmembrane G-protein-coupled receptors mediates their biologic effects. Based on the position of the NH2-terminal cysteine residues, chemokine receptors are grouped into 4 classes. To date, 18 different chemokine receptors (CCR1–CCR10, CXCR1–CXCR6, XCR1 and CX3CR1) have been identified in humans.8

Recent evidence indicates that tumor cells express distinct, tumor type-specific, nonrandom patterns of chemokine receptors and that signaling through these receptors is crucial for chemotactic migration, invasion and cancer metastasis.9, 10, 11, 12 The most comprehensive of these recent studies has examined expression levels of all chemokine receptors in breast cancer.9 The analysis revealed CXCR4 and CCR7 to be the chemokine receptors most highly upregulated in breast cancer cells. Intriguingly, the specific ligands for these receptors exhibit peak expression in lung, liver, bone marrow and lymph nodes, which represent preferential destinations for breast cancer metastasis. As proof that these correlations were functionally significant, Muller et al.9 also showed that neutralizing antibodies directed against the chemokine receptor CXCR4 reduced the number of lung and lymph node metastases in SCID mice carrying xenografts of human breast tumors. These observations imply a critical role for specific chemokines and their receptors in cancer metastasis.

Although the general role of chemokine receptors in tumor metastasis has been evaluated previously, there is no study that has investigated the interaction of chemokines and their receptors in bladder cancer metastasis. Current evidence indicates that different receptors are upregulated in different tumors. Therefore, we performed a systematic analysis of the expression levels of all 18 chemokine receptors in bladder cancer. Our study identified CXCR4 as the most highly expressed chemokine receptor in bladder tumor cells. Furthermore, our data show a correlation between CXCR4 expression level and tumor stage and grade. The results establish a functional role for these receptors using in vitro assays of bladder cancer cell migration and invasion along a chemokine gradient.

Material and methods

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cell lines

Seven cell lines from transitional cell carcinomas of the bladder, RT4, 5637, T24, HT1376, J82, UMUC3 and TCCSUP, as well as 1 breast cancer cell line, DU-4475, and 1 osteosarcoma cell line, SaOS-2, were originally obtained from the American Type Culture Collection (Rockville, MD, USA). Cell lines were cultured in 75 cm2 flasks in RPMI-1640 supplemented with 10% heat-inactivated FBS, 10 U/ml penicillin and 10 mg/ml streptavidin (Sigma, St. Louis, MO, USA).

Primary normal urothelial cells were derived from a cystectomy specimen in the Department of Urology, Saarland University, Homburg, Germany. Primary cells were established as detailed previously.13 Urothelial cells were cultured in DMEM/F-12, supplemented with 10% FBS, 10 ng/ml epidermal growth factor (Sigma), 10 ng/ml cholera toxin (Sigma), 4 mg/ml insulin (Sigma) and 1 μg/ml hydrocortisone.

Tumor specimens

A total of 28 fresh frozen tumor samples were analyzed, including stages pTa (n = 8), pT1 (n = 6), pT2 (n = 2), pT3 (n = 7) and pT4 (n = 5). An initial hematoxylin-eosin (H&E)-stained frozen section was reviewed to assess tumor quality and content. Normal and necrotic tissue was excluded by trimming of the frozen block. A tumor sample was considered suitable for study if the proportion of tumor cells was higher than 70%.

A bladder cancer tissue microarray (TMA) contained 62 elements of different tumor stages with 14 pTa, 3 pT1, 14 pT2, 18 pT3 and 13 pT4. The grading system classified 12 tumors as low grade and 46 as high grade. Additionally, 5 tissue samples of normal urothelium were examined for CXCR4 immunoreactivity. Breast cancer tissue samples served as CXCR4 positive controls and normal breast tissue as a negative control.

Fresh frozen tumor samples and the TMA were obtained from the tissue bank of the UCSF Cancer Center in accordance with institutional guidelines on the use of human tissue. The histology of all bladder tumors was transitional cell carcinoma. Tumors were staged according to the American Joint Committee on Cancer (AJCC) tumor node metastasis (TNM) classification of malignant tumors14 and graded according to the World Health Organization and International Society for Urological Pathology (WHO/ISUP) classification.15

Isolation of total RNA and multiplex real-time PCR

Fresh frozen tissue samples were placed directly in Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA from cell lines and tissue samples was isolated using the RNeasy mini kit (Qiagen, Valencia, CA, USA) following standard protocols. Gene-specific primers (Biosearch Technologies, Novato, CA, USA) for multiplex RT-PCR and TaqMan were designed using Primer Express software (Perkin Elmer, Foster City, CA, USA) based on sequencing data from the National Center for Biotechnology Information databases. All primer sequences (CCR1–CCR10, CXCR1– CXCR6, XCR1 and CX3CR1) and composition of multiplex mixtures used in our study are available from our website: http://asthmagenomics.ucsf.edu. Quantitative PCR analysis was performed as described previously.16 All forward and reverse TaqMan primers (F/R) were optimized and transcript quantifications run in duplicates with minus RT cDNA controls on an ABI Prizm 7900 Sequence Detection System (PE Applied Biosystems, Foster City, CA, USA). Raw data from ABI Prizm7900 were processed into Excel (Microsoft, Redmond, WA, USA) spreadsheets using software that automated proper baseline selection and Ct (threshold cycle of PCR) calculation for each of the genes on a 384-well plate as described. Results were calculated as absolute copy number/20 ng of RNA by reference to the average copy number of 2 housekeeping genes (Ubiquitin, PP2A).

Western blotting

Cells were lysed in RIPA buffer containing a cocktail of proteinase inhibitors (Complete Roche, Mannheim, Germany). Protein quantification of the lysates was performed by protein assay reagent from Biorad (Hercules, CA, USA). Typically, 10 μg of total protein was used for Western analyses. Lysates were electrophoresed under nonreducing conditions in 12% TRIS-HCl Ready Gels (Biorad) and transferred to Hybond nitrocellulose paper (Amersham Pharmacia, Piscataway, NJ, USA). Nonspecific binding sites were blocked by incubating blots in 5% nonfat milk in PBS containing 0.1% Tween-20 (PBST; Sigma). For CXCR4 detection, blots were incubated with a primary rabbit anti-human polyclonal antibody (2 μg/ml; Imgenex, San Diego, CA, USA) overnight at 4°C. Blots were washed in PBST and incubated with horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hr at room temperature. Proteins were visualized by enhanced chemiluminescence (ECL Western blotting kit; Amersham) and exposed to Hyperfilm (Amersham). All experiments were carried out in triplicate.

Flow cytometric analysis

TCCSUP cells were treated or not with CXCL12 (100 ng/ml; R&D Systems, Minneapolis, MN, USA) for 24 hr. Cells were adjusted to a concentration of 4 × 105 cells/50 μl in PBS with 0.25% BSA and 0.02% sodium azide. Nonspecific binding sites were blocked with 10% normal human serum. TCCSUP cells were incubated with either anti-CXCR4-phycoerythrin (clone 12G5) monoclonal antibody (10 μg/ml) or the appropriate isotype control antibody (10 μg/ml) from BD PharMingen (San Diego, CA, USA) for 1 hr at 4°C. After the final wash, cells were fixed in 2% formaldehyde prior to fluorescence-activated cell-sorting (FACS) analysis. TCCSUP cells were analyzed on a FACSscan (Becton Dickinson, Mountain View, CA, USA). Flow cytometry data were analyzed using Cellquest software (Becton Dickinson).

Immunohistochemistry

The formalin-fixed and paraffin-embedded TMA was stained using a standard indirect avidin-biotin HRP method. The TMA was deparaffinized and rehydrated. Endogenous peroxidase was quenched with 3% hydrogen peroxide. Antigen retrieval was performed using citrate buffer (Antigen Retrieval Citra; BioGenex, San Ramon, CA, USA) and the microwave method.17 Nonspecific binding sites were blocked with 2% normal goat serum in PBS. The TMA was incubated with mouse anti-human CXCR4 monoclonal antibody (Clone 12G5; R&D Systems) for 1 hr at room temperature at a dilution of 1:500. The TMA was washed and incubated with biotinylated goat anti-mouse Ig (Caltag Laboratories, Burlingame, CA, USA) and streptavidin-peroxidase complex (Vector Laboratories, Burlingame, CA, USA). Peroxidase activity was detected using 3,3′-diaminobenzidine (DAB) (Vector Laboratories) with 0.5% hydrogen peroxide after 4 min. The TMA was counterstained with hematoxylin, dehydrated and mounted. Negative controls were performed by replacing the primary antibody with a mouse IgG-matched isotype control antibody. The intensity of cytoplasmic staining for CXCR4 and the percentage of tumor cells that stained positively were estimated by 2 different observers without prior knowledge of the patient disease status. Cytoplasmic staining intensity in bladder tumor cells was compared to that in breast cancer cells, which served as CXCR4 positive controls. Staining intensity was recorded on an ordered scale: 0 = no staining, +1 = weak staining and +2 = strong staining. Staining was rated as positive for each section, when a minimum of 20% of cells were judged to be positive by the 2 independent observers.

In vitro cell migration and invasion assay

Chemotactic migration of bladder cancer cells (TCCSUP) and normal urothelial cells (BN48) was investigated using 24-well cell culture chambers containing inserts with 8 μm pores (BD PharMingen). Invasion of bladder tumor cells was assayed in similar chambers that also contained a reconstituted extracellular matrix (ECM) membrane (Matrigel with 8 μm pore membrane; BD PharMingen). Cells were resuspended in chemotaxis buffer (DMEM/0.1% BSA/12 mM HEPES), 5 × 104 cells/500 μl was added to the upper chamber and 500 μl of chemotaxis buffer as a control was placed in the lower chamber. Migration and invasion assays of normal urothelial and tumor cells were supported by a chemokine CXCL12 gradient (0–200 ng/ml) (R&D Systems) established by placing chemokine in the lower chamber. After incubation for 24 hr at 37°C and 5% CO2, cells on the upper surface were wiped off with a cotton swab. Migrating and invading cells on the lower surface of the membrane were stained with H&E. Cells from 3 different fields were counted under the light microscope using a magnification of ×200. To evaluate specificity of the chemotactic and chemoinvasive effects, bladder cancer cells were preincubated with either anti-human CXCR4 monoclonal antibody (2 μg/ml, clone 12G5; R&D Systems) or mouse IgG-matched isotype control antibody (2 μg/ml; BD PharMingen). Antibody-coated cells were added to the upper chamber and incubated for 24 hr in the presence of CXCL12 (100 ng/ml) in the lower chamber. All experiments were repeated 3 times with triplicate samples.

Actin-polymerization assay

Bladder tumor cells (TCCSUP) were seeded on Labtech 8-well chamber slides precoated with 0.01% poly-L-lysine solution (Sigma). Tumor cells were serum-starved over night and then stimulated with 100 ng/ml CXCL12 (R&D Systems) at various time points. Cells were placed at 4°C and fixed for 20 min in 3% paraformaldehyde in PBS, permeabilized for 5 min with 0.2% Triton X-100, incubated with 5 mU/ml Alexa Fluor 594 phalloidin (Molecular Probes, Inc., Eugene, OR) for 60 min, washed with PBS and mounted in Vectashield aqueous mountant, including DAPI (Vector Laboratories). Fluorescence microscopy was performed using a Nikon Eclipse, E600 microscope.

Statistical analysis

Since most variables did not comply with normal Gaussian distribution as determined by the Kolmogorov-Smirnov test,18, 19 a nonparametric Mann-Whitney U-test20 or Kruskal-Wallis test21 was applied in order to compare independent samples of continuous variables. All p-values were based on 2-sided tests and the threshold to accept statistical significance was set at the alpha level 0.05. Analyses were performed with the statistical software package SPSS, version 10.0 (SPSS, Chicago, IL, USA).

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

CXCR4 chemokine receptor is expressed in bladder tumor cell lines

To evaluate whether chemokine receptors and their corresponding ligands are involved in bladder cancer metastasis, we assayed for the presence of all 18 chemokine receptors (CCR1–CCR10, CXCR1–CXCR6, XCR1 and CX3CR1) in 7 bladder cancer cell lines and 1 sample of normal primary urothelial cells using TaqMan real-time PCR. Our goals were to identify chemokine receptors that were (i) significantly upregulated in bladder cancer cells vs. normal urothelial cells and (ii) consistently upregulated across multiple cell lines, rather than aberrant expression levels in just 1 or 2 cell lines. CXCR4 was the only chemokine receptor whose mRNA expression levels were upregulated in all bladder cancer cell lines, but not expressed in normal urothelial cells (Fig. 1a and b). We then compared the protein expression levels of CXCR4 in bladder tumor cells and normal urothelial cells. The breast cancer cell line DU-4475 served as a CXCR4 positive control and the osteosarcoma cell line SaOS-2 as a negative control. Western blotting analysis detected substantial CXCR4 protein expression in all investigated bladder cancer cell lines, whereas the normal urothelial cell line expressed only a very faint protein band (Fig. 2a). In order to investigate CXCR4 surface protein expression on bladder cancer cells, flow cytometry was performed on unstimulated and CXCL12-stimulated TCCSUP bladder cancer cells. Only a few unstimulated TCCSUP cells expressed the CXCR4 receptor on the cell surface. In contrast, 33% of the total TCCSUP population expressed surface CXCR4 after exposure to CXCL12, 100 ng/ml for 24 hr (Fig. 2b).

thumbnail image

Figure 1. Expression of chemokine receptors in bladder cancer cell lines and normal urothelial cell line. (a) Quantitative, multiplex real-time PCR analysis of all known chemokine receptors (CCR1–CCR10, CXCR1–CXCR6, XCR1 and CX3CR1) was performed in 7 bladder cancer cell lines—RT4 and 5637 (tumor grade G1); T24, HT1376, J82 and UMUC3 (grade G3); and TCCSUP (grade G4)—and in a primary normal urothelial cell line, BN48. CXCR4 mRNA was expressed in all bladder cancer cell lines with the highest level in the T24 cell line. In contrast, normal urothelial cells exhibited no CXCR4 mRNA expression. Values are expressed as the absolute number of copies of the target gene in 20 ng of total RNA. (b) Analysis of the fold increase of CXCR4 mRNA expression levels in bladder cancer cell lines over normal urothelial cells. CXCR4 was the only chemokine receptor whose expression levels were consistently upregulated across multiple bladder cancer cell lines vs. normal urothelial cells.

Download figure to PowerPoint

thumbnail image

Figure 2. CXCR4 protein expression in bladder cancer cell lines. (a) Western blot analysis of CXCR4 was performed on cell lysates (10 μg) of 7 bladder cancer cell lines (RT4, 5637, T24, HT1376, J82, UMUC3 and TCCSUP) and in 1 normal urothelial cell line using an anti-human CXCR4 polyclonal antibody. Results showed that all bladder cancer cell lines express CXCR4 (40 kDa). Normal urothelial cell lines displayed a very faint protein band. The breast cancer cell line DU-4475 served as a positive control and the osteosarcoma cell line SaOS-2 as a negative control. (b) Surface protein expression of CXCR4 in unstimulated vs. CXCL12-stimulated bladder cancer cells (TCCSUP). Cells were evaluated by FACS. FACS histograms show the mean fluorescence intensity of unstimulated and stimulated (CXCL12, 100 ng/ml) TCCSUP cells stained with PE-conjugated anti-CXCR4 mAb (shaded histograms) or rat IgG2a isotype control antibody (open histogram). The percentage of cells within the marker-expressing surface CXCR4 is displayed above the histograms.

Download figure to PowerPoint

CXCR4 chemokine receptors are expressed in primary bladder cancer tissues in a tumor stage-dependent manner

Quantitative PCR analysis of the 18 chemokine receptors on 28 primary bladder cancer tissue samples revealed that the highest mRNA levels of CXCR4 occurred in invasive and locally advanced bladder cancer (pT2–pT4), while superficial bladder cancer (pTa and pT1) displayed low CXCR4 mRNA levels (p = 0.0006; Fig. 3a). A more detailed analysis of each tissue sample showed that all specimens from superficial bladder cancer (pTa and pT1) displayed low mRNA levels of CXCR4. In the group of advanced stage tumors (pT2–pT4), 6 of 14 (43%) samples had high CXCR4 expression levels (Fig. 3b). In comparison, CCR7 mRNA expression levels were equivalent in both superficial and invasive bladder cancer (Fig. 3A).

thumbnail image

Figure 3. Expression of chemokine receptors in bladder cancer tissue samples. (a) Quantitative, multiplex real-time PCR analysis of all chemokine receptors (CCR1–CCR10, CXCR1–CXCR6, XCR1 and CX3CR1) was performed in 28 primary bladder cancer tissue samples. mRNA levels from individual samples in groups pTa (n = 8) and pT1 (n = 6) were averaged and compared to the mRNA levels of samples in groups pT2 (n = 2), pT3 (n = 7) and pT4 (n = 5). The highest mRNA levels of CXCR4 occurred in invasive and locally advanced bladder cancer (pT2–T4), while superficial bladder cancer (pTa and pT1) displayed low CXCR4 mRNA levels. CCR7 mRNA showed equal levels in superficial as well as invasive bladder cancer without differentiation of the tumor stage. Values are expressed as absolute number of copies of target gene from 20 ng of total RNA. Means and standard errors are plotted. *, difference between groups (p = 0.0006). (b) mRNA expression levels of CXCR4 in bladder cancer tissue samples stratified for each tumor stage. All specimens from superficial bladder cancer (pTa and pT1) displayed low levels of CXCR4. In the group of advanced tumor stages (pT2–pT4), 6 of 14 (43%) samples revealed high CXCR4 mRNA expression levels.

Download figure to PowerPoint

To examine CXCR4 protein expression in a large group of tumor specimens, we carried out CXCR4 immunostaining on a bladder cancer TMA containing 62 different tumor specimens as well as 5 samples of normal urothelium. Normal urothelium showed either no protein expression (n = 3) or a very faint staining exclusively on the umbrella cell layer, but not in the intermediate or basal layer (n = 2) (Fig. 4). All bladder cancer specimens showed at least some positive staining. Superficial bladder tumors (pTa and pT1) generally showed weak (+) staining of CXCR4 in the cytoplasm and nucleus (Figs. 4 and 5). In contrast, 15 of 45 (33%) cases of invasive cancer (pT2–pT4) showed high levels (++) of CXCR4 expression in the cytoplasm and nucleus, whereas 30 (67%) invasive tumor samples displayed low levels (+) of CXCR4 expression (Figs. 4 and 5). In the group of invasive bladder cancers with high CXCR4 protein expression, 4 samples that were classified as high-grade and high-stage (pT3) tumors displayed heterogenous staining. Specifically, CXCR4-positive cells were segregated in focal areas of the tumors and the staining intensity varied among individual cells.

thumbnail image

Figure 4. Immunohistochemistry of CXCR4 in normal urothelium as well as in superficial and invasive bladder cancer. Tissue sections from normal urothelium as well as superficial and invasive bladder cancer were stained with either anti-CXCR4 monoclonal antibody (a, c, e) or a mouse IgG-matched isotype control antibody (b, d, f) using the indirect avidin-biotin HRP method. Sections were counterstained with hematoxylin. Normal urothelium displayed no protein expression for CXCR4 (a). Superficial bladder tumor (pTa) showed weak staining of CXCR4 in the cytoplasm as well as nuclear staining in a subgroup of cells (c). In contrast, an invasive bladder tumor (pT3) showed homogenous staining with high expression of CXCR4 in the cytoplasm as well as the nucleus in most bladder cancer cells (e). Scale bar = 100 μm.

Download figure to PowerPoint

thumbnail image

Figure 5. Quantification of CXCR4 immunostaining in bladder cancer. CXCR4 protein expression in bladder tumors was rated as low (+) or high (++) cytoplasmic staining. High-level staining (++) required a minimum of 20% of cells exhibiting strong staining.

Download figure to PowerPoint

Stratifying bladder cancer samples according to the grading system, all 11 low-grade tumors displayed a weak (+) protein expression. Of the 51 high-grade tumors, 11 cases (22%) showed strong (++) CXCR4 expression and 35 samples (78%) displayed weak (+) staining. The relationship between the staining patterns of bladder cancer and histopathologic features is shown in Figure 5.

CXCR4 chemokine receptor and its ligand CXCL12 mediate enhanced directional migration of bladder cancer cells

To approach the question of whether the CXCR4 chemokine receptor has any functional significance in bladder cancer cell metastasis, we examined the chemotaxis of bladder cancer cells and normal urothelial cells along a CXCL12 chemokine gradient using 8 μm pore membranes in a modified Boyden chamber system. For this purpose, we chose an undifferentiated bladder cancer cell line TCCSUP (grade 4), which was taken from a patient who subsequently developed metastases.22 Bladder cancer cell line TCCSUP and primary cells from normal urothelium were exposed to a gradient of CXCL12 ranging from 0–200 ng/ml (Fig. 6a). CXCL12 exposure resulted in increased directional migration of bladder cancer cells in a dose-dependent manner. The optimal chemotactic response to CXCL12 was found at a concentration of 100 ng/ml, which induced a 2-fold increase (p = 0.041) of migration compared to unstimulated bladder tumor cells. In contrast, normal urothelial cells did not respond to CXCL12 and lacked chemotactic migration (Fig. 6a). To confirm that the migration effect was due to the presence of the cognate chemokine receptor CXCL12, bladder tumor cells were preincubated with either CXCR4-blocking antibody or an isotype-matched control antibody. The CXCR4-blocking antibody, but not the isotype-matched control, significantly reduced the number of migrating cells (p = 0.014; Fig. 6B). In summary, CXCL12-mediated chemotaxis of bladder tumor cells was blocked by a CXCR4 antibody, indicating the specificity of the chemokine ligand/receptor interaction.

thumbnail image

Figure 6. CXCL12/CXCR4 interaction-stimulated chemotactic migration of bladder cancer cells. (a) Chemotaxis of CXCR4-positive bladder cancer cells (TCCSUP) and normal urothelial cells (BN48) was investigated using Boyden chambers with 8 μm pore membrane inserts. Cell suspensions (5 × 104 cells/500 μl) were added to the upper chamber. A CXCL12 gradient was achieved by adding various concentrations of CXCL12 (0–200 ng/ml) to the lower chamber. After incubation for 24 hr, migrated cells on the lower surface of the membrane were stained with H&E and counted by light microscopy in 3 different fields at a magnification of ×200. The optimal chemotactic response to CXCL12 was observed at a concentration of 100 ng/ml. Normal urothelial cells did not respond to CXCL12. *, significantly different from control (no chemokine; p = 0.041). (b) Bladder cancer cells were preincubated with either anti-human CXCR4 monoclonal antibody or mouse IgG isotype antibody. Antibody-coated cells were added to the upper chamber and incubated for 24 hr in the presence of CXCL12 (100 ng/ml) in the lower chamber. Exposure to CXCR4-blocking antibody, but not an isotype-matched control, significantly reduced the number of migrating cells. Results are means of independent experiments performed in triplicate; Bars, SD (n = 3). **, significantly different from control (no chemokine; p = 0.047); ***, significantly different from CXCL12 alone (p = 0.014).

Download figure to PowerPoint

CXCR4 chemokine receptor and its ligand CXCL12 mediate enhanced invasion of bladder cancer cells

To examine the possibility that CXCR4 and its ligand CXCL12 could play a role in bladder cancer cell invasion, we examined the ability of bladder cancer cells to invade a reconstituted ECM membrane (Matrigel) in the Boyden chamber assay system. In the presence of CXCL12 (100 ng/ml), the number of invading cells increased significantly (p = 0.005) compared to tumor cells without chemokine ligand stimulation (Fig. 7a).

thumbnail image

Figure 7. CXCL12/CXCR4 interaction-stimulated invasion of bladder cancer cells through ECM. (a) TCCSUP bladder tumor cells (5 × 104 cells/500 μl) were incubated for 24 hr on a transwell insert coated with ECM proteins (Matrigel). Differing concentrations of CXCL12 (0–200 ng/ml) were placed in the lower chamber of the transwell plate. H&E-stained invading cells were counted under a light microscope in 3 different fields at a magnification of ×200. The highest number of invading tumor cells through the ECM membrane was found with a CXCL12 concentration of 100 ng/ml. *, significantly different from control (no chemokine; p = 0.005). (b) To confirm specificity of chemokine ligand/receptor interaction, bladder tumor cells were preincubated with anti-human CXCR4 monoclonal antibody or matched isotype control antibody. The number of invading cells in response to CXCL12 (100 ng/ml) was significantly reduced after CXCR4 blocking. No effect was observed with an IgG isotype antibody. Results are means of independent experiments performed in triplicate; Bars, SD (n = 3). **, significantly different from control (no chemokine; p = 0.036); ***, significantly different from CXCL12 alone (p = 0.026).

Download figure to PowerPoint

The specificity of the CXCL12/CXCR4 interaction was confirmed by preincubating cells with either CXCR4-blocking antibody or an isotype-matched control. Neutralizing anti-CXCR4 significantly impaired the invasion of bladder tumor cells by 56% (p = 0.026). In contrast, bladder tumor cells supplemented with isotype-matched control antibody were not affected (Fig. 7b).

CXCL12 induces actin polymerization in bladder cancer cells

An extensive cytoskeletal reorganization of tumor cells involving actin polymerization is a prerequisite for cell migration and invasion.23 We monitored cytoskeletal changes in bladder tumor cells (TCCSUP) in response to 100 ng/ml CXCL12 at various time points. Alexa Fluor 594 phalloidin staining of CXCL12-treated cells exhibited a distinct cytoskeletal redistribution of F-actin stress fibers, beginning 10 min after CXCL12 exposure (Fig. 8b) and persisting for 40 min. In comparison, untreated bladder tumor cells contained only a few cytoplasmatic stress fibers and there was no evidence of pseudopodia formation (Fig. 8a).

thumbnail image

Figure 8. CXCL12 induces actin polymerization in bladder cancer cells. TCCSUP bladder tumor cells were serum-starved overnight and then stimulated with 100 ng/ml CXCL12 (R&D Systems). Actin cytoskeleton was visualized by Alexa Fluor 594 phalloidin and fluorescence microscopy. CXCL12-stimulated cells exhibited a distinct cytoskeletal redistribution of F-actin stress fibers and pseudopodia beginning 10 min after CXCL12 exposure (b). In contrast, untreated bladder tumor cells contained only a few cytoplasmatic stress fibers and no pseudopodia (a). Scale bar = 20 μm.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Systemic tumor progression is the prevalent form of bladder cancer recurrence after radical cystectomy. Pelvic lymph nodes are the preferential sites of metastases, followed by widespread metastases to the lung, liver and bone marrow. Despite advances in systemic polychemotherapy, metastatic bladder cancer continues to be incurable.2, 3 Hence, it is important to understand mechanisms underlying bladder cancer metastasis and to identify novel drug targets for the inhibition of metastatic spread.

Several recent studies have demonstrated that tumor metastasis is not a random process, but rather mimics specific mechanisms of cell migration such as leukocyte trafficking and homing by the chemokine system.24 Tumor cells of different origins have been shown to express distinct, tumor-specific patterns of chemokine receptors; signaling through these receptors is thought to be crucial for chemotactic migration, invasion and cancer metastasis.9, 10, 11, 12 The aim of our study was to investigate the role of chemokines and their receptors in promoting bladder cancer metastasis.

Eighteen chemokine receptors were screened using both quantitative PCR and Western blotting in 7 bladder cancer cell lines and a primary normal urothelial cell line. Our analysis identified CXCR4 as the only chemokine receptor whose mRNA expression levels were consistently upregulated in all bladder cancer cell lines, but not expressed in normal urothelial cells. The consistent expression across all our tumor cell lines reinforced the idea that CXCR4 might play a prominent role in bladder cancer metastasis. Western blot analysis confirmed the protein expression of CXCR4 in all investigated bladder cancer cell lines. While mRNA expression levels in normal urothelial cells were almost undetectable, Western blot analysis displayed a very faint protein band under the conditions of our experiments. The quantitative discrepancy between RNA and protein levels can be best explained by the fact that RNA and protein levels are regulated by distinct mechanisms. These involve control at the level of both synthesis and stability and are discussed in Wang et al.25

Consistent with our findings in bladder cancer cell lines, quantitative PCR analysis of 28 primary bladder cancer tissue samples also revealed that CXCR4 was the only chemokine receptor whose mRNA expression levels correlated with tumor stage. High mRNA levels of CXCR4 were seen in invasive and locally advanced bladder cancer (pT2–pT4). In contrast, superficial bladder cancer (pTa and pT1), which is clinically characterized by rare metastatic spread, displayed the lowest CXCR4 mRNA expression levels.26 In comparison, CCR7 mRNA expression levels did not correlate with bladder tumor stage. Immunohistochemic analysis of a bladder cancer TMA was consistent with the mRNA expression data for CXCR4. Superficial and low-grade bladder tumors showed low cytoplasmic staining intensity, whereas invasive and high-grade tumors were associated with high levels of cytoplasmic CXCR4 protein expression. In some immunoreactive carcinoma cells, the nuclei were also positively stained. Nuclear staining has also been described in breast cancer.17

The significance of the role of CXCR4 will have to be determined in a large cohort of bladder tumor patients undergoing long-term observation for disease outcome. Only such a study will help determine the prognostic value of CXCR4 mRNA and protein expression. Interestingly, a recent study investigating tissue samples from clear cell renal cell carcinoma found a significant correlation between strong CXCR4 staining and poor tumor-specific survival using multivariate analysis.27 Monitoring CXCR4 expression in patients with bladder cancer may improve current tumor staging systems and provide new concepts for adjuvant chemotherapy.

Based on the results in bladder cancer tissue samples (Fig. 3a), it would also be interesting to investigate the protein expression of CCR7 in a broader range of primary bladder tumor samples and to explore the functionality of the CCR7/CXCL21 axis. It is possible that CCR7 also plays a role in migration and metastasis of bladder cancer cells.

Consistent with its role in chemotactic and invasive responses, the CXCR4/CXCL12 axis has been associated with actin polymerization and pseudopodia formation, both of which are crucial steps in metastasis.12 CXCL12, which is the only known ligand for CXCR4, is expressed at high levels in organs such as lymph nodes, lung, liver and bone marrow.9 These organs are the preferential sites for bladder cancer metastasis, consistent with the importance of the CXCR4/CXCL12 axis.

Not only do our results establish a correlation between CXCR4 expression and tumor aggressiveness, but they also suggest that this relationship is based on the ability of CXCR4 to be recruited to the cell surface upon stimulation by CXCL12 (as shown by FACS analysis) and the ability of CXCR4 to stimulate cell motility. In tumor cells, motility is associated with extensive actin polymerization and cytoskeletal reorganization.23 We observed that the corresponding ligand CXCL12 stimulated these cytoskeletal changes as well as induced the migration of bladder cancer cells in Boyden chambers. This effect was blocked by anti-CXCR4 antibody.

Importantly, CXCR4-positive bladder cancer cells were able to cross Boyden chamber membranes even when the membranes were precoated with ECM in the form of Matrigel. Similar invasive behavior has also been reported previously for small-cell lung, breast, and prostate cancer cells.9, 11, 12 ECM invasion is thought to be enhanced by local production of ECM-degrading enzymes, including metalloproteinases (MMPs). Inoue et al.28 demonstrated that bladder cancer cells overexpressing CXCL8 also showed enhanced MMP9 activity. It has also been shown that CXCL12 enhances MMP2 activity in rhabdomyosarcoma cell lines.29 Thus, CXCR4 may coordinate cytoskeletal and proteolytic responses that culminate in tumor growth and metastasis.

In addition to its role in tumor cells, CXCR4 is a major coreceptor for HIV. Several compounds have been found to block HIV binding to CXCR4. One such compound is a bicyclam derivative AMD3100 (AnorMED, Langley, BC, Canada).30 In a phase I clinical trial, AMD3100 was well tolerated without any significant toxicity.31 This suggests that AMD3100 or similar compounds may potentially be used to interfere with tumor progression and metastasis in bladder cancer.

In summary, we have identified CXCR4 as the only chemokine receptor whose expression level in bladder tumor cells correlates with the advancement of tumor stage. The chemokine CXCL12 has an important chemoattractant effect on CXCR4-positive bladder cancer cells in stimulating their migration and invasion. Based on our results, the CXCR4/CXCL12 axis appears to be a crucial step in the metastasis of bladder cancer and is a potential therapeutic target.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

We are grateful to Mrs. H. Angeli for her expert technical assistance.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  • 1
    Stein JP, Lieskovsky G, Cote R, Groshen S, Feng AC, Boyd S, Skinner E, Bochner B, Thangathurai D, Mikhail M, Raghavan D, Skinner DG. Radical cystectomy in the treatment of invasive bladder cancer: long-term results in 1,054 patients. J Clin Oncol 2001; 19: 66675.
  • 2
    Saxman SB, Propert KJ, Einhorn LH, Crawford ED, Tannock I, Raghavan D, Loehrer PJ, Trump D. Long-term follow-up of a phase III intergroup study of cisplatin alone or in combination with methotrexate, vinblastine, and doxorubicin in patients with metastatic urothelial carcinoma: a cooperative group study. J Clin Oncol 1997; 15: 25649.
  • 3
    Lehmann J, Retz M, Stockle M. Is there standard chemotherapy for metastatic bladder cancer? Quality of life and medical resources utilization based on largest to date randomized trial. Crit Rev Oncol Hematol 2003; 47: 1719.
  • 4
    Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, Kitamura Y, Yoshida N, Kikutani H, Kishimoto T. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 1996; 382: 6358.
  • 5
    Bogenrieder T, Herlyn M. Axis of evil: molecular mechanisms of cancer metastasis. Oncogene 2003; 22: 652436.
  • 6
    Vicari AP, Caux C. Chemokines in cancer. Cytokine Growth Factor Rev 2002, 13: 14354.
  • 7
    Bernardini G, Ribatti D, Spinetti G, Morbidelli L, Ziche M, Santoni A, Capogrossi MC, Napolitano M. Analysis of the role of chemokines in angiogenesis. J Immunol Methods 2003; 273: 83101.
  • 8
    Zlotnik A, Yoshie O. Chemokines: a new classification system and their role in immunity. Immunity 2000; 12: 1217.
  • 9
    Muller A, Homey B, Soto H, Ge N, Catron D, Buchanan ME, McClanahan T, Murphy E, Yuan W, Wagner SN, Barrera JL, Mohar A, et al. Involvement of chemokine receptors in breast cancer metastasis. Nature 2001; 410: 5056.
  • 10
    Scotton CJ, Wilson JL, Milliken D, Stamp G, Balkwill FR. Epithelial cancer cell migration: a role for chemokine receptors? Cancer Res 2001; 61: 49615.
  • 11
    Taichman RS, Cooper C, Keller ET, Pienta KJ, Taichman NS, McCauley LK. Use of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to bone. Cancer Res 2002; 62: 18327.
  • 12
    Burger M, Glodek A, Hartmann T, Schmitt-Graff A, Silberstein LE, Fujii N, Kipps TJ, Burger JA. Functional expression of CXCR4 (CD184) on small-cell lung cancer cells mediates migration, integrin activation, and adhesion to stromal cells. Oncogene 2003; 22: 8093101.
  • 13
    Zwergel T, Kakirman H, Schorr H, Wullich B, Unteregger G. A new serial transfer explant cell culture system for human prostatic cancer tissues preventing selection toward diploid cells. Cancer Genet Cytogenet 1998; 101: 1623.
  • 14
    Sobin LH, Fleming ID. TNM classification of malignant tumors, 5th edition (1997). Sobin LH, Fleming ID. Union Internationale Contre le Cancer and the American Joint Committee on Cancer. Cancer 1997; 80: 18034.
  • 15
    Epstein JI, Amin MB, Reuter VR, Mostofi FK. The World Health Organization/International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder. Bladder Consensus Conference Committee. Am J Surg Pathol 1998; 22: 143548.
  • 16
    Dolganov GM, Woodruff PG, Novikov AA, Zhang Y, Ferrando RE, Szubin R, Fahy JV. A novel method of gene transcript profiling in airway biopsy homogenates reveals increased expression of a Na+–K+–Cl– cotransporter (NKCC1) in asthmatic subjects. Genome Res 2001; 11: 147383.
  • 17
    Kato M, Kitayama J, Kazama S, Nagawa H. Expression pattern of CXC chemokine receptor-4 is correlated with lymph node metastasis in human invasive ductal carcinoma. Breast Cancer Res 2003; 5: 14450.
  • 18
    Kolmogoroff A. Confidence limits for an unknown distribution function. Ann Math Stat 1941; 12: 4613.
  • 19
    Smirnoff N. Tables for estimating the goodness of fit of empirical distributions. Ann Math Stat 1948; 19: 27981.
  • 20
    Mann HB, Whitney DR. On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat 1947; 18: 5060.
  • 21
    Kruskal WH, Wallis WA. Use of ranks in one-criterion variance analysis. J Am Stat Assoc 1953; 47: 583621.
  • 22
    Nayak SK, O'Toole C, Price ZH. A cell line from an anaplastic transitional cell carcinoma of human urinary bladder. Br J Cancer 1977; 35: 14251.
  • 23
    Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat Rev Cancer 2003; 3: 36274.
  • 24
    Rossi D, Zlotnik A. The biology of chemokines and their receptors. Annu Rev Immunol 2000; 18: 21742.
  • 25
    Wang Y, Newton DC, Robb GB, Kau CL, Miller TL, Cheung AH, Hall AV, VanDamme S, Wilcox JN, Marsden PA. RNA diversity has profound effects on the translation of neuronal nitric oxide synthase. Proc Natl Acad Sci U S A 1999; 96: 1215055.
  • 26
    Helpap B, Schmitz-Drager BJ, Hamilton PW, Muzzonigro G, Galosi AB, Kurth KH, Lubaroff D, Waters DJ, Droller MJ. Molecular pathology of non-invasive carcinomas. Virchows Arch 2003, 442: 30916.
  • 27
    Staller P, Sultikova J, Lisztwan J, Moch H, Oakeley EJ, Krek W. Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL. Nature 2003; 425: 30711.
  • 28
    Inoue K, Slaton JW, Kim SJ, Perrotte P, Eve BY, Bar-Eli M, Radinsky R, Dinney CPN. Interleukin 8 expression regulates tumorigenicity and metastasis in human bladder cancer. Cancer Res 2000; 60: 22909.
  • 29
    Libura J, Drukala J, Majka M, Tomescu O, Navenot JM, Kucia M, Marquez L, Peiper SC, Barr FG, Janowska-Wieczorek A, Ratajczak MZ. CXCR4-SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion. Blood 2002; 100: 2597606.
  • 30
    Donzella GA, Schols D, Lin SW, Este JA, Nagashima KA, Maddon PJ, Allaway GP, Sakmar TP, Henson G, De Clercq E, Moore JP. AMD3100, a small molecule inhibitor of HIV-1 entry via the CXCR4 co-receptor. Nat Med 1998; 4: 727.
  • 31
    Hendrix CW, Flexner C, MacFarland RT, Giandomenico C, Fuchs EJ, Redpath E, Bridger G, Henson GW. Pharmacokinetics and safety of AMD-3100, a novel antagonist of the CXCR-4 chemokine receptor, in human volunteers. Antimicrob Agents Chemother 2000; 44: 166773.