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Peripheral blood dendritic cells and vascular endothelial growth factor in oral squamous cell carcinoma: correlation analysis and in vitro study
Int. J. Oral Maxillofac. Surg. 2010; 39: 713–720 doi:10.1016/j.ijom.2009.10.025, available online at http://www.sciencedirect.com
Research Paper Molecular Oncology
Peripheral blood dendritic cells and vascular endothelial growth factor in oral squamous cell carcinoma: correlation analysis and in vitro study
Z.-Y. Wang1,a, P.-H. Shi1,a, X.-F. Huang1, Z.-C. Hua2, Y.-Y. Hou3, W. Han1, Q.-G. Hu1 1 Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, Affiliated to Medical School, Nanjing University, Nanjing 210008, PR China; 2The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China; 3Immunology Lab, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
Z.-Y. Wang, P.-H. Shi, X.-F. Huang, Z.-C. Hua, Y.-Y. Hou, W. Han, Q.-G. Hu: Peripheral blood dendritic cells and vascular endothelial growth factor in oral squamous cell carcinoma: correlation analysis and in vitro study. Int. J. Oral Maxillofac. Surg. 2010; 39: 713–720. # 2010 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Vascular endothelial growth factor (VEGF) may cause functional deficiency in dendritic cells (DCs) in vitro. The roles of peripheral blood dendritic cells (PBDCs) and VEGF in patients with oral squamous cell carcinoma (OSCC) are not well understood. The authors analysed the correlation between VEGF and PBDC in 81 OSCC patients. They assessed the effect of VEGF on DC function in vitro. VEGF levels were significantly increased in OSCC patients compared with control subjects (P < 0.05), but PBDC levels were significantly lower (P < 0.05). VEGF expression in TNM I–II (67%) and T1–T2 (74%) was significantly lower, compared with TNM III–IV (88%, P < 0.05) and T3–T4 (89%, P < 0.05). Increased VEGF expression in primary tumours was significantly correlated with elevated serum VEGF levels, but reduced PBDC levels. In vitro cultured DC exposed to VEGF showed significantly decreased expression of functional proteins, enhanced endocytosis activity, and elicited weaker proliferation of T cells, compared with that of free VEGF (P < 0.01). These findings suggest that decreased PBDC and elevated VEGF occur in OSCC patients. Higher VEGF levels may affect precursor cells, resulting in decreased numbers of functional DC.
A defect in the host anti-tumour immune response is one of several mechanisms that allow tumours to evade immune system surveillance. An effect of such defects is the failure to mount an effective antitumour response induced by host bone 0901-5027/070713 + 08 $36.00/0
marrow-derived antigen-presenting cells (APCs) responsible for presenting tumour-specific antigens9. Among APCs, dendritic cells (DCs) play a crucial role in anti-tumour immunity by initiating the primary immune response in naı¨ve T
Key words: dendritic cells; oral squamous cell carcinoma; vascular endothelial growth factor; immunology. Accepted for publication 12 October 2009 Available online 14 April 2010
cells20. Several groups have reported defective DC function in malignant tumours of the gastrointestinal tract14, oral a
These authors contributed equally to this work.
# 2010 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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cavity6,8, breast and lung1. Defective DC function is correlated with cytokines secreted by the tumour. Among these cytokines, vascular endothelial growth factor (VEGF) is a key factor in physiological and pathological angiogenesis. VEGF regulates multiple biological responses in endothelial cells, including cell proliferation, migration, survival and production of vasoactive mediators. VEGF is produced by almost all tumour cells, including oral squamous cell carcinoma (OSCC)3, and is found in the serum of cancer patients11 and in tumour tissues24. In vitro studies suggest that VEGF may inhibit DC development and differentiation5,17. A recent OSCC study suggested that secreted VEGF might promote escape from tumour immunity by inhibiting the differentiation of CD1a+ DCs from progenitor cells and increasing the numbers of dysfunctional CD83+ DCs10. The roles of peripheral blood dendritic cells (PBDCs) and VEGF in OSCC patients are not clear. In the present study, the authors detected PBDC and levels of serum VEGF in OSCC patients, analysed VEGF expression in oral primary tumours, and correlated the findings with clinical disease stages. DCs were cultured in vitro with exogenous VEGF to explore the effect on their development and differentiation. Materials and methods Patients
Ethical approval was given for 105 patients to enter the study; there were 81 patients with OSCC in the experimental group and 24 patients with tumour-like lesions, including cysts, papillomas and fibromas, in the ‘no-cancer’ control group. There was no statistical difference in age between the two groups. All 81 patients in the experimental group had histologically confirmed OSCC and had not undergone chemotherapy, radiotherapy or biotherapy. The patients did not have unrelated diseases including diabetes, haematological disease, autoimmune diseases or other malignant tumours at the time of diagnosis. Blood samples were collected from patients before treatment, and tissue samples were collected postoperatively. The patient group consisted of 38 males and 43 females with an average age of 57 years (range: 34–82 years). The patients’ clinical stages, based on the 2002 UICC TNM staging system, are described in Table 1. None of these patients had metastasis, except for lymph node invasion.
Table 1. Clinical stage of 81 OSCC patients. Clinical stage
Number of patients
I (T1N0M0) II (T2N0M0) III (T2N1M0, T3N0M0, T3N1M0) IV (T1N2M0, T2N2M0, T3N2M0, T4N0M0, T4N1M0, T4N2M0)
15 22 17 (8, 6, 3) 27 (2, 5, 3, 12, 3, 2)
Total patients
81
Clinical stage was classified according to the UICC 2002 TNM staging system.
Blood was collected from the control patients with tumour-like lesions. This group included nine males and 15 females; their mean age was 55 years (range: 36–72 years). Specimen sampling
Seven millilitres of peripheral venous blood was collected in the morning, after overnight fasting and before any therapeutic intervention. To measure the PBDC and leukocyte populations, 1 ml of the blood sample was transferred to a tube pretreated with ethylenediamine tetraacetic acid (EDTA). For cytokine measurements, the residual 5 ml blood was gathered in a serum separator tube and centrifuged at 3000 rpm for 10 min. Serum was extracted and stored at 20 8C until further analysis. Fifty-seven primary tumour samples were collected from excised tumour specimens from the 81 OSCC patients. Tissue samples were immediately immersed in 10% formalin overnight. Fixed samples were embedded in paraffin and 4 mm tissue sections were mounted onto slides. PBDC measurement
Hundred microlitres of the anticoagulated blood was co-cultured with 20 ml of the following antibodies (Biolegend, USA): fluorescein isothiocyanate (FITC)-conjugated anti-CD33, antigen-presenting cell (APC)-conjugated anti-HLA-DR, and phycoerythrin (PE)-conjugated antiCD14 and -CD16, at room temperature in the dark for 30 min, as previously described25. After treatment with fluorescence-activated cell sorter (FACS) lysis solution (BD Biosciences, USA), the samples were washed with phosphate-buffered saline (PBS) and centrifuged twice for 5 min at 450 g to remove the red cells. The PBDC population was identified and analysed with flow cytometry. Serum VEGF assessment
Serum VEGF was detected using a quantitative enzyme-linked immunosorbent
assay (ELISA) according to the manufacturer’s instruction (Biosource, USA). Briefly, samples (100 ml/well) were incubated in 96-well plates precoated with a monoclonal anti-human VEGF. An enzymatic labelling detection instrument (TECAN) read the optical density (OD) at 450 nm. Serum VEGF concentration (pg/ml) corresponding to the OD value of each sample was determined from the manufacturer’s standard curve card. Quantification of VEGF expression in primary tumour tissue
Immunohistochemical staining was performed to quantify VEGF in primary tumour samples. Sample slides were heated at 60 8C for 6 h, and were incubated in hydrogen peroxide for 10–15 min at room temperature to quench endogenous peroxidase activity. Non-specific binding was blocked with rabbit serum for 5 min at room temperature. Slides were stained for 30 min at room temperature with murine monoclonal anti-VEGF (NeoMarkers, USA). Negative control slides were carried out by omitting the primary antibodies. A positive reaction was indicated by a score, as previously described26. The score was established corresponding to the sum of: the percentage of positive cells (0, 0% immunopositive cells; 1, <25% positive cells; 2, 26– 50% positive cells; and 3, >50% positive cells); and the staining intensity (0, negative; 1, weak; 2, moderate; and 3, high). The sum of the percentage of positive cells and the staining intensity reached a maximum score at 6. Scores between 0 and 2 were regarded as negative, scores of 3 and 4 as weak, and scores between 5 and 6 as strongly positive. Effect of VEGF on cultured DCs in vitro
Peripheral blood mononuclear cell (PBMC) isolation and in vitro DC culture: Up to 30 ml leukocyte suspension, a blood component isolated from 200 ml of whole blood from a healthy donor, was diluted with 30 ml Ca2+/Mg2+-free PBS (pH 7.2). The sample was centrifuged through
Vascular endothelial GF and peripheral blood dendritic cells in OSCC Ficoll/Hypaque (density, 1.077 g/ml; Shanghai Shisheng Cytobiotechnological Corp. China) at 2000 rpm, 25 8C for 20 min. The PBMCs in the resulting interface layer were extracted, and washed with PBS three times (10 min, 1500 rpm). After the last centrifugation, the cells were suspended in RPMI 1640 medium (Gibco, USA) supplemented with 10% foetal bovine serum (FBS) (Gibco, USA) in 24-well culture plates for 2 h at 37 8C. The supernatant including nonadherent cells was removed and adherent monocytes were divided into two groups for continuous culture. One group was cultured in medium supplemented with 100 ng/ml rhGM-CSF (granulocyte macrophage colony-stimulating factor), 40 ng/ml rhIL-4 (interleukin (IL)-4), 20 ng/ml rhTNF-a (tumour necrosis factor alpha), and 75 ng/ml rhVEGF 165 (Peprotech, UK). The other group served as the control and was cultured with all of the above cytokines except for VEGF 165. Both culture groups were pulsed with Tca8113 antigen, which was prepared as described by WANG27. Tca8113 cells are human tongue squamous cell carcinoma and were
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Table 2. PBDC and serum VEGF levels in OSCC patients and control subjects (x SD). Group n DC/PBMC (%) DC count (106/l) Serum VEGF (pg/ml)
OSCC 81
Control 24
0.47 0.19 7.25 3.15 785.73 227.91
0.77 0.21 11.44 3.11 325.70 117.54
t-value
P
6.691 5.738 11.584
<0.01 <0.01 <0.01
PBDCs (CD14 , CD16 , CD33+, and HLA-DR+ cells), including DC count and a percentage of DC/PBMC were detected by flow cytometry. Serum VEGF was assayed by quantitative ELISA. All values shown are mean and standard deviation. Independent sample t-tests were employed in all analyses. P < 0.05 was considered significant.
obtained from the Ninth Hospital of Shanghai. Culture medium was replaced every 3 days with fresh complete medium. Analysis of DC phenotypes: On day 8 of culture, the cells were harvested and suspended in PBS for analysis. A 100 ml cell suspension was stained directly with the following murine anti-human monoclonal antibodies: FITC-conjugated anti-CD14, CD83, -CD86 and -HLA-DR (5 ml), PEconjugated anti-CD1a, -CD40, -CD54 (20 ml) and -CD80 (5 ml) (Biolegend, USA). Murine isotype FITC- and PE-conjugated IgG antibodies served as negative controls. Cells were analysed on a Becton
Dickinson FACS machine using Cell Quest data acquisition and analysis software. At least 10,000 events were evaluated for each marker. Mixed leukocyte reaction (MLR): Allogeneic PBMCs from another healthy donor were isolated as described. From the PBMCs, B cells were removed by immunomagnetic CD19 microbeads (Dynal Biotech ASA, Oslo, Norway) according to the manufacturer’s instruction, and T lymphocyte cells were harvested. Allogeneic T cells (1 105 cells/ well) were used as effector cells in 96-well flat bottom plates. Cultured DCs (1 104
Fig. 1. Serum VEGF correlated with the level of PBDCs (P < 0.01) in OSCC patients, but no correlation in control subjects (P > 0.05).
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Fig. 2. VEGF expression in primary tumour: immunohistochemical staining using primary antibodies recognising VEGF, shown staining patterns of various classifications in primary tumours. (A) Strong-positive VEGF expression; (B) positive VEGF expression; (C) negative VEGF expression; and (D) negative control. Magnification, 100.
cells/well) were added to the T cells. 4 h before the end of the 96 h incubation, 20 ml Methylthiazoletetrazolium (MTT) (5 mg/ ml) was added to each well. At 96 h, the supernatant was replaced with 150 ml/well dimethyl sulphoxide (DMSO; Shanghai Shisheng Cytobiotechnological Corp.,
China) for 20 min. An enzymatic labelling detection instrument (TECAN) was used to read OD 490 nm. DCs alone (104 cells/ well) were used as a blank control. DC endocytosis assay: Cultured DCs (105 cells/well) were suspended in culture medium in a 96-well plate, and neutral red
Table 3. VEGF expression in 57 cases of primary OSCC. VEGF (n) + TNM stage I and II III and IV T stage T1 and T2 T3 and T4 N stage N0 N1, N2 and N3 Total
n
Positive rate
x2
P
11.06
<0.05
6.73
<0.05
5.76
>0.05
++
solution (0.075%, 100 ml) was incubated at 37 8C for 1 h. DCs were washed three times with warm PBS, and 200 ml of cell lysis solution (0.1 mmol/l ice acetic acid/anhydrous ethyl alcohol) was added and incubated at room temperature for 30 min. The OD 540 was detected using the enzymatic labelling detection instrument.
Statistical analysis
8 4
13 11
3 18
24 33
66.7% 87.9%
10 2
19 5
10 11
39 18
74.3% 88.9%
10 2
16 8
9 12
35 22
71.4% 90.9%
12
24
21
57
78.9%
VEGF expression in primary tumours is visualized by immunohistochemical staining using primary antibodies recognising VEGF. The overall degree of staining in the sections was graded as previously described. TNM stage is indicated. All analyses were performed using the x2 test. P < 0.05 was considered significant.
Independent sample t-tests were used for the comparison between OSCC and control groups of PBDC and serum VEGF variation. One-way ANOVA was used for mean comparisons between groups for VEGF expression intensity. The x2 test was used for positive rate analysis of VEGF expression. Correlations were measured using Spearman’s correlation coefficient test. For all tests, a P-value < 0.05 was considered to be statistically significant. All statistical data were calculated using SPSS 11.5 for Windows software.
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Vascular endothelial GF and peripheral blood dendritic cells in OSCC Table 4. Correlation between VEGF expression in primary tumour, PBDC and serum VEGF (x SD). VEGF expression in tumour tissue (n) (12) Serum VEGF (pg/ml) DC count (106/l) DC/PBMC (%)
490.83 61.62 9.83 3.62 0.58 0.15
+(24)
++(21)
748.11 158.17 6.94 2.16 0.49 0.17
997.24 290.76 5.39 2.79 0.35 0.13
Intergroup differences: Dunnett’s T3-test (P-value) versus + 0.000 0.063 0.341
+ versus ++ 0.000 0.131 0.013
versus ++ 0.004 0.005 0.001
Correlation r-value 0.780 0.450 0.505
P <0.01 <0.01 <0.01
PBDCs were identified by flow cytometry. The level of serum VEGF was assayed by quantitative ELISA. VEGF expression in primary tumours is shown in immunohistochemical staining. All values shown are mean and standard deviation. All analyses were performed using the Dunnett’s T3test. P < 0.05 was considered significant.
Results PBDC and serum VEGF levels
The results showed that PBDC levels, including the absolute numbers of DC and the percent of DC/PBMC (n = 81, 7.25 3.15 106/l, 0.47 0.19%), were significantly lower in the blood of OSCC patients compared with those in the control group (n = 24, 11.44 3.11 106/l, 0.77 0.21%) (P < 0.05, Table 2). Serum VEGF levels were significantly increased in OSCC patients (n = 81, 764.33 227.91 pg/ml) compared with control subjects (n = 24, 325.70 117.54 pg/ml) (P < 0.05, Table 2). Correlation analysis showed that serum VEGF was significantly and adversely correlated with DC counts (Spearman’s test, n = 81, r = 0.662, P < 0.01) and DC/PBMC (Spearman’s test, n = 81, r = 0.652, P < 0.01) in OSCC patients, but not in control subjects (n = 24, r = 0.302, P = 0.151; r = 0.305, P = 0.147, respectively, Fig. 1). VEGF expression in primary tumours and correlation with TNM stage
VEGF immunohistochemical staining patterns in tumour tissues are shown in Fig. 2, showing negative, positive and strong-
positive VEGF expression, as well as negative control. Table 3 describes the effect of tumour classification, lymph node metastasis, and TNM stage on VEGF expression in OSCC tissues. The total positive rate was 79% (45/57). The positive rate of VEGF expression (67%) in TNM stages I and II was significantly lower (P < 0.05) than that in TNM stage III and IV (88%), and the rate of T1 and T2 (74%) was significantly lower compared with T3 and T4 (89%, P < 0.05, Table 3). VEGF expression in tumours from patients without lymph node metastasis was not significantly different from that in tumours from patients with lymph node metastasis (P > 0.05). Correlations between VEGF in primary tumours, serum VEGF and PBDC
As described in Table 4, there was a statistically significant difference in serum VEGF in patients with negative, positive, and strong-positive VEGF expression in tumour tissues (P < 0.01). Increased serum VEGF was significantly correlated with upregulated VEGF expression in tumour tissues from OSCC patients (Spearman’s test, n = 57, r = 0.780, P < 0.01). DC counts were gradually decreased along with intensity of tumour VEGF
Fig. 3. Phenotypic marker expression of the control and experimental groups: the experimental DCs cultured with VEGF showed significantly decreased expression of CD1a, CD83, CD40, CD80, CD86 and HLA-DR surface antigens, compared with the control group (P < 0.01). No statistically significant difference was found between the experimental and control groups (P > 0.05) for CD54 expression. The error bar represents the standard deviation for three samples.
expression (negative, 9.83; positive, 6.94; strong positive, 5.39), but a significant difference was observed only in DC counts between the negative and strong-positive VEGF expression of patients (P < 0.01). Decreased DC/PBMC correlated with upregulated VEGF expression in tumour tissues (Spearman’s test, n = 57, r = 0.450, P < 0.01). The DC/PBMC of the patients with strongly positive VEGF expression (0.35%) in tumours was significantly lower than that in patients with negative VEGF expression (0.58%, P < 0.01) and with positive VEGF expression (0.49%, P < 0.05). There was no difference in DC/PBMC between patients with negative and positive tumour VEGF expression. Increased DC/PBMC correlated with down-regulated VEGF expression in OSCC tissue (Spearman’s test, n = 57, r = 0.505, P < 0.01).
Effect of VEGF on cultured DCs in vitro
After 8 days in culture, the cells in the experimental group showed significantly decreased expression of CD1a, CD83, CD40, CD80, CD86 and HLA-DR, compared with the control group (P < 0.01). No statistically significant difference was observed in CD54 expression between the experimental and control groups (P > 0.05, Fig. 3). An allogeneic primary MLR was carried out to determine whether VEGF was capable of inhibiting the induction of a primary T-cell response. The DC cultured with GM-CSF, IL-4, TNF-a, and VEGF (0.285 0.015), elicited weaker proliferation of allogeneic T cells (P < 0.01), compared with the cells treated with GMCSF, IL-4, and TNF-a (0.318 0.024, Fig. 4). Endocytosis analysis was performed by adding neutral red solution to the cultured DCs. The results show that the endocytosis activity of the DCs cultured with external VEGF (0.184 0.026) was significantly enhanced (P < 0.01), compared with cells cultured without external VEGF (0.155 0.014, Fig. 5).
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Fig. 4. DCs cultured with VEGF elicited weaker (P < 0.01) proliferation of allogeneic T cells, compared with the cells cultured without added VEGF. The error bar represents the standard deviation for three samples.
Fig. 5. Endocytosis activity of DCs cultured with external VEGF was significantly enhanced (P < 0.01), compared with the cells cultured without external VEGF. The error bars represent the standard deviation for three samples.
Discussion
Dendritic cells are the most potent professional APCs with the capacity to elicit cellular immunity. One mature DC can stimulate 100–3000 T cells in vitro23. Several studies have shown that decreases in DC number and the attenuated functional nature of DCs may inhibit the DC/ T-cell interaction, bringing about an impaired T-cell response with tumour escape from immunological control22,21. The authors’ previous study28 found that the number of infiltrating DCs in the tumour tissues of OSCC patients was decreased, but this did not explain what
happened to the systemic immunity of OSCC patients. The aim of the present study was to examine the PBDC levels of OSCC patients. The results show that in OSCC patients, PBDC levels, including absolute numbers of DC and the percent of DC/PBMC, were significantly lower than in control patients. These results suggest that the tumour infiltrating DCs as well as the PBDCs of OSCC patients are decreased, resulting in a limited T-cell response. Many immunosuppressive cytokines are excreted from tumours, including several associated with functional maturation of DC such as IL-102, transforming
and growth factor (TGF)-b17,29, VEGF5,17. VEGF was assayed because it is a primary mediator of angiogenesis, and can be secreted by most solid tumours3. In addition, VEGF expression may have prognostic significance for patients with head and neck squamous cell carcinoma12. The VEGF expression in OSCC samples was 79%. Overall survival data for these OSCC patients is not presented, but the authors demonstrated that TNM and T stage had a significant impact on VEGF expression in primary OSCC lesions, and up-regulated VEGF expression is correlated with increased levels of serum VEGF. These results suggest that overexpression of serum VEGF might be a prognostic factor for OSCC. The results did not support an association between VEGF expression and lymph node status. A possible explanation may be that the antibody used in this study was against the whole VEGF family, whereas only VEGFC and VEGF-D are specifically implicated in tumour lymphangiogenesis and lymph node metastasis13,16. Serum VEGF and focal VEGF was significantly increased in OSCC patients. Only strongly positive VEGF expression in the foci of OSCC samples had a significant impact on the PBDC count and DC/PBMC. VEGF in OSCC foci may indirectly affect the PBDC levels of OSCC patients. Consistent with the authors’ study, LISSONI et al. found that increased serum VEGF levels are associated with reduced PBDCs in patients with various solid malignancies15. These results suggest that the increased serum level of VEGF in cancer patients might be due to increased secretion of the cytokine by cancer cells, which might prevent myeloid precursor cells from evolving into functional DCs. To determine whether VEGF secreted by OSCC can inhibit the development of DC, the authors carried out phenotypic analysis, MLR, and endocytosis assays on DCs from peripheral blood. Exogenous VEGF was used as a substitute for the supernatant of tumour cell cultures, and to eliminate potential interference from other tumour-secreted factors. The numbers of CD1a+ and CD83+ DCs cultured with VEGF was significantly lower than the cells without VEGF, and the expression of CD80, CD86, and CD40 was decreased. Cells cultured with VEGF showed a significantly impaired ability to stimulate Tcell proliferation. VEGF-treated DCs showed an increased ability to take up neutral red dye. This is probably a reflection of immature status, because mature DCs usually lose this function concurrent
Vascular endothelial GF and peripheral blood dendritic cells in OSCC with the ability to present antigens18. GAB4 RILOVICH et al. observed that the splenic DCs isolated from mice treated with VEGF in vivo had significantly higher levels of FITC-dextran uptake than control mice. This suggests that VEGF may exert potent effects on DC precursor cells, resulting in inhibited functional DC development. VEGF binding to VEGF-R on the surface of haematogenic cells may prevent NF-kB activation in haematopoietic progenitor cells, blocking DC development19. When immunotherapy with mature DCs against cancer is considered, treatment aiming at VEGF signalling may reverse the VEGF-mediated DC defect and induce the effective anti-tumour immunity. In conclusion, the authors found that decreased PBDC in OSCC patients correlates with increased serum VEGF, which may be caused by the tumour. VEGF may exert effects on precursor cells, resulting in the decreased DC antigen presentation functions. Overcoming VEGF-mediated DC defects may have significant clinical implications for OSCC and other cancer patients. Competing interests
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None declared. Funding
This study was supported by the Chinese National Nature Science Foundation (30772441) to Dr. Qin-Gang Hu. Ethical approval
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The study was approved by the Ethics Committee of the Nanjing Stomatological Hospital, and all patients gave informed consent before participation. Acknowledgement. The authors would like to thank Pei-Shan Lu (Jiangsu Province Center for Disease Prevention and Control) for his technical assistance.
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Research Paper Molecular Oncology
Peripheral blood dendritic cells and vascular endothelial growth factor in oral squamous cell carcinoma: correlation analysis and in vitro study
Z.-Y. Wang1,a, P.-H. Shi1,a, X.-F. Huang1, Z.-C. Hua2, Y.-Y. Hou3, W. Han1, Q.-G. Hu1 1 Department of Oral and Maxillofacial Surgery, Nanjing Stomatological Hospital, Affiliated to Medical School, Nanjing University, Nanjing 210008, PR China; 2The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China; 3Immunology Lab, Medical School & State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
Z.-Y. Wang, P.-H. Shi, X.-F. Huang, Z.-C. Hua, Y.-Y. Hou, W. Han, Q.-G. Hu: Peripheral blood dendritic cells and vascular endothelial growth factor in oral squamous cell carcinoma: correlation analysis and in vitro study. Int. J. Oral Maxillofac. Surg. 2010; 39: 713–720. # 2010 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. Vascular endothelial growth factor (VEGF) may cause functional deficiency in dendritic cells (DCs) in vitro. The roles of peripheral blood dendritic cells (PBDCs) and VEGF in patients with oral squamous cell carcinoma (OSCC) are not well understood. The authors analysed the correlation between VEGF and PBDC in 81 OSCC patients. They assessed the effect of VEGF on DC function in vitro. VEGF levels were significantly increased in OSCC patients compared with control subjects (P < 0.05), but PBDC levels were significantly lower (P < 0.05). VEGF expression in TNM I–II (67%) and T1–T2 (74%) was significantly lower, compared with TNM III–IV (88%, P < 0.05) and T3–T4 (89%, P < 0.05). Increased VEGF expression in primary tumours was significantly correlated with elevated serum VEGF levels, but reduced PBDC levels. In vitro cultured DC exposed to VEGF showed significantly decreased expression of functional proteins, enhanced endocytosis activity, and elicited weaker proliferation of T cells, compared with that of free VEGF (P < 0.01). These findings suggest that decreased PBDC and elevated VEGF occur in OSCC patients. Higher VEGF levels may affect precursor cells, resulting in decreased numbers of functional DC.
A defect in the host anti-tumour immune response is one of several mechanisms that allow tumours to evade immune system surveillance. An effect of such defects is the failure to mount an effective antitumour response induced by host bone 0901-5027/070713 + 08 $36.00/0
marrow-derived antigen-presenting cells (APCs) responsible for presenting tumour-specific antigens9. Among APCs, dendritic cells (DCs) play a crucial role in anti-tumour immunity by initiating the primary immune response in naı¨ve T
Key words: dendritic cells; oral squamous cell carcinoma; vascular endothelial growth factor; immunology. Accepted for publication 12 October 2009 Available online 14 April 2010
cells20. Several groups have reported defective DC function in malignant tumours of the gastrointestinal tract14, oral a
These authors contributed equally to this work.
# 2010 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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cavity6,8, breast and lung1. Defective DC function is correlated with cytokines secreted by the tumour. Among these cytokines, vascular endothelial growth factor (VEGF) is a key factor in physiological and pathological angiogenesis. VEGF regulates multiple biological responses in endothelial cells, including cell proliferation, migration, survival and production of vasoactive mediators. VEGF is produced by almost all tumour cells, including oral squamous cell carcinoma (OSCC)3, and is found in the serum of cancer patients11 and in tumour tissues24. In vitro studies suggest that VEGF may inhibit DC development and differentiation5,17. A recent OSCC study suggested that secreted VEGF might promote escape from tumour immunity by inhibiting the differentiation of CD1a+ DCs from progenitor cells and increasing the numbers of dysfunctional CD83+ DCs10. The roles of peripheral blood dendritic cells (PBDCs) and VEGF in OSCC patients are not clear. In the present study, the authors detected PBDC and levels of serum VEGF in OSCC patients, analysed VEGF expression in oral primary tumours, and correlated the findings with clinical disease stages. DCs were cultured in vitro with exogenous VEGF to explore the effect on their development and differentiation. Materials and methods Patients
Ethical approval was given for 105 patients to enter the study; there were 81 patients with OSCC in the experimental group and 24 patients with tumour-like lesions, including cysts, papillomas and fibromas, in the ‘no-cancer’ control group. There was no statistical difference in age between the two groups. All 81 patients in the experimental group had histologically confirmed OSCC and had not undergone chemotherapy, radiotherapy or biotherapy. The patients did not have unrelated diseases including diabetes, haematological disease, autoimmune diseases or other malignant tumours at the time of diagnosis. Blood samples were collected from patients before treatment, and tissue samples were collected postoperatively. The patient group consisted of 38 males and 43 females with an average age of 57 years (range: 34–82 years). The patients’ clinical stages, based on the 2002 UICC TNM staging system, are described in Table 1. None of these patients had metastasis, except for lymph node invasion.
Table 1. Clinical stage of 81 OSCC patients. Clinical stage
Number of patients
I (T1N0M0) II (T2N0M0) III (T2N1M0, T3N0M0, T3N1M0) IV (T1N2M0, T2N2M0, T3N2M0, T4N0M0, T4N1M0, T4N2M0)
15 22 17 (8, 6, 3) 27 (2, 5, 3, 12, 3, 2)
Total patients
81
Clinical stage was classified according to the UICC 2002 TNM staging system.
Blood was collected from the control patients with tumour-like lesions. This group included nine males and 15 females; their mean age was 55 years (range: 36–72 years). Specimen sampling
Seven millilitres of peripheral venous blood was collected in the morning, after overnight fasting and before any therapeutic intervention. To measure the PBDC and leukocyte populations, 1 ml of the blood sample was transferred to a tube pretreated with ethylenediamine tetraacetic acid (EDTA). For cytokine measurements, the residual 5 ml blood was gathered in a serum separator tube and centrifuged at 3000 rpm for 10 min. Serum was extracted and stored at 20 8C until further analysis. Fifty-seven primary tumour samples were collected from excised tumour specimens from the 81 OSCC patients. Tissue samples were immediately immersed in 10% formalin overnight. Fixed samples were embedded in paraffin and 4 mm tissue sections were mounted onto slides. PBDC measurement
Hundred microlitres of the anticoagulated blood was co-cultured with 20 ml of the following antibodies (Biolegend, USA): fluorescein isothiocyanate (FITC)-conjugated anti-CD33, antigen-presenting cell (APC)-conjugated anti-HLA-DR, and phycoerythrin (PE)-conjugated antiCD14 and -CD16, at room temperature in the dark for 30 min, as previously described25. After treatment with fluorescence-activated cell sorter (FACS) lysis solution (BD Biosciences, USA), the samples were washed with phosphate-buffered saline (PBS) and centrifuged twice for 5 min at 450 g to remove the red cells. The PBDC population was identified and analysed with flow cytometry. Serum VEGF assessment
Serum VEGF was detected using a quantitative enzyme-linked immunosorbent
assay (ELISA) according to the manufacturer’s instruction (Biosource, USA). Briefly, samples (100 ml/well) were incubated in 96-well plates precoated with a monoclonal anti-human VEGF. An enzymatic labelling detection instrument (TECAN) read the optical density (OD) at 450 nm. Serum VEGF concentration (pg/ml) corresponding to the OD value of each sample was determined from the manufacturer’s standard curve card. Quantification of VEGF expression in primary tumour tissue
Immunohistochemical staining was performed to quantify VEGF in primary tumour samples. Sample slides were heated at 60 8C for 6 h, and were incubated in hydrogen peroxide for 10–15 min at room temperature to quench endogenous peroxidase activity. Non-specific binding was blocked with rabbit serum for 5 min at room temperature. Slides were stained for 30 min at room temperature with murine monoclonal anti-VEGF (NeoMarkers, USA). Negative control slides were carried out by omitting the primary antibodies. A positive reaction was indicated by a score, as previously described26. The score was established corresponding to the sum of: the percentage of positive cells (0, 0% immunopositive cells; 1, <25% positive cells; 2, 26– 50% positive cells; and 3, >50% positive cells); and the staining intensity (0, negative; 1, weak; 2, moderate; and 3, high). The sum of the percentage of positive cells and the staining intensity reached a maximum score at 6. Scores between 0 and 2 were regarded as negative, scores of 3 and 4 as weak, and scores between 5 and 6 as strongly positive. Effect of VEGF on cultured DCs in vitro
Peripheral blood mononuclear cell (PBMC) isolation and in vitro DC culture: Up to 30 ml leukocyte suspension, a blood component isolated from 200 ml of whole blood from a healthy donor, was diluted with 30 ml Ca2+/Mg2+-free PBS (pH 7.2). The sample was centrifuged through
Vascular endothelial GF and peripheral blood dendritic cells in OSCC Ficoll/Hypaque (density, 1.077 g/ml; Shanghai Shisheng Cytobiotechnological Corp. China) at 2000 rpm, 25 8C for 20 min. The PBMCs in the resulting interface layer were extracted, and washed with PBS three times (10 min, 1500 rpm). After the last centrifugation, the cells were suspended in RPMI 1640 medium (Gibco, USA) supplemented with 10% foetal bovine serum (FBS) (Gibco, USA) in 24-well culture plates for 2 h at 37 8C. The supernatant including nonadherent cells was removed and adherent monocytes were divided into two groups for continuous culture. One group was cultured in medium supplemented with 100 ng/ml rhGM-CSF (granulocyte macrophage colony-stimulating factor), 40 ng/ml rhIL-4 (interleukin (IL)-4), 20 ng/ml rhTNF-a (tumour necrosis factor alpha), and 75 ng/ml rhVEGF 165 (Peprotech, UK). The other group served as the control and was cultured with all of the above cytokines except for VEGF 165. Both culture groups were pulsed with Tca8113 antigen, which was prepared as described by WANG27. Tca8113 cells are human tongue squamous cell carcinoma and were
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Table 2. PBDC and serum VEGF levels in OSCC patients and control subjects (x SD). Group n DC/PBMC (%) DC count (106/l) Serum VEGF (pg/ml)
OSCC 81
Control 24
0.47 0.19 7.25 3.15 785.73 227.91
0.77 0.21 11.44 3.11 325.70 117.54
t-value
P
6.691 5.738 11.584
<0.01 <0.01 <0.01
PBDCs (CD14 , CD16 , CD33+, and HLA-DR+ cells), including DC count and a percentage of DC/PBMC were detected by flow cytometry. Serum VEGF was assayed by quantitative ELISA. All values shown are mean and standard deviation. Independent sample t-tests were employed in all analyses. P < 0.05 was considered significant.
obtained from the Ninth Hospital of Shanghai. Culture medium was replaced every 3 days with fresh complete medium. Analysis of DC phenotypes: On day 8 of culture, the cells were harvested and suspended in PBS for analysis. A 100 ml cell suspension was stained directly with the following murine anti-human monoclonal antibodies: FITC-conjugated anti-CD14, CD83, -CD86 and -HLA-DR (5 ml), PEconjugated anti-CD1a, -CD40, -CD54 (20 ml) and -CD80 (5 ml) (Biolegend, USA). Murine isotype FITC- and PE-conjugated IgG antibodies served as negative controls. Cells were analysed on a Becton
Dickinson FACS machine using Cell Quest data acquisition and analysis software. At least 10,000 events were evaluated for each marker. Mixed leukocyte reaction (MLR): Allogeneic PBMCs from another healthy donor were isolated as described. From the PBMCs, B cells were removed by immunomagnetic CD19 microbeads (Dynal Biotech ASA, Oslo, Norway) according to the manufacturer’s instruction, and T lymphocyte cells were harvested. Allogeneic T cells (1 105 cells/ well) were used as effector cells in 96-well flat bottom plates. Cultured DCs (1 104
Fig. 1. Serum VEGF correlated with the level of PBDCs (P < 0.01) in OSCC patients, but no correlation in control subjects (P > 0.05).
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Fig. 2. VEGF expression in primary tumour: immunohistochemical staining using primary antibodies recognising VEGF, shown staining patterns of various classifications in primary tumours. (A) Strong-positive VEGF expression; (B) positive VEGF expression; (C) negative VEGF expression; and (D) negative control. Magnification, 100.
cells/well) were added to the T cells. 4 h before the end of the 96 h incubation, 20 ml Methylthiazoletetrazolium (MTT) (5 mg/ ml) was added to each well. At 96 h, the supernatant was replaced with 150 ml/well dimethyl sulphoxide (DMSO; Shanghai Shisheng Cytobiotechnological Corp.,
China) for 20 min. An enzymatic labelling detection instrument (TECAN) was used to read OD 490 nm. DCs alone (104 cells/ well) were used as a blank control. DC endocytosis assay: Cultured DCs (105 cells/well) were suspended in culture medium in a 96-well plate, and neutral red
Table 3. VEGF expression in 57 cases of primary OSCC. VEGF (n) + TNM stage I and II III and IV T stage T1 and T2 T3 and T4 N stage N0 N1, N2 and N3 Total
n
Positive rate
x2
P
11.06
<0.05
6.73
<0.05
5.76
>0.05
++
solution (0.075%, 100 ml) was incubated at 37 8C for 1 h. DCs were washed three times with warm PBS, and 200 ml of cell lysis solution (0.1 mmol/l ice acetic acid/anhydrous ethyl alcohol) was added and incubated at room temperature for 30 min. The OD 540 was detected using the enzymatic labelling detection instrument.
Statistical analysis
8 4
13 11
3 18
24 33
66.7% 87.9%
10 2
19 5
10 11
39 18
74.3% 88.9%
10 2
16 8
9 12
35 22
71.4% 90.9%
12
24
21
57
78.9%
VEGF expression in primary tumours is visualized by immunohistochemical staining using primary antibodies recognising VEGF. The overall degree of staining in the sections was graded as previously described. TNM stage is indicated. All analyses were performed using the x2 test. P < 0.05 was considered significant.
Independent sample t-tests were used for the comparison between OSCC and control groups of PBDC and serum VEGF variation. One-way ANOVA was used for mean comparisons between groups for VEGF expression intensity. The x2 test was used for positive rate analysis of VEGF expression. Correlations were measured using Spearman’s correlation coefficient test. For all tests, a P-value < 0.05 was considered to be statistically significant. All statistical data were calculated using SPSS 11.5 for Windows software.
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Vascular endothelial GF and peripheral blood dendritic cells in OSCC Table 4. Correlation between VEGF expression in primary tumour, PBDC and serum VEGF (x SD). VEGF expression in tumour tissue (n) (12) Serum VEGF (pg/ml) DC count (106/l) DC/PBMC (%)
490.83 61.62 9.83 3.62 0.58 0.15
+(24)
++(21)
748.11 158.17 6.94 2.16 0.49 0.17
997.24 290.76 5.39 2.79 0.35 0.13
Intergroup differences: Dunnett’s T3-test (P-value) versus + 0.000 0.063 0.341
+ versus ++ 0.000 0.131 0.013
versus ++ 0.004 0.005 0.001
Correlation r-value 0.780 0.450 0.505
P <0.01 <0.01 <0.01
PBDCs were identified by flow cytometry. The level of serum VEGF was assayed by quantitative ELISA. VEGF expression in primary tumours is shown in immunohistochemical staining. All values shown are mean and standard deviation. All analyses were performed using the Dunnett’s T3test. P < 0.05 was considered significant.
Results PBDC and serum VEGF levels
The results showed that PBDC levels, including the absolute numbers of DC and the percent of DC/PBMC (n = 81, 7.25 3.15 106/l, 0.47 0.19%), were significantly lower in the blood of OSCC patients compared with those in the control group (n = 24, 11.44 3.11 106/l, 0.77 0.21%) (P < 0.05, Table 2). Serum VEGF levels were significantly increased in OSCC patients (n = 81, 764.33 227.91 pg/ml) compared with control subjects (n = 24, 325.70 117.54 pg/ml) (P < 0.05, Table 2). Correlation analysis showed that serum VEGF was significantly and adversely correlated with DC counts (Spearman’s test, n = 81, r = 0.662, P < 0.01) and DC/PBMC (Spearman’s test, n = 81, r = 0.652, P < 0.01) in OSCC patients, but not in control subjects (n = 24, r = 0.302, P = 0.151; r = 0.305, P = 0.147, respectively, Fig. 1). VEGF expression in primary tumours and correlation with TNM stage
VEGF immunohistochemical staining patterns in tumour tissues are shown in Fig. 2, showing negative, positive and strong-
positive VEGF expression, as well as negative control. Table 3 describes the effect of tumour classification, lymph node metastasis, and TNM stage on VEGF expression in OSCC tissues. The total positive rate was 79% (45/57). The positive rate of VEGF expression (67%) in TNM stages I and II was significantly lower (P < 0.05) than that in TNM stage III and IV (88%), and the rate of T1 and T2 (74%) was significantly lower compared with T3 and T4 (89%, P < 0.05, Table 3). VEGF expression in tumours from patients without lymph node metastasis was not significantly different from that in tumours from patients with lymph node metastasis (P > 0.05). Correlations between VEGF in primary tumours, serum VEGF and PBDC
As described in Table 4, there was a statistically significant difference in serum VEGF in patients with negative, positive, and strong-positive VEGF expression in tumour tissues (P < 0.01). Increased serum VEGF was significantly correlated with upregulated VEGF expression in tumour tissues from OSCC patients (Spearman’s test, n = 57, r = 0.780, P < 0.01). DC counts were gradually decreased along with intensity of tumour VEGF
Fig. 3. Phenotypic marker expression of the control and experimental groups: the experimental DCs cultured with VEGF showed significantly decreased expression of CD1a, CD83, CD40, CD80, CD86 and HLA-DR surface antigens, compared with the control group (P < 0.01). No statistically significant difference was found between the experimental and control groups (P > 0.05) for CD54 expression. The error bar represents the standard deviation for three samples.
expression (negative, 9.83; positive, 6.94; strong positive, 5.39), but a significant difference was observed only in DC counts between the negative and strong-positive VEGF expression of patients (P < 0.01). Decreased DC/PBMC correlated with upregulated VEGF expression in tumour tissues (Spearman’s test, n = 57, r = 0.450, P < 0.01). The DC/PBMC of the patients with strongly positive VEGF expression (0.35%) in tumours was significantly lower than that in patients with negative VEGF expression (0.58%, P < 0.01) and with positive VEGF expression (0.49%, P < 0.05). There was no difference in DC/PBMC between patients with negative and positive tumour VEGF expression. Increased DC/PBMC correlated with down-regulated VEGF expression in OSCC tissue (Spearman’s test, n = 57, r = 0.505, P < 0.01).
Effect of VEGF on cultured DCs in vitro
After 8 days in culture, the cells in the experimental group showed significantly decreased expression of CD1a, CD83, CD40, CD80, CD86 and HLA-DR, compared with the control group (P < 0.01). No statistically significant difference was observed in CD54 expression between the experimental and control groups (P > 0.05, Fig. 3). An allogeneic primary MLR was carried out to determine whether VEGF was capable of inhibiting the induction of a primary T-cell response. The DC cultured with GM-CSF, IL-4, TNF-a, and VEGF (0.285 0.015), elicited weaker proliferation of allogeneic T cells (P < 0.01), compared with the cells treated with GMCSF, IL-4, and TNF-a (0.318 0.024, Fig. 4). Endocytosis analysis was performed by adding neutral red solution to the cultured DCs. The results show that the endocytosis activity of the DCs cultured with external VEGF (0.184 0.026) was significantly enhanced (P < 0.01), compared with cells cultured without external VEGF (0.155 0.014, Fig. 5).
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Fig. 4. DCs cultured with VEGF elicited weaker (P < 0.01) proliferation of allogeneic T cells, compared with the cells cultured without added VEGF. The error bar represents the standard deviation for three samples.
Fig. 5. Endocytosis activity of DCs cultured with external VEGF was significantly enhanced (P < 0.01), compared with the cells cultured without external VEGF. The error bars represent the standard deviation for three samples.
Discussion
Dendritic cells are the most potent professional APCs with the capacity to elicit cellular immunity. One mature DC can stimulate 100–3000 T cells in vitro23. Several studies have shown that decreases in DC number and the attenuated functional nature of DCs may inhibit the DC/ T-cell interaction, bringing about an impaired T-cell response with tumour escape from immunological control22,21. The authors’ previous study28 found that the number of infiltrating DCs in the tumour tissues of OSCC patients was decreased, but this did not explain what
happened to the systemic immunity of OSCC patients. The aim of the present study was to examine the PBDC levels of OSCC patients. The results show that in OSCC patients, PBDC levels, including absolute numbers of DC and the percent of DC/PBMC, were significantly lower than in control patients. These results suggest that the tumour infiltrating DCs as well as the PBDCs of OSCC patients are decreased, resulting in a limited T-cell response. Many immunosuppressive cytokines are excreted from tumours, including several associated with functional maturation of DC such as IL-102, transforming
and growth factor (TGF)-b17,29, VEGF5,17. VEGF was assayed because it is a primary mediator of angiogenesis, and can be secreted by most solid tumours3. In addition, VEGF expression may have prognostic significance for patients with head and neck squamous cell carcinoma12. The VEGF expression in OSCC samples was 79%. Overall survival data for these OSCC patients is not presented, but the authors demonstrated that TNM and T stage had a significant impact on VEGF expression in primary OSCC lesions, and up-regulated VEGF expression is correlated with increased levels of serum VEGF. These results suggest that overexpression of serum VEGF might be a prognostic factor for OSCC. The results did not support an association between VEGF expression and lymph node status. A possible explanation may be that the antibody used in this study was against the whole VEGF family, whereas only VEGFC and VEGF-D are specifically implicated in tumour lymphangiogenesis and lymph node metastasis13,16. Serum VEGF and focal VEGF was significantly increased in OSCC patients. Only strongly positive VEGF expression in the foci of OSCC samples had a significant impact on the PBDC count and DC/PBMC. VEGF in OSCC foci may indirectly affect the PBDC levels of OSCC patients. Consistent with the authors’ study, LISSONI et al. found that increased serum VEGF levels are associated with reduced PBDCs in patients with various solid malignancies15. These results suggest that the increased serum level of VEGF in cancer patients might be due to increased secretion of the cytokine by cancer cells, which might prevent myeloid precursor cells from evolving into functional DCs. To determine whether VEGF secreted by OSCC can inhibit the development of DC, the authors carried out phenotypic analysis, MLR, and endocytosis assays on DCs from peripheral blood. Exogenous VEGF was used as a substitute for the supernatant of tumour cell cultures, and to eliminate potential interference from other tumour-secreted factors. The numbers of CD1a+ and CD83+ DCs cultured with VEGF was significantly lower than the cells without VEGF, and the expression of CD80, CD86, and CD40 was decreased. Cells cultured with VEGF showed a significantly impaired ability to stimulate Tcell proliferation. VEGF-treated DCs showed an increased ability to take up neutral red dye. This is probably a reflection of immature status, because mature DCs usually lose this function concurrent
Vascular endothelial GF and peripheral blood dendritic cells in OSCC with the ability to present antigens18. GAB4 RILOVICH et al. observed that the splenic DCs isolated from mice treated with VEGF in vivo had significantly higher levels of FITC-dextran uptake than control mice. This suggests that VEGF may exert potent effects on DC precursor cells, resulting in inhibited functional DC development. VEGF binding to VEGF-R on the surface of haematogenic cells may prevent NF-kB activation in haematopoietic progenitor cells, blocking DC development19. When immunotherapy with mature DCs against cancer is considered, treatment aiming at VEGF signalling may reverse the VEGF-mediated DC defect and induce the effective anti-tumour immunity. In conclusion, the authors found that decreased PBDC in OSCC patients correlates with increased serum VEGF, which may be caused by the tumour. VEGF may exert effects on precursor cells, resulting in the decreased DC antigen presentation functions. Overcoming VEGF-mediated DC defects may have significant clinical implications for OSCC and other cancer patients. Competing interests
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None declared. Funding
This study was supported by the Chinese National Nature Science Foundation (30772441) to Dr. Qin-Gang Hu. Ethical approval
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The study was approved by the Ethics Committee of the Nanjing Stomatological Hospital, and all patients gave informed consent before participation. Acknowledgement. The authors would like to thank Pei-Shan Lu (Jiangsu Province Center for Disease Prevention and Control) for his technical assistance.
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