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. 2005 Jul;242(1):55–63. doi: 10.1097/01.sla.0000168555.97710.bb

Macrophage Migration Inhibitory Factor Stimulates Angiogenic Factor Expression and Correlates With Differentiation and Lymph Node Status in Patients With Esophageal Squamous Cell Carcinoma

Yi Ren *, Simon Law *, Xin Huang *, Ping Yin Lee *, Michael Bacher †, Gopesh Srivastava ‡, John Wong *
PMCID: PMC1357705  PMID: 15973102

Abstract

Objective:

The objectives of this study were: 1) to examine the expression of macrophage migration inhibitory factor (MIF) in esophageal squamous cell carcinoma (ESCC); 2) to see if a relationship exists between MIF expression, clinicopathologic features, and long-term prognosis; and 3) to ascertain the possible biologic function of MIF in angiogenesis.

Summary Background Data:

MIF has been linked to fundamental processes such as those controlling cell proliferation, cell survival, angiogenesis, and tumor progression. Its role in ESCC, and the correlation of MIF expression and tumor pathologic features in patients, has not been elucidated.

Methods:

The expression of MIF in tumor and nontumor tissues was examined by immunohistochemical staining. Concentrations of MIF, vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) in patients’ sera and in the supernatant of tumor cells culture were examined by ELISA. Correlations with clinicopathologic factors were made.

Results:

In 72 patients with ESCC, intracellular MIF was overexpressed in esophagectomy specimens. The expression of MIF correlated with both tumor differentiation and lymph node status. The median survival in the low-MIF expression group (<50% positively stained cancer cells on immunohistochemistry) and high expression group (≥50% positively stained cancer cells) was 28.3 months and 15.8 months, respectively (P = 0.03). The 3-year survival rates for the 2 groups were 37.7% and 12.1%, respectively. MIF expression was related to microvessel density; increased MIF serum levels also correlated with higher serum levels of VEGF. In addition, in vitro MIF stimulation of esophageal cancer cell lines induced a dose-dependent increase in VEGF and IL-8 secretion.

Conclusions:

These results demonstrate, for the first time, that human esophageal carcinomas express and secrete large amounts of MIF. Through its effects on VEGF and IL-8, MIF may serve as an autocrine factor in angiogenesis and thus play an important role in the pathogenesis of ESCC.

Macrophage migration inhibitory factor (MIF) is overexpressed in esophageal squamous cell carcinoma. MIF expression correlated with tumor differentiation, lymph node metastasis, and survival. MIF produced by tumor cell, acting as an autocrine factor, stimulates the expression of VEGF and IL-8 and therefore enhances angiogenesis and tumor growth.

Esophageal cancer is the sixth most common cancer in the world, with a particularly high incidence among Chinese. The 2 main histopathologic types of esophageal cancer are squamous cell carcinoma (ESCC) and adenocarcinoma. In Asian countries, most are squamous. Surgical resection remains the standard treatment of resectable disease. In experienced centers, the mortality from resection is low,1,2 and a 5-year survival rate of up to 40% is achieved after apparently curative resections. In unselected patients, however, the overall 5-year survival is less than 10%, largely because of late disease presentation. Various studies have attempted to find markers that, by acting as useful adjuncts to morphologic and clinical features, could be used to predict the behavior of esophageal tumors.3–7 However, to date, none has been proven to be sufficiently reliable to be of practical use in the clinical setting.

Macrophage migration inhibitory factor (MIF), originally identified as a product of activated lymphocytes, has since been found to play an important role in the host response to endotoxic shock,8–10 joint inflammation,11 glomerulonephritis,12 transplantation rejection,13,14 and atherosclerosis.15 MIF normally circulates at basal levels in the blood. In response to various stimuli, additional MIF is secreted by the anterior pituitary gland and activated monocytes/macrophages.16 In addition to its potent effects on the immune system, several reports have linked MIF to fundamental processes that control cell proliferation, differentiation, angiogenesis, and tumor progression.17 Hudson et al18 have shown that MIF could inactivate the tumor suppression activity of p53. Recently, overexpression of MIF was reported in human melanoma,19 breast carcinoma,20 metastatic prostate cancer,21 lung adenocarcinoma,22 and hepatocellular carcinoma.23 The discovery of MIF in these cancers led to our hypothesis that MIF could also play an important role in esophageal cancer carcinogenesis and could serve as an early prognostic indicator.

Two angiogenic factors, vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), have received much attention with regard to their roles in tumor carcinogenesis. These factors are often produced by tumor cells.24–26 MIF has also been shown to stimulate angiogenesis,27 but a possible relationship between MIF and the production of VEGF and IL-8 by tumor cells remains to be confirmed.

The aims of this study were 1) to examine the expression of MIF in ESCC; 2) to see if a relationship exists between MIF expression, clinicopathologic features and long-term prognosis; and 3) to ascertain if MIF is related to VEGF or IL-8 secretions.

MATERIALS AND METHODS

Reagents

All reagents were obtained from Sigma (St. Louis, MO) unless otherwise stated.

Patients and Specimens

This study included 72 Chinese patients who had esophageal cancer and had undergone esophagectomy in the Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Hong Kong. All patients had ESCC of the thoracic esophagus and no history of other malignancy. All patients had surgical resection only, and none had neoadjuvant treatments. To determine whether MIF was an important prognostic factor in esophageal cancer, only patients who had survived the esophagectomy were recruited. Tumor staging was performed according to the TNM classification.28 The grades of tumor differentiation (well, moderately, or poorly differentiated) were determined using the World Health Organization criteria. Histologic sections of the resected surgical specimens were retrieved and reviewed. Representative blocks from each specimen (both tumor and nontumor tissue away from the primary tumor) were chosen for immunohistochemical study.

Blood samples were collected, before surgical resection, from patients, and from healthy subjects (N = 15) for controls. Sera were collected by centrifuging the whole blood at 3000 rpm for 15 minutes, and the sera aliquots were stored at −80°C until used in ELISA assay.

Immunohistochemical Staining

One-color immunohistochemical staining using mouse monoclonal antibodies (mAbs) to human MIF, VEGF (BD PharMingen, Inc., San Diego, CA) and CD34 (Dako, Glostrup, Denmark) was performed as described previously.12 Paraffin sections were incubated with mAbs overnight at 4°C. Sections were then washed with phosphate-buffered saline (PBS, pH 7.4). Endogenous peroxidase was inactivated in 3% H2O2 in methanol, and then incubated with peroxidase-conjugated goat antimouse IgG (Dako). After washing with PBS, sections were incubated with mouse peroxidase antiperoxidase complexes (Dako) and developed with 3,3-diaminobezidine to produce a brown color.

Assessment of Immunohistochemical Staining

Examination of MIF staining was done at high power (×400) using a standard light microscope. MIF-positive cells were counted by monitoring at least 500 tumor cells in each field for at least 10 randomly selected fields. MIF and VEGF expression was graded semi-quantitatively into 5 categories according to the percentage of positively stained cancer cells (Table 1): −, <5% positively stained cancer cells; +, 5% to 25% positively stained cancer cells; ++, 26% to 50% positively stained cancer cells; +++, 51% to 75% positively stained cancer cells; ++++, 76% to 100% positively stained cancer cells.

TABLE 1. Characteristics of 72 Patients and Correlation Between the Expression of MIF and Clinical Classification: Results of Immunohistochemical Staining

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In the multivariate analyses for factors affecting survival, patients were divided into 2 groups: low MIF expression (<50% positively stained tumor cells) and high MIF expression (≥50% positively stained tumor cells).

Evaluation of Microvessel Density (MVD)

Tissue sections were immunostained with human CD34 mAb. The tissue sections were scanned by light microscopy at low power field (×40) and the vessel count in areas with the highest number of capillaries was assessed at high power field (HPF, ×200). The average counts of 5 fields were determined. The MVD were counted by 2 investigators who had no knowledge of MIF and VEGF expression of the tumors.

Primary Epithelial Cell Culture

Normal epithelial cells were established from normal human esophageal biopsies in patients with benign diseases who underwent endoscopy. The tissue specimens were obtained fresh from esophageal tissue removed by biopsy. The biopsy specimens were washed with PBS and minced with scissors into very small pieces of 0.5 to 1 mm3. The tissue fragments were incubated with 1.5 units/mL dispase (Roche Molecular Biochemicals) at 4°C overnight, and the epithelium was dissected away and incubated with trypsin (Invitrogen, San Diego, CA). The reaction was stopped with soybean trypsin inhibitor and centrifuged. The pellet was cultured in keratinocyte-SFM medium (KSFM) (Invitrogen) supplemented with 40 μg/mL bovine pituitary extract (Invitrogen), 1.0 ng/mL epithelial growth factor (Invitrogen), 100 units/mL penicillin (Gibco, Grand Island, NY), 100 μg/mL streptomycin (Gibco), 5 μg/mL gentamycin, and 100 units/mL nyastatin (Invitrogen). After 2 and 3 passages, cells showed the morphology of epithelial cells and more than 95% of cells were cytokeratin positive.

Determination of MIF Expression in Tumor Cell Lines and Primary Epithelial Cells

Two previously reported human cell lines, HKESC-1 and HKESC-2, were established from primary ESCC locally.29,30 Both cell lines grew as adherent monolayers. Cell lines were maintained in Minimum Essential Medium (MEM, Gibco) with 0.1 mmol/L nonessential amino acids, containing 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/mL, Gibco), and streptomycin (100 μg/mL, Gibco).

Cell surface MIF was evaluated by immunofluorescence staining with anti-MIF mAb (R&D Systems, Minneapolis, MN) and FITC-conjugated secondary Ab. After incubation for 45 minutes at 4°C and followed by washing with PBS, cells were either fixed in 1% paraformaldehyde/PBS or resuspended in PBS containing 0.5% propidium iodide to exclude dead cells. Labeled cells were assayed by a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) that automatically and simultaneously measured the fluorescence of individual cells identified by their size-dependent light-scattering properties.

Intracellular MIF expression was evaluated following cell membrane staining. Cells were fixed in 4% paraformaldehyde/PBS for 10 minutes and then treated with 1% saponin for 20 minutes at room temperature. The treated cells were incubated with anti-MIF mAb and secondary mAb conjugated with FITC, both for 45 minutes at 20°C. After a final wash, cells were resuspended in 1% paraformaldehyde/PBS. Analysis of MIF expression was performed with FACScan as described above.

Measurement of the Levels of MIF, VEGF, and IL-8 in Patients’ Sera and Cell Culture Supernatants

Levels of MIF, VEGF and IL-8 in: 1) sera of patients and healthy subjects, and 2) supernatants of tumor cell lines and primary epithelial cells were measured using ELISA kits (R&D Systems). Tumor cells and primary epithelial cells (5 × 104) were cultured in 24-well plates in MEM with 10% FBS or KSFM, respectively, for 24 hours and then washed twice with PBS, and further cultured for 24 hours in medium without sera. The culture supernatants of cells were collected after each experiment, and centrifuged at 1000 rpm for 10 minutes to remove cells and debris. ELISA was then performed.

Cell Stimulation Study Using MIF

Tumor cells and primary epithelial cells (5 × 104) were seeded in 24-well plates, and cultured in MEM with 10% FBS or KSFM, respectively, for 24 hours. Cells were washed with PBS and treated in MEM with 1% BSA and incubated with various concentrations (ranging from 1.0 ng/mL to 200.0 ng/mL) of recombinant human MIF for 24 hours. The culture supernatants were collected, and the angiogenic cytokines VEGF and IL-8 were then assayed by ELISA.

Statistical Analysis

Statistical tests including the Mann-Whitney U, χ2, and Kendall tau-b tests were used. Survival was estimated by the Kaplan-Meier method, and the differences were analyzed by the log-rank test. Multivariate analysis was performed by using the Cox proportional hazards regression model to evaluate various prognostic factors. A P value of less than 0.05 indicated that the difference was significant. All statistical analyses were performed using the computer software Statistical Package for the Social Sciences 10.0 (SPSS Inc., Chicago, IL).

RESULTS

Patient demographics, tumor differentiation, and disease stage distributions are shown in Table 1. There were 64 men and 8 women; the mean age was 63.2 years (range, 39–89 years).

Expression of MIF in ESCC Tissues

Tumor and nontumor tissues were examined under hematoxylin and eosin staining by standard histopathologic criteria. MIF was expressed in all specimens of ESCC. MIF staining in tumor tissue was heterogeneous (Fig. 1A and C). MIF protein was also detected in the vessel endothelia and some infiltrating cells such as macrophages and lymphocytes in nontumor regions (data not shown). MIF expression however was absent in all nontumor specimens.

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FIGURE 1. Immunohistochemical staining of ESCC. The expression of MIF, VEGF, and CD34 is stained brown. The purple color is counterstain, which indicates cell nuclei. A, MIF in well-differentiated tumor (original magnification ×100). B, MIF in nontumor tissue (original magnification ×100). C, MIF in poorly differentiated tumor (original magnification ×200). D, VEGF in tumor tissue (original magnification ×200). E, MVD in tumor tissue (original magnification ×100).

Correlation Between MIF and VEGF Immunoreactivity and MVD

Tumor sections were immunohistochemically stained for the expression of MIF and VEGF (Fig. 1A, C, and D). The results showed that MIF expression positively correlated with VEGF expression (r = 0.78, P = 0.021). Specific staining of capillary vessels by anti-CD34 was observed in all tumor specimens (Fig. 1E). The median tumor MVD was 47.4/HPF (range, 29.4–106.6). The MVD was significantly higher in tumors with high expression of MIF than in tumors with low expression of MIF (r = 0.82, P = 0.0001; Fig. 2).

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FIGURE 2. Box plots showing higher tumor MVD in tumors with high MIF expression than in tumors with low MIF expression (P = 0.0001).

MIF Expression and Clinicopathologic Features

The relationships between MIF expression in esophageal carcinoma tissues and clinicopathologic features are shown in Table 1. No statistically significant association was observed between MIF expression and age, gender, T-stage, or M-stage. MIF expression, however, correlated with both tumor differentiation (P = 0.003) and lymph node status (P = 0.021). Poorly differentiated esophageal carcinomas expressed a higher level of MIF compared with well-differentiated tumors. Higher MIF expression was also more frequently observed in patients with lymph node metastasis. Correlation with R-category of resection was of borderline significance (P = 0.055).

MIF Expression and Survival

Patients were divided into 2 groups, low MIF expression (<50% positively stained cancer cells) and high MIF expression (≥50% positively stained cancer cells). Kaplan-Meier survival curves for these 2 groups are shown in Figure 3. The median survival time for patients with low MIF expression and those with high MIF expression was 28.3 months and 15.8 months, respectively (P = 0.03). The 3-year survival rates for the 2 groups were 37.7% and 12.1%, respectively.

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FIGURE 3. Kaplan-Meier survival curves of patients in low-MIF expression group versus high-MIF expression group. Low-MIF expression group: <50% positively stained cancer cells; high-MIF expression group: >50% positively stained cancer cells, P = 0.03.

Multivariate analysis was carried out to identify independent prognostic factors for long-term survival for the following variables: pTNM stage distribution, R-category of resection, tumor differentiation, pT-stage, pN-stage, pM-stage, and level of MIF expression (indicated as high or low groups). Only pTNM stage was an independent factor for survival (hazard ratio, 2.02; 95% confidence interval, 1.350–3.027, P < 0.001).

MIF Concentration in the Sera of Patients With ESCC

The serum MIF level of patients with ESCC was significantly higher than that of healthy controls (Table 2; Fig. 4). The levels of serum VEGF and IL-8 were also significantly higher in patients with esophageal carcinoma compared with controls. Furthermore, there was a positive correlation between serum MIF concentration and the level of VEGF (P = 0.001) in sera of patients with esophageal carcinoma. Serum concentration of MIF, however, was not related to clinicopathologic features or survival.

TABLE 2. Serum Levels of Cytokines in Patients With ESCC

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FIGURE 4. Serum concentrations of MIF in patients with ESCC and healthy controls.

Detection of MIF in ESCC Cell Lines and Normal Epithelial Cells

Expressions of MIF in HKESC-1 and HKESC-2 were assessed by flow cytometry. MIF expression was predominantly identified in the cytoplasm of tumor cells, while there was no expression on the cell surface (Fig. 5). By contrast, MIF expression in normal primary epithelial cells was only weakly positive in the cytoplasm of cells (Fig. 5).

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FIGURE 5. MIF expression in esophageal tumor cell lines and normal epithelial cells detected by flow cytometry. Cell surface MIF expression (left panel) and intracellular expression (right panel) were evaluated by inmmunofluorescence staining with anti-MIF mAb. MIF was predominantly identified in the cytoplasm of HKESC-1 and HKESC-2 cell lines.

The levels of secreted MIF protein in the supernatants of cultured tumor cell lines and normal primary epithelial cells were determined by ELISA. The concentrations of MIF (± SD) in the supernatants of primary epithelial cells, HKESC-1 and HKESC-2 cultures were 0.78 ± 1.0, 13.6 ± 1.5, and 8.6 ± 0.89 ng/mL, respectively (Fig. 6). Although normal epithelial cells expressed MIF, the level of secreted MIF by normal epithelial cells was much less than that of tumor cells.

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FIGURE 6. Secretion of MIF by esophageal tumor cell lines and normal epithelial cells. Cells were cultured in 24-well plates at 5 × 104 cells for 24 hours. The culture supernatants were collected, and MIF was quantified by ELISA. Culture medium alone was used as control. Values represent the mean of 3 experiments conducted in triplicate and are expressed as mean ± SD (**P < 0.001).

Effect of MIF on Stimulation of VEGF and IL-8 Secretion

Esophageal tumor cell lines HKESC-1 and HKESC-2 secreted high levels of IL-8 (± SD) (HKESC-1, 944.5 ± 76.4 pg/mL; HKESC-2, 580.9 ± 130.2 pg/mL) and VEGF (HKESC-1, 14.9 ± 1.0 ng/mL; HKESC-2, 9.1 ± 1.1 ng/mL) in the absence of exogenous MIF stimulation. MIF at concentrations of up to 200 ng/mL in HKESC-1 and HKESC-2 cells induced a significant dose-dependent increase in IL-8 and VEGF secretion (Fig. 7). However, MIF had no effect on stimulating the production of IL-8 and VEGF in normal epithelial cells (Fig. 7). Furthermore, the production of IL-8 and VEGF by ESCC cell lines was inhibited by neutralizing MIF mAb, but not by control antibody (Fig. 8).

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FIGURE 7. MIF induces the secretion of VEGF and IL-8 by esophageal tumor cell lines and normal epithelial cells. Cells were cultured in 24-well plates at 5 × 104 cells. The cells were treated with recombinant human MIF at concentrations as indicated for 24 hours. The culture supernatants were collected and IL-8 and VEGF were measured by ELISA. Values represent the mean of 3 experiments conducted in triplicate and are expressed as mean ± SD.

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FIGURE 8. Effect of anti-MIF neutralizing mAb on production of IL-8 (A) and VEGF (B) by ESCC cell lines HKESC-1 and HKESC-2. Cells were cultured in 24-well plates at 5 × 104 cells. The cells were treated with anti-MIF neutralizing mAb (100 μg/mL) or isotype control mAb (100 μg/mL) for 24 hours. IL-8 and VEGF in the supernatants were measured by ELISA (mean ± SD, n = 3). *P < 0.05.

DISCUSSION

The present study demonstrates, for the first time, that MIF is overexpressed in esophageal cancer and that MIF can stimulate the secretion of angiogenic factors VEGF and IL-8 from tumor cells. MIF overexpression also correlated with tumor differentiation, microvessel density, lymph nodalstatus, and long-term survival in patients with esophageal cancer.

Expression of MIF has been reported in various tumor cells,20–23 but it has not been studied in esophageal cancer. In this study, it was shown that high levels of MIF expression were found in tumor specimens, as well as in patients’ sera. In addition, esophageal cancer cell lines secreted large amounts of MIF. In comparison, MIF expression in fresh isolated normal epithelial cells was much less than that of ESCC tumor cells. However, the mechanism by which tumor cells up-regulate MIF expression remains unknown.

The extent of overexpression of MIF correlated with tumor differentiation; poorly differentiated tumors had a higher percent of stained cells. It has been shown that the rate of apoptosis is lower in poorly differentiated esophageal tumors31 and that the tumor suppression protein p53 activity is impaired in all esophageal tumor cell lines.32 It is known that MIF can exert a role in tumorigenesis by inactivating p53,18 which in turn can affect the rate of tumor cell apoptosis. Therefore, it is possible that the low rate of apoptosis observed in poorly differentiated esophageal tumor could be related to inactivation of p53 caused by MIF overexpression. Down-regulation of tumor cell apoptosis may be the hallmark of immune privilege for the tumor, and thus causing worse patient outcome.33 This hypothesis requires further study.

Lymphatic spread in ESCC is an early event, and lymph node status is a strong prognostic factor of esophageal cancer.34 MIF expression was also found to correlate with lymph node metastasis in the present study; more widespread MIF expression appeared to correlate with higher prevalence of N1 disease. On univariate analysis, MIF expression also correlated with survival. On multivariate analysis, however, only pTNM stage was of prognostic significance. It is likely that pTNM stage was a much stronger predictive factor and overshadowed other variables, including MIF. Unfortunately, the finding that the level of serum MIF did not correlate with clinicopathologic features or survival indicates that serum MIF alone is not a useful marker for clinical decision making. MIF may be a local regulator rather than a systemic mediator in ESCC. MIF is secreted by tumor cells and acts as a local autocrine factor. The serum level may be an “over-spilled” phenomenon; thus, its measurement may not be directly related to clinical parameters. A combination of MIF with other biomarkers, such as VEGF,35–37 or Fas,33 could be explored as prognostic markers in the future.

Angiogenesis, the formation of new blood vessels, is essential for tumor growth and spread. Angiogenesis is controlled by angiogenic factors that are secreted mostly by tumor cells. Among the angiogenic factors, VEGF and IL-8 can be considered as a growth factor and as an interleukin specific to neovascularization, respectively. Several studies have shown that a high VEGF level correlates with tumor progression, poor treatment response, lymph node metastasis, and worse survival in patients with ESCC.37,38 Studies have also identified elevated IL-8 and VEGF in both culture cell lines and tumor specimens. These tumor-derived angiogenic factors therefore appear to play important roles in the pathogenesis of cancer.24,25,39

One established activity of MIF is to stimulate angiogenesis.27 Immuno-neutralization of MIF during the initial stage of lymphoma growth had profound effects on tumor size.40 This effect was not a result of the inhibition of tumor cell proliferation but was related to the inhibition of endothelial cell growth. It was shown that MIF was an essential factor for tumor-initiated endothelial cell proliferation,41 and, ultimately, tumor neovascularization.40 The mechanism by which MIF stimulates endothelial cell growth remains obscure, but one possibility is that it is an upstream regulator of VEGF and IL-8.

It was found that VEGF expression in tumor cells significantly correlated with tumor MVD in ESCC.42 Our results showed that tumors with high MIF expression correlated with MVD in tumor specimens, suggesting that MIF is an important regulator of angiogenesis in ESCC. To elucidate the possible pathway through which MIF is involved in the angiogenesis of ESCC, we evaluated its relationship with VEGF. Our study revealed a positive correlation between MIF and VEGF expression in the tumor tissues. Furthermore, in our study, the levels of MIF, IL-8, and VEGF were increased in sera from patients with ESCC, and there was a positive correlation between serum MIF and serum VEGF levels. We hypothesized from these results that MIF may contribute to regulate the expression of angiogenic factors. Our data showed that MIF increased the secretion of VEGF and IL-8 in our human cell lines in a dose-dependent manner. The enhanced production of VEGF and IL-8 was partially inhibited by anti-MIF neutralizing antibody. Our results provide a foundation for the hypothesis that MIF participates in angiogenesis via autocrine mechanisms. Ongoing studies are in progress to determine the mechanisms by which MIF regulates IL-8 and VEGF production.

CONCLUSION

Our results showed that MIF was overexpressed in esophageal cancer and that MIF overexpression correlated with loss of cell tumor differentiation, and lymph node metastases. From our in vitro studies, it is proposed that tumor-derived MIF, acting as an autocrine factor, enhances the production of VEGF and IL-8, and hence, angiogenesis and tumor growth. Future studies may determine whether blocking the action of MIF would benefit tumor control via angiogenesis inhibition or other mechanisms. If so, neutralizing antibodies to MIF or antagonists to MIF expression or function may prove to be effective agents for extending the survival of patients with esophageal cancer.

Footnotes

Supported by research grants from the University of Hong Kong (CRCG 10204522/05058/21700/301/01) and DAAD-Project HongKong-German Joint Research Collaboration.

Reprints: Yi Ren, PhD, Department of Surgery, University of Hong Kong Medical Centre, Queen Mary Hospital, Hong Kong. E-mail: yren@hkucc.hku.hk.

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