Cell culture
The human OSCC cell lines, CAL27, SCC9 and SCC25 (American Type Culture Collection, ATCC, Manassas, USA), were maintained at 37 °C in a humidified atmosphere of 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Shanghai, China) for the first two cell lines and Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) (Gibco, Shanghai, China) for the last cell line and supplemented both with 1% (v/v) penicillin/streptomycin and 10% (v/v) fetal bovine serum (FBS, Gibco, Shanghai, China) for growth.
Gene Expression Assay
To identify a target gene of interest for subsequent experiments, we used qRT-PCR technology to screen the expression of 14 target genes in OSCC cells. These tumor-targeted genes were obtained by analysis of gene expression profiling database through Oncomine™ Platform (http://www.oncomine.org/resource/login.html), including ABCA1, ABCA5, ABCB6, ABCC3, ABCC4, ABCC5, ABCC10, CD20, CD22, CD47, ERBB2, PIGR, GPNMB and NECTIN4 (Table 1).
Table 1
Amplification primers for quantitative real-time PCR.
Gene name | NCBI accession | Forward (5’−3’) | Reverse (5’−3’) |
ABCA1 | NC_000009.12 | ACGACCACCATGTCAATCCT | AGCATGTCAAACAGCACGTT |
ABCA5 | NC_000017.11 | GCTGGCTGTTCAAAATCATGTG | GACTCCAGCTCCTTCCAAAG |
ABCB6 | NC_000002.12 | GATCAAGTTCAGGCACAGCC | AACCTGCTGGCCCAAGTC |
ABCC3 | NC_000017.11 | TTCTGGGACTCCAACCTGTC | TAGAGCAAGTAGCAGGGCAG |
ABCC4 | NC_000013.11 | TGGCGAATTGTTAGCTGTGG | CAGGGCTGCTGAGACACATA |
ABCC5 | NC_000003.12 | GGGAGAGAACCAGCACTTCT | TGCATGGAGGCATCAAGAGA |
ABCC10 | NC_000006.12 | CCAACAAGACAGTGCTGACC | TACCACTCTCCCCGCTTGTA |
CD20(MS4A1) | NC_000011.10 | AGCTGGCATCGTTGAGAATG | TGTTTCAGTTAGCCCAACCAC |
CD22 | NC_000019.10 | CATACCACGAACTGGAGATGC | TGACGTTCTCATAGTCGCCC |
CD47 | NC_000003.12 | CATGGCCCTCTTCTGATTTC | GGAGGTTGTATAGTCTTCTGATTGG |
ERBB2 | NC_000017.11 | CTGGCCTGCCTCCACTT | ATTGGGCATGGACTCAAACG |
PIGR | NC_000001.11 | CAGATCAACGGGAGAGAAGG | ACTCTTCGTGGAGATGGCT |
GPNMB | NC_000007.14 | TCACGAGCACCCTGATTTCT | ACACCAAGAGGGAGATCACA |
NECTIN4 | NC_000001.11 | ATGGGGACACTTTGGGCTTT | GTCTTCCTGGGGGTCAAGAA |
GAPDH | NC_000012.12 | AGGTCGGTGTGAACGGATTTG | TGTAGACCATGTAGTTGAGGTCA |
RNA was isolated from OSCC cells lysates using the Trizol method (Thermo Fisher Scientific, Waltham, MA, USA) after cells were incubated in a humidified atmosphere with 5% CO2 at 37 °C, and total RNA samples were quantified with the Nanodrop machine (Agilent Technologies, Santa Clara, CA, USA). Next, the cDNA was generated with SuperRT cDNA Synthesis Kit (CWbiotech, Beijing, China), and UltraSYBR One Step qRT-PCR Kit (Low ROX, CWbiotech, Beijing, China) was then used to quantify the expression of a target gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene for normalization as previously described [21], and gene expression was determined by the 2−△△CT method. qRT-PCR primer sequences are listed in Table 1.
Detection Of Plasma Igg Levels
According to the results of qRT-PCR analysis, a linear peptide antigen of human CD47 was designed using a computational epitope prediction software (http://www.iedb.org) based on the features of the target proteins such as hydrophilicity, flexibility, surface accessibility and antigenicity [19]; plasma IgG against the extracellular domain of the CD47 protein was detected with in-house ELISA assay [22].
Human plasma samples were collected from healthy blood donors by the Blood Center of Dongguan, Guangdong Province, China. Pooled plasma from more than 100 randomly selected individuals was used as a quality control (QC) for relative quantification of plasma anti-CD47 IgG levels. This work was approved by the local Ethics Committee based in Dongguan and conformed to the requirements of the Declaration of Helsinki. Briefly, PBS containing 0.5% bovine serum albumin (BSA) was prepared as an analysis buffer and added to each negative control (NC) well, and a positive control (PC) sample was added to each PC well. The detection was performed according to the instructions of the ELISA kit, which was provided by Hailanshen Biotechnology Ltd, Qingdao, China. Optical density (OD) measurement was done on a microplate reader at 450 nm within 10 minutes at a reference wavelength of 620 nm and the specific binding ratio (SBR) was used to represent plasma anti-CD47 IgG levels. Plasma with the highest SBR value from two healthy donors was mixed as anti-CD47 IgG-positive plasma; anti-CD47 IgG negative plasma was taken from two healthy donors with the lowest SBR value and mixed properly as described in our previous study [15, 18, 21, 23, 24]. SBR calculation is as follows: SBR = (OD sample – OD NC)/(OD PC – OD NC).
Cell Proliferation Assay
96-well plates were used to seed three OSCC cell lines in 100 µl/well with a density of 3 × 104 cells/ml, 5 × 104 cells/ml and 5 × 104 cells/ml, respectively, and then cultured in complete medium for 24 h under the same conditions as mentioned above; the medium containing 20% human plasma either negative or positive for anti-CD47 IgG antibodies was then used to culture OSCC cells for 48 h under the same conditions as indicated above [21]. Cell counting kit-8 (CCK-8) (Vazyme, Nanjing, China) was used to detect cell viability. Briefly, 10 µl CCK-8 solution was mixed with complete medium at a ratio of 1:10 and then added to each well. After incubation at 37 °C for 2 h, the optical density (OD) was measured at a wavelength of 450 nm. Cell viability measurements were used to present data and calculated as follows: Cell viability = (OD positive-OD blank)/(OD negative-OD blank).
Analysis Of Apoptosis
6-well dishes were used to seed three OSCC cell lines seeded in 2 ml/dish with a density of 2 × 105 cells/ml, 2.5 × 105 cells/ml and 3 × 105 cells/ml, respectively, for initial culture for 24 h as mentioned above. Cells were then treated with the medium containing 20% human plasma either positive or negative for anti-CD47 IgG and harvested at 48 h; the apoptosis rate was evaluated using the Annexin V-FITC/PI Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the instructions from the manufacturer. In brief, 5 µl Annexin V-FITC and 5 µl PI were added to the buffer and incubated at room temperature for 15 min in the dark. Cells were analyzed by flow cytometry (BD FACSCanto) within 1 h. Annexin V-FITC +/PI- staining and Annexin V-FITC +/PI + staining were used to define early apoptosis and late apoptosis.
Transwell Assay
Cell invasion assays were performed in Transwell chambers (24-well format; 8 mm pore size; Corning, NY, USA) coated with Matrigel (1 mg/ml; Corning Incorporated, Corning, NY, USA) in triplicate. After treatment with the medium containing 20% human plasma either positive or negative for anti-CD47 IgG for 48 h, OSCC cells (5 × 105 cell/well) were planted into the upper chamber and 1000 µl complete culture was added to the lower chamber, and then incubated in a humidified atmosphere with 5% CO2 at 37 °C for additional 48 h. After that, polyoxymethylene and Giemsa (Salarbio, Beijing, China) were used for the fixation and staining of invading cells on the underside of the membrane. Five visual fields were randomly selected and the average number of invading cells was calculated with an inverted microscope (Olympus Corporation, Tokyo, Japan, × 200).
Statistical analysis
All experimental data were expressed as mean ± standard deviation (SD). Student’s t-test (two-tailed) and one-way analysis of variation were applied to examine the differences in cell viability, percentage of apoptotic cells and cell invasion/metastasis between OSCC cells treated with anti-CD47 IgG positive and negative plasma as well as in gene expression. P < 0.05 (*) and P < 0.01 (**) were considered to be statistically significant. All experiments were repeated at least three times.