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.

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 °C 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 μm tissue sections were mounted onto slides.

PBDC measurement

Hundred microlitres of the anticoagulated blood was co-cultured with 20 μl of the following antibodies (Biolegend, USA): fluorescein isothiocyanate (FITC)-conjugated anti-CD33, antigen-presenting cell (APC)-conjugated anti-HLA-DR, and phycoerythrin (PE)-conjugated anti-CD14 and -CD16, at room temperature in the dark for 30 min, as previously described . 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 μl/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 °C 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 described . 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 Ca ²⁺ /Mg ²⁺ -free PBS (pH 7.2). The sample was centrifuged through Ficoll/Hypaque (density, 1.077 g/ml; Shanghai Shisheng Cytobiotechnological Corp. China) at 2000 rpm, 25 °C 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 °C. The supernatant including non-adherent 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-α (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 W ang . Tca8113 cells are human tongue squamous cell carcinoma and were 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 μl cell suspension was stained directly with the following murine anti-human monoclonal antibodies: FITC-conjugated anti-CD14, -CD83, -CD86 and -HLA-DR (5 μl), PE-conjugated anti-CD1a, -CD40, -CD54 (20 μl) and -CD80 (5 μl) (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 × 10 ⁵ cells/well) were used as effector cells in 96-well flat bottom plates. Cultured DCs (1 × 10 ⁴ cells/well) were added to the T cells. 4 h before the end of the 96 h incubation, 20 μl Methylthiazoletetrazolium (MTT) (5 mg/ml) was added to each well. At 96 h, the supernatant was replaced with 150 μl/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 (10 ⁴ cells/well) were used as a blank control.

DC endocytosis assay : Cultured DCs (10 ⁵ cells/well) were suspended in culture medium in a 96-well plate, and neutral red solution (0.075%, 100 μl) was incubated at 37 °C for 1 h. DCs were washed three times with warm PBS, and 200 μl 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

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 χ ² 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.

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 × 10 ⁶ /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 × 10 ⁶ /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 ).

Serum VEGF correlated with the level of PBDCs ( P < 0.01) in OSCC patients, but no correlation in control subjects ( P > 0.05).

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).

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.

Table 3

VEGF expression in 57 cases of primary OSCC.

	VEGF ( n )			n	Positive rate	χ 2	P
	−	+	++
TNM stage						11.06	<0.05
I and II	8	13	3	24	66.7%
III and IV	4	11	18	33	87.9%
T stage						6.73	<0.05
T1 and T2	10	19	10	39	74.3%
T3 and T4	2	5	11	18	88.9%
N stage						5.76	>0.05
N0	10	16	9	35	71.4%
N1, N2 and N3	2	8	12	22	90.9%
Total	12	24	21	57	78.9%

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