The human esophageal squamous cell carcinoma (ESCC) cell lines Eca109 (CCTCC, Wuhan, China) and Kyse510 (CCTCC, Wuhan, China) were cultured in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco), penicillin (100 U/ml), and streptomycin (100 g/ml). HEEC cell lines were generously provided by Renchang Zhao from the Thoracic Surgery team of Qilu Hospital and cultured in DMEM medium. Eca109 and Kyse510 cells were transfected with recombinant lentiviruses and positive clones were selected with puromycin (4 g/ml). Quantitative RT-PCR (qRT-PCR) and western blot analyses were used to assess the efficacy of lentiviral transfection. CD155 overexpression lentiviruses (NM 006505) were obtained from Shanghai Genechem Co., Ltd. CD155 gene knockdown lentiviruses (LPP-CS-HSH058084-LVRU6GP-01-200) were obtained from GeneCopoeia (Inc, Rockville, MD, USA. The symbols OE- and sh- means overexpression and knockdown, respectively. OE-NC, control group for overexpression; OE-CD155, overexpression of CD155; sh-NC, control group for knockdown; sh-CD155 knockdown of CD155.
Clinical samples and information
A paraffin-embedded EC tissue chip (Catalogue no. Esc968) was purchased from Shanghai Superbiotek Pharmaceutical Technology Co., Ltd.. (Shanghai, China).
RNAfast200 (Cat no. 20010, fastagen) was used to purify the total RNA from cells according to instructions from the manufacturer. qRT-PCR was conducted with SYBR Green Premix Pro Taq HS qPCR Kit (AG11701, ACCURATE BIOTECHNOLOGY, Hunan, China) according to instructions from the manufacturer and using the Bio-Rad Single-Color Real-Time PCR system (Bio-Rad, Hercules, California, USA). The primers (Accurate Biology, China) were designed and synthesized as follows: CD155: 5’-GCGTAGAGGATGAAGGCAACT-3’ (forward primer); CD155: 5’-CAAGCACTCGGAGCCAGATAT-3’ (reverse primer); GAPDH: 5’-GCACCGTCAAGGCTGAGAAC-3’ (forward primer); GAPDH: 5’-TGGTGAAGACGCCAGTGGA-3’ (reverse primer). Relative CD155 expression was calculated using the 2-∆∆CT method.
Total protein extract was isolated from cells lysed in pre-cooled RIPA buffer with protease inhibitors (BestBio, China), according to instructions from the manufacturer. Protein concentration in each extract was determined using a BCA protein assay kit (Beyotime Biotechnology, Wuhan, China). Proteins from the extract were separated according to size in SDS-PAGE acrylamide gel (10% – 15%) and subsequently transferred to a Poly (vinylidene fluoride) (PVDF) membrane (Millipore). After protein transfer, the membrane was blocked using 5% non-fat milk or bovine serum albumin for 2 h. The blocked membrane was incubated overnight with the primary antibodies (Table 2) at 4° C. The membrane was then incubated with secondary anti-rabbit or anti-mouse antibodies (ZSGB-BIO) at room temperature for 2 h. Protein bands in the membranes were detected using an ECL kit (Millipore). Images of the blots were obtained using a CCD imager (Tanon-5200, Tanon Science & amp; Technology).
Cell viability assays
A cell counting kit-8 assay (CCK8; Bioss company, China) was used to evaluate the proliferation of Eca109 and Kyse510 cells. Cells (2000/well) were seeded into 96-well plates. After incubation for 0, 24, 48, 72, 96, and 120 h at 37 °C, CCK8 assays were conducted according to instructions from the manufacturer. The optical density (OD) of each well was measured using an Infinite M200 PRO microplate reader (Tecan) with the 450 nm wavelength.
A wound-scratch assay was performed on Eca109 and Kyse510 cells grown to confluence. Cells were seeded in six-well plates and cultured in serum-free culture medium. When the cells reached 100% confluency, a scratch line over the cell layer was made using a gun head perpendicular to the plate and washed twice with PBS to remove the floating cells. Gaps were observed and photographed at 0 h and 24 h after scratching. The cell free area of the scratch was calculated using ImageJ software. The percentage of scratch healing was quantified as: (0 h scratch area-24 h scratch area) / 0 h scratch area × 100. For each experiment, at least three scratched fields were recorded, and all wound-scratch assays were performed in triplicate (× 10 magnification).
Equal numbers of cells from different groups were first grown into transwell chambers (3422, Corning, NY, USA), which were incubated with serum-free 1640 and supplemented with serum-free 1640 to a volume of 200 ul. Then 800ul of 1640 medium containing 20% fetal bovine serum was added to the 24-well plate. The chambers were placed into the 24-well plate and incubated for 28-32 hours. At the end of incubation, the chambers were removed and fixed in methanol and stained with aqueous crystal violet solution. Finally, randomly selected fields of view were photographed (100X, Olympus, Tokyo, Japan) and counted for statistical purposes. Statistical analysis was performed with GraphPad Prisme 8.
Clonal efficiency assay
Cells (1000 per well) were seeded in 6-well plates, grown overnight, and then irradiated with 6 Gy of X-ray. After irradiation, cells were grown for two weeks. Cell clones were fixed and stained with crystal violet. The stained cells and colonies (>50 cells/colony) were photographed and counted. The relative clone formation ability was calculated as ability % = (mean experimental clone number/mean control clone number) × 100. The survival rate of clonogenic cells was used to calculate the sensitization enhancement rate (SER) induced by CD155 knockdown. Survival curves were obtained using GraphPad Prism 8.0. Mean lethal dose (D0) and quasi-threshold dose (Dq) were derived by fitting survival curves to a single-shot multi-target model (y=1-[1-e(-kx)]N). SER of D0 (SERD0) and Dq (SERDq) were used to assess radioresistance.
EC tissue was fixed with 10% formalin, embedded in paraffin and then cut into 3 μm sections. Sections were dried at 80 °C for 15 min, dewaxed in xylene, rinsed in ethanol at various concentrations, and rehydrated in double-distilled water. Antigens were unmasked by microwaving the sections in 10 mmol/L of citrate buffer (pH 6.0) for 15 min. The sections were then incubated with hydrogen peroxide to block peroxidase and incubated overnight at 4 °C with anti-CD155 antibody (1:200; Abcam, ab267788). Negative controls were incubated with PBS instead of the primary antibody. The sections were then incubated with horseradish peroxidase (HRP)-labeled streptavidin and the biotinylated secondary antibody. The sections were stained with DAB and counterstained with hematoxylin. We selected sections randomly (×200 magnification) and invited two pathologists to evaluate and score themindependently. The intensity of the dye color and number of positive cells were used to assess the scores. The intensity was graded as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (intense staining). On the other hand, the percentage of positive cells was divided into four categories: 0 (5%), 1 (5%–25%), 2 (25%–50%), 3 (51%–75%), and 4 (>75%). The final score was the product of these two scores. If the multiplied score was more than 8, the degree of expressiveness was considered as "high", else it was characterized as "low."
In vivo experiments
All animal procedures were performed according to the protocol approved by the Ethics Committee of Qilu Hospital of Shandong University. For this study, female nude mice were strictly fed according to the guidelines of the institution. For the tumor xenograft model of EC, female NU/NU nude mice were randomly divided into six groups of five mice each, and labeled according to the type of cells and treatment they were subjected sh-NC (xenografted with cells expressing the shControl), sh-CD155 (xenografted with cells expressing the shCD155), sh-NC+R (xenografted with cells expressing the shControl and subjected to radiation after), sh-CD155+R (xenografted with cells expressing the shCD155 and subjected to radiation after), OE-NC (xenografted with cells overexpressing the control construct), and OE-CD155 (xenografted with cells overexpressing CD155). For xenografting, cells were resuspended in saline and subcutaneously injected into the groin region of nude mice. The experimental nude mice were anesthetized and exposed to X-rays. The sh-NC+R and sh-CD155+R groups were administered 6 Gy radiation 10, 12, and 14 days after injection. Tumor volume (V) was measured every three days in two dimensions (a, b; a>b) and calculated according to the following formula: V=a×b²/2. The mice were sacrificed 28 days after injection. In metastasis mouse models, twelve four-week-old female NU/NU nude mice were randomly divided into four groups, each containing three mice. CD155 was knocked down and overexpressed in GFP-tagged Eca109 cells. The cells were injected intravenously into nude mice (n = 3). The mice of the metastasis mouse model were sacrificed 60 days after injection. Eca109 expressing GFP xenograft growth was pictured with bioluminescent imaging using an In Vivo Imaging System (IVIS) Spectrum (Perkin-Elmer; Waltham, MA). The lungs and the livers of the mice also underwent bioluminescence imaging.
Hematoxylin-eosin (HE) staining
HE staining was performed according to a general protocol. In short, after dewaxing and rehydration, the tissue sections were incubated with hematoxylin solution (ZSGB-BIO, China) for 5 min and fractionated in 1% acid alcohol solution (1% hydrochloric acid in 75% ethanol), and then rinsed with distilled water. The sections were then stained with the eosin solution (ZSGB-BIO, China) for 2 min, followed by dewatering in graded alcohol and clearing in xylene. Finally, the tissue sections were photographed under a microscope.
Cells were grown overnight on coverslips and fixed with methanol. After washing 3 times with PBS, the cells were permeabilized with 0.2% Triton X100 in PBS and closed with goat serum for 1 h at room temperature. The coverslips were then incubated with YAP (14074T) antibody overnight at 4 °C, followed by incubation with fluorescently coupled secondary antibody for 1 h at room temperature. After washing 3 times with PBS, the slides were stained with DAPI for 15 min, followed by washing with PBS.Finally，the coverslip is fixed to the slide with anti-fluorescence quencher reagent. The coverslip is fixed to the slide with anti-fluorescence quencher reagent. The coverslip is fixed to the slide with ProLong Gold Antifade reagent. The slides were then examined with microscope and the YAP was analyzed for fluorescence intensity by ImageJ software (n=3).
For cell experiments, data are presented as the mean ± SD and were analyzed by a two-tailed unpaired t-test using Prism 8.0. The interaction between radiation and CD155 knockdown was analyzed using the Bliss independence model . Based on the inhibition rate (IR), the combined percentage inhibition, IRR+sh-CD155, was predicted using the following formula: IRR+sh-CD155=IRR+IRsh-CD155−IRR×IRsh-CD155, where IRR and IRsh-CD155 were the IR of mono-treatment with radiation and sh-CD155, respectively. Then, the actual combined percentage inhibition, IRR+sh-CD155, was compared with IRR+sh-CD155(Thero.). IRR+sh-CD155>, =, and < IRR+sh-CD155(Thero.) indicated synergistic, additive, and antagonistic effects, respectively. Overall survival (OS) and progression-free survival (PFS; the time from treatment to disease progression or death from any cause) were assessed using data represented as mean ± standard deviation. GraphPad Prism 8 was used for statistical and survival analyses. Survival analysis p-values were based on log-rank tests. Statistical significance was defined when p < 0.05.
Analysis of human PC datasets
Data analysis was performed using the cBioPortal Cancer Genomic and Oncomine websites.