Tissue samples
180 paired tumors, adjacent esophageal mucosa (within 2 cm from tumor) and distant normal mucosa (5 cm from tumor) from 60 surgical resected ESCC specimens without any prior radio- or chemotherapy were collected from the Cancer Hospital of Shantou University Medical College. 21 normal esophageal tissues at different ages in paraffin embedded tissue blocks were obtained from The Judicial Critical Center, Shantou University Medical College, written informed consents were received from relative of the deceased. The research was approved by the ethical board of Shantou University Medical College, ethic license number was SUMC-2015-15.
Cell culture and H2O2, TPA treatment
Immortalized normal esophageal epithelial cell NE6 were a gift kindly given by Prof. George Tsao from Hong Kong University in 2008. NE6 has been authenticated by DNA STR analysis in June of 2020 in the lab of IGE BIOTECH INC., LTD. (Guangzhou, China), resulted as a unique and none contaminated cell line. NE6 were cultured in mixed medium with an equal amount mixture of Epilife and Defined Keratinocyte-SFM (Gibco, Thermo Fisher), supplemented with growth factor (Def Ker Growth Supp 1 ml, Gibco, Thermo Fisher) and penicillin/streptomycin. Cultured at 37 °C in humidified incubator with 5% CO2.
NE6 were treated by 1 μM H2O2 for 48 h, fresh culture medium containing 1 μM H2O2 was changed every 24 h. Cell proliferation was determined by CCK8 assay after 48 h. RNA and protein were extracted at the same time.
TPA (12-O-Tetradecanoylphorbol-13-acetate) was used for the malignant transformation of NE6. Briefly, NE6 was cultured in medium with 10 ng/ml of TPA (12-O-Tetradecanoylphorbol-13-acetate) for 2 weeks, medium was changed every 3 days. After the first round of TPA treatment, NE6 were cultured in normal medium for 2 weeks. Then, the second round of TPA treatment was conducted for another 2 weeks. Totally, NE6 cells were treated by 10 ng/ml TPA for 4 weeks. After TPA treatment, 5% FBS were supplemented in culture medium.
Histological classifications
Tissues embedded in paraffin were sectioned at 4 μm. H.E. staining was performed for histopathological observation. Normal epithelium was defined as esophageal epithelium with 1–3 basal cell layers. During hyperplasia, over three basal cells layers should be observed or with papillae height over 2/3 of epithelium [14]. Cancer and precancerous lesions diagnosis were given based on the 2010 WHO tumors categorization of the digestive system [20]. For precancerous lesions: low-grade intra-epithelial neoplasia (LGIEN) was defined as when dysplastic epithelial cells were confined to the lower half of the whole epithelium; high-grade intra-epithelial neoplasia (HGIEN) was defined as when dysplastic epithelial cells occupied over half of the whole epithelium. ESCC patients were arranged into Stage I, II, III and IV, according to the 7th Edition of the Union for International Cancer Control-American Joint Committee on Cancer TNM staging system [21].
Inflammation classification
The degree of chronic inflammation was classified into four degree according to the density and the location of chronic inflammatory cells (lymphocyte and macrophage). None, less than 10 inflammatory cells in every high power area restricted to the lamina propria; mild, less than 100 inflammatory cells per high power area restricted to the lamina propria, without infiltration in the epithelium; moderate, less than 300 inflammatory cells per high power area in lamina propria, less than 20 presented in esophageal epithelium; severe, more than 300 inflammatory cells per high power area and more than 20 infiltrated inflammatory cells within epithelium.
Immunohistochemistry
Antibodies: CD98hc mouse monoclonal antibody was purchased from Santa Cruz#sc-376815 (Dallas, USA), CK14 mouse monoclonal antibody was purchased from Abcam#ab7800 (Cambridge, UK), CK6 rabbit monoclonal antibody was purchased from HUABIO#ET1611-70 (Boston, USA), Ki67 rabbit monoclonal antibody was purchased from MAIXIN Biotechnology #Kit-0005 (Fuzhou, China).
4 μm slides were deparaffinized in xylene, rehydrated through graded ethanol to water. Antigen retrieval was conducted in pressure cooker at 125 °C for 3 min. Endogenous peroxidase was blocked by 3% H2O2 for 10 min RT. Primary antibodies were applied and incubated at 4 °C in a humidified chamber overnight, followed by incubated with secondary antibody for 30 min at 37 °C. Peroxidase substrate DAB was used for developing. Slides were observed by Olympus BX43 equipped with DP21 digital camera, photographed by CellSens Entry 1.8.
Expression of CD98hc in esophageal epithelium was scored on the percentage taken up by positive stained epithelial cells: score = 0, only the basal layer was of positive; 1, over one layer of the positive cells—25% of the entire epithelium; 2, 26–50%; 3, 51–75%; 4, 76–100%.
The expression of Ki67 in esophageal epithelium was scored based on the ratio of the positive epithelial cells: 0, only one or two layers were positive; 1, more than two layers of positive cells—1/3 of the entire epithelium; 2: 1/3–2/3; 3, > 2/3; 4, the entire epithelium was positive.
Cell proliferation assay
7500 cells/well in 0.1 ml medium were seeded in 96-well plate, after 48 h, exhausted medium was replaced by 0.1 ml fresh medium, 0.01 ml CCK8 reagents (MCE) were spiked into the medium. The OD value were acquired by iMark plate reader (Bio-Rad). The cell survival rate was calculated according to manufacture guidance.
Clonal formation assay
100 cells were seeded in 6-well plate, medium was changed every 3 days. After 10 days, cells were fixed by methanol, stained by Giemsa. Photos were taken by digital camera, cell area were measure by ImageJ software 1.52a.
RT-qPCR analysis
Cellular RNA was obtained by RNeasy Mini kit (Qiagen), gDNA were removed and RNA were reverse transcript to cDNA by Takara#RR047A, qPCR was performed by Takara#RR820A. The primers used were as follows: GAPDH-F 5′-ACAACTTTGGTATCGTGGAAGG, GAPDH-R 5′-GCCATCACGCCACAGTTTC; SLC3A2-F 5′-TGAATGAGTTAGAGCCCGAGA, SLC3A2-R 5′-GTCTTCCGCCACCTTGATCTT.
Statistical analyses
Statistical analyses were conducted by SPSS 22.0 software (SPSS, Chicago, IL) and GraphPad Prism7. Spearman's rank correlation coefficient analysis was employed for assessing the relevance among chronic inflammation level, esophageal histological severity, expression of CD98hc, expression of Ki67, and histological severity. Chi-square Test and Fisher’s Exact Test were applied on the correlation between CD98hc expression and clinicopathological characteristics. For CCK8 and RT-qPCR results, Shapiro–Wilk normality test and paired T test was utilized in the analysis of the significance of differences. p value < 0.05 was considered as statistically significant.