Patients and samples
A total of 122 patients with primary OSCC treated between January 2014 and November 2014 in the Department of Oral Maxillofacial-Head and Neck Oncology, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, were enrolled. Patients fulfilling the following criteria were included in the study: (1) primary OSCC confirmed by pathology after surgery; (2) no radiotherapy, chemotherapy, or immunotherapy before surgery; and (3) complete clinicopathological data available. Paraffin-embedded tumor tissue sections prepared for immunohistochemistry of BDCA-2.
Subsequently, from January 2018 to July 2018, we collected an additional 60 tumor tissue samples from OSCC patients that met the above criteria. Six patients (three males and three females, mean age of 57.2 years old) who underwent alveoloplasty were selected as controls. The collected tumor samples and normal oral mucosal epithelium were quickly washed in phosphate-buffered saline (PBS) thrice and divided into three portions, one of which was soaked in formalin, another was soaked in high-glucose Dulbecco's Modified Eagle Medium (DMEM; Gibco, Invitrogen, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, France) and the last was immediately frozen in liquid nitrogen and stored at -80 °C for further analysis. No significant differences were noted in age, gender, alcohol use, or smoking habits between the control and OSCC groups. This study was approved by the Shanghai Ninth People’s Hospital IRB and informed signed consent was obtained from each patient.
Single‑cell suspension preparation
Surgical human primary OSCC tissue samples were washed several times with PBS and carefully minced to small pieces in sterile serum-free high-glucose DMEM (supplemented with 100 U/mL penicillin, 1 mM glutamine, and 100 U/mL streptomycin). Tumor tissue was digested with collagenase type VIII (1.5 mg/mL; Sigma, USA) and DNase type I (1 mg/mL; Sigma, USA) for 120 min at 37 °C with gentle agitation. The resulting cell suspensions were washed in PBS, resuspended in PBS containing trypsin/EDTA, and filtered through a 40-mm nylon cell strainer (Falcon, Becton Dickinson Labware) into cold DMEM containing 10% FBS.
Isolation of pDC and preparation of pDC‑conditioned medium (CM)
pDC were isolated using magnetically activated cell sorting with the BDCA-4 dendritic cell isolation kit from Miltenyi Biotec according to manufacturer’s instructions [21]. pDC was labeled with anti-BDCA-4 antibody coupled to colloidal paramagnetic microbeads and passed through magnetic separation columns twice (LS and RS columns; Miltenyi Biotec). The purity of the isolated pDC (APC-conjugated anti-CD123+ and PERCP-conjugated anti-MHC II+) was﹥90%. Viability was﹥95% as determined using the trypan blue exclusion test. Isolated pDC were seeded at a final concentration of 5 × 105 cells/mL and cultured overnight in serum-free DMEM at 37 °C and 5% CO2 to prepare pDC‑conditioned medium (pDC-CM). pDC were then collected by centrifugation at 3000 rpm for further experiments, and pDC-CM were collected and stored at -80 °C for in vitro cancer cell line stimulation and in vivo studies.
Cell culture and stimulation
For cell line stimulation, cells were seeded at a density of 4 × 105 and cultured in DMEM supplemented with 10% FBS in a humidified atmosphere of 5% CO2, at 37 °C for 24 h. The cells were washed twice with 1× PBS and starved in 1% FBS-supplemented growth medium overnight. Next, cells were stimulated with different concentrations of pDC-CM, with or without anti-TNF-α antibodies or TNF-α (50 ng/mL; R&D Systems, USA) for 72 h. Experiments were repeated a minimum of three times.
The siRNA targeting CXCR-4 was purchased from Gene Pharma (Shanghai, China). The OSCC cell lines were cultured in 6-well cell culture plates and transfected with the siRNA using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol.
Immunohistochemical staining (IHC)
Formalin-fixed, paraffin-embedded tissue blocks from OSCC patients were used. IHC was performed on 4-mm-thick routinely processed paraffin sections, according to a standard protocol. After deparaffinizing and rehydrating, tissue sections were incubated in sodium citrate buffer and heated for antigen retrieval. anti-CXCR-4 antibody (1:150; Rabbit; Proteintech, USA), or anti-CXCR-7 antibody (1:200; Rabbit; Proteintech, USA) was applied to tissue sections overnight at 4 °C. All sections were then incubated with a corresponding secondary antibody for 30 min at 23-25 °C. Immunohistochemical evaluations were performed independently by two pathologists who were not informed of the clinicopathological profile of the patients.
CXCR-4/7 expressions were quantified using a visual grading system based on the extent of staining (percentage of positive cells graded on a scale from 0 – 4: 0, < 5%; 1, 5 – 25%; 2, 25 – 50%; 3, 50 – 75%; 4, > 75%) and the intensity of staining (graded on a scale of 0 – 3: 0, none; 1, weak staining; 2, moderate staining; 3, strong staining). Five high-power representative field (400×) were evaluated. A weighted score was assigned to each case by multiplying the score for the percentage of positive cells by the staining intensity score. Cases with a weighted score < 1 were considered low; > 1 and < 4 were classified as middle; and all others were considered to be high. The score was assigned by two pathologists.
Immunofluorescent staining
Tissue sections were deparaffinized and rehydrated with xylene, gradient ethanol, and distilled water. After rinsing thrice with PBS, the tissue sections were blocked with 3% BSA for 30 min and incubated with anti-BDCA-2 antibody (10 µg/mL; goat; R&D Systems, USA) overnight at 4 °C. Tissue sections were then washed and incubated for 30 min with an Alexa Fluor 488-conjugated anti-mouse IgG F(abʹ) 2 fragment (1:200; Invitrogen, USA). Cells were co-stained with 4′,6-diamidino-2-phenylindole (1:300; Invitrogen) to detect nuclei, after which they were observed and imaged using a TCS SP2 laser-scanning confocal microscope (Leica Microsystems, Germany). Based on BDCA-2 staining, the pDC infiltrates were assessed based on the same criteria applied in IHC analysis. Based on BDCA-2 staining, pDC infiltrates were considered low if < 10 pDC per high-power field (HPF, 400×) were observed, high if > 10 pDC per HPF were observed [22].
Flow cytometry
Flow cytometric analysis was performed as previously described by Hartmann et al [21]. Briefly, cells were incubated at 4 °C for 30 min in PBS with 0.1% BSA and 0.01% NaN3 in the presence of appropriately diluted labeled monoclonal antibodies, which were purchased from BD Pharmingen (USA) and used as follows: APC-conjugated anti-CD123+ and PERCP-conjugated anti-MHC II+. The cells were subsequently analyzed using a FACSCalibur and CellQuest FACS analysis software (BD Biosciences, Erembodegem-Aalst, Belgium).
Cytokine production analysis
The Bio-Plex Cytokine Assay (Bio-Rad, Munich, Germany) was used for detection of IL-6, IL-8, and TNF-α. An IFN-α module set from Bender Med Systems (Vienna, Austria) was used to detect IFN-α in cell culture supernatants, according to the manufacturer’s instructions. These cytokine assays allowed for the multiplexed quantitative measurement of multiple cytokines in a small volume of cell culture supernatant. The protein array was analyzed by a dedicated microplate reader system (Bio-Plex Array Reader; Bio-Rad) and data were calculated by the Bio-Plex Manager software. Experiments were repeated a minimum of three times.
Quantitative real‑time PCR (qPCR)
The preparation of total RNA and cDNA was performed as previously described [23]. CXCR-4 (primer sequences: upstream, 5´-ACTACACCGAGGAAATGGGCT-3´; downstream, 5´-GCTACCCACAATGCCAGTTAAGAAGA-3´), CXCR-7 (primer sequences: upstream, 5´-TCTGCATCTCTTCGACTACTCA-3´; downstream, 5´-GTAGAGCAGGACGCTTTTGTT-3´), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; primer sequences: upstream, 5´- GGAGCGAGATCCCTCCAAAAT-3´, downstream, 5´- GGCTGTTGTCATACTTCTCATGG-3´) mRNA were detected by real-time PCR using the TaqManTM Gene Expression Assay (Applied Biosystems, Life Technologies Life Technologies, USA). Gene-specific products were measured continuously by an ABI PRISM 7000 Sequence Detection System (Applied Biosystems) over 40 cycles. Experiments were repeated a minimum of three times.
Western blotting (WB)
Cells were treated with lysis buffer (PBS containing 1% Triton X-100, protease inhibitor cocktail, and 1 mmol/L phenylmethylsulfonyl fluoride) at 4 °C for 30 min. Equal concentrations of protein from the cytoplasm and nucleus were subjected to SDS-PAGE. Following transfer to a Immobilon-P Transfer Membrane, successive incubations with anti-CXCR-4 (1:500; Rabbit; Proteintech, USA), CXCR-7 (1:500; Rabbit; Proteintech, USA), p65 (0.5 µg/mL; Abcam, USA), p-p65 (0.5 µg/mL; Abcam, USA), and p-Iĸb (0.6 µg/mL; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) antibodies or anti-GAPDH antibody (Sangon Biotech) were performed, followed by corresponding horseradish peroxidase-conjugated secondary antibody (Sangon Biotech) incubation. The immunoreactive proteins were then detected using the ECL system (NCM Biotech, Suzhou, China). Bands were scanned using a densitometer (GS-700; Bio-Rad) and quantification was performed via Quantity One 4.6.3 software (Bio-Rad). Experiments were repeated a minimum of three times.
Cell proliferation assay
Cell proliferation assays were performed using Cell Counting Kit-8 (CCK-8; Dojindo, USA). Cells were plated in 96-well plates at 3 × 104 cells per well and incubated for five days. Each day, 10 uL CCK-8 solution was added to each well. The OD value was read at 450 nm, 2 h after CCK-8 addition, according to the manufacturer instructions, and the cell viability was measured at intervals of 24 h. Experiments were repeated a minimum of three times.
Colony formation assay
Oral cancer cells (CAL 27, HN 30) were seeded at 1,000 cells per well in a 6-well plate and incubated overnight at 37 °C. The cells were treated with different concentrations of pDC-CM containing 10% FBS. Control wells were treated with DMEM containing 10% FBS alone. The plates were incubated for 14 days; during this period, the medium was changed twice per week with the appropriate concentration of pDC-CM. The plates were washed with ice-cold PBS, colonies were fixed with methanol for 15 min, stained with 2% crystal violet, and counted. Colonies consisting of ≥ 50 counts were scored. Experiments were repeated a minimum of three times.
Scratch wound healing assay
Cells were treated with different concentration of pDC-CM after creating a wound across the cell monolayer with a plastic tip. Cell migration into the wound surface was then measured every 6 h. Experiments were repeated a minimum of three times.
Cell migration and invasion assay
The cell migration assay was performed with transwell chambers (0.8 μm pores; Merck Millipore, USA), while the cell invasion assay was implemented by coating the upper surface of the transwell chambers with Matrigel (BD Biosciences, USA). Cells (3×104) were resuspended in serum-free DMEM and different concentrations of pDC-CM were added to the interior of the inserts. DMEM containing 20% FBS was added to the lower chamber as a chemoattractant. Cells were incubated for 48 h at 37 °C in a CO2 incubator (5% CO2). Cells that migrated or invaded through the membrane were fixed and stained with crystal violet. Images of five randomly selected fields containing fixed cells were captured, and cells were counted. Experiments were repeated a minimum of three times.
Tumorigenesis assay in nude mice
To evaluate the tumor-promoting role of pDC-CM in vivo, a xenograft model was implemented in 4-week-old BALB/c nude mice (male). Briefly, a total of 1 × 106 Cal 27 cells in 100 μL serum-free DMEM were subcutaneously injected into the left or right buttock. Mice were maintained under pathogen-free conditions and were handled in accordance with the Guidelines for Animal Experimentation of Shanghai Jiao Tong University. Five days after the injections, all mice were divided randomly into two groups (five mice per group) and treated intraperitoneally with pDC-CM or PBS every three days. The tumor volume was measured every three days using the following formula: tumor volume = length × width × width / 2. After treatment for 64 days, the animals were sacrificed, and the tumor volume and tumor weight were measured.
LN metastasis assay in nude mice
The footpads of 140 BALB/c nude mice (male) were inoculated with 20 μL PBS suspensions containing 106 oral cancer cells (Cal 27). One week after inoculation, the nude mice were randomly divided into three groups and treated every three days with pDC-CM containing anti-TNF-α antibodies or PBS (intraperitoneal). LN metastasis was observed for each group every week. This study protocol was approved by the Ethical Committee on Animal Research of the Shanghai Jiao Tong University.
Statistical analysis
Data are presented as mean ± standard deviation (SD) of at least three independent experiments. Comparisons of independent samples were performed using Student's t-test or nonparametric tests when appropriate (SPSS 11.0, Chicago). Correlation analyses were done using the Chi-square test or Spearman’s rank correlation coefficient. Statistical significance was defined as P < 0.05 for all analyses.