Fig 1: Knockdown of CD155 enhances radiosensitivity of EC. A Colony formation analysis of Eca109 and Kyse510 cells stably expressing sh-CD155 or sh-NC after exposure to 0, 3, 6, and 9 Gy of radiation. B Survival curves were obtained based on the clonal efficiency assay. Data are presented as the mean ± SD of independent experiments (n = 3). C D0, Dq, SERD0, and SERDq were calculated using the single-hit multitarget model. D, E Eca109, and Kyse510 cells expressing sh-NC or sh-CD155 were exposed to radiation, and cell viability was analyzed using the CCK8 assay. Left: CCK8 assay results; middle: statistical analysis of CCK8 assay; right: IR of cell viability was calculated and presented as mean ± SD (n = 3). **** P < 0.0001, two-tailed unpaired t-test. F wound-scratch assays were imaged immediately after the initial scratch (T0) and 24 h later. G Quantification of the relative cell migration in the wound-scratch assay. H–L Tumors from different groups (sh-NC, sh-NC + R, sh-CD155, and sh-CD155 + R) were xenografted in nude mouse and collected 28 days later. H Tumor images. I Tumor growth curve in nude mice, p-values were calculated using a two-tailed unpaired test. J Tumor volumes were recorded every three days and are expressed as the mean ± standard deviation (n = 5). K Tumor weight was measured in each case. L The IR on xenograft growth was calculated based on the tumor volume and shown as mean ± SD (n = 5). *, ** p < 0.05, *** p < 0.001, **** p < 0.0001, two-tailed unpaired t-test. M Representative IHC images showing CD155 in xenografts from Eca109 cells (magnification, ×100). N Statistical analysis for metastasis in nude mice shown in (M). *, ** p < 0.05, *** p < 0.001, **** p < 0.0001
Fig 2: CD155 expression levels in EC are upregulated after X-ray irradiation. A, B Analysis of CD155 expression levels in Eca109 and Kyse510 cultured cells after X-ray irradiation. A mRNA levels were analyzed using RT-PCR. B Protein levels were measured using western blot analysis. C Changes in the expression of ?-H2AX were detected by western blotting when the esophageal cancer cell lines were irradiated at 6 Gy within 24 h. D The expression of CD155 was detected via western blotting after the addition of the ATM inhibitor, KU55933. E Immunohistochemistry (IHC) analysis of CD155 protein expression in tumors of irradiated and non-irradiated nude mice. F Statistical analysis of IHC results presented in E. Data are expressed as the mean ± SD. *p < 0.05, ** p < 0.01 and *** p < 0.001
Fig 3: CD155 overexpression is correlated with poor prognosis in EC. A Analysis of CD155 protein levels in esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) using gene expression data available in Oncomine. Expression levels of CD155 in normal (left plot) and cancer tissue (right plot) were plotted using GraphPad Prism software. The y-axis represents median ratio. B Progression-free survival (PFS) analysis in patients with high and low CD155 expression levels. Image obtained from the cBioPortal. C Kaplan-Meier analysis of survival in patients with ESCC with low and high CD155 expression. D, E Analysis of CD155 expression levels in HEEC, Eca109 and Kyse510 cultured cells. D mRNA levels were analyzed using qRT-PCR. E Protein levels were measured using western blot F Immunohistochemistry (IHC) analysis of CD155 protein levels in ESCC tissues and adjacent normal tissues. Data are expressed as mean ± SD. *p < 0.05, ** p < 0.01 and *** p < 0.001
Fig 4: Expression of CD155 affects the proliferation and migration of EC. A, B CD155 expression in stably transfected cells. A RT–PCR was used to analyze mRNA levels. B Western blot revealed CD155 protein levels. C Cell viability was analyzed using the CCK8 assay. D Migration rates were measured using the wound-scratch assay method. Left: Image of one wound-scratch assay. Right: Quantitative analysis of the wound-scratch assays performed. E Image of xenografted nude mouse model and respective xenografted tumor. F Tumor volume curves of xenograft tumors overexpressing CD155 and NC (n = 5 for each condition). G, H analysis of xenograft tumors on the day of tumor removal. The volume (G) and weight (H) of each tumor was measured. I, J Immunohistochemistry (IHC) analysis of CD155 protein expression in the tumor tissues of nude mice. I Images of the tissues. J quantitative analysis of the images was performed using Image Pro Plus, and statistical analysis was performed using GraphPad Prism 8.0. K, L CD155 was knocked down and overexpressed in GFP-tagged Eca109 cells. The cells were injected intravenously into nude mice (n = 3). Two months later, the mice and the lungs and liver of the mice underwent bioluminescence imaging. K HE staining of leg metastasis (100 X magnification). L Representative images of HE staining of the lung and liver metastases (100 X magnification). Scale bar, 100 µm. (M) Showing the statistical analysis for the counts of metastases imaged in vivo in nude mice in Fig. 3K. *, ** p < 0.05, *** p < 0.001, **** p < 0.0001
Fig 5: CD155 stimulates EC cellular proliferation and migration through the Hippo signaling pathway. A Western blot analysis of CD155, pLATS1, pTAZ, TAZ, pYAP, and YAP in cells overexpressing or depleted of CD155. B Immunofluorescence analysis of YAP in cells overexpressing or depleted of CD155. red, YAP; blue, DAPI. C Quantification of immunofluorescence staining using ImageJ software. D Analysis of the nucleoplasmic ratio of B using ImageJ. E, F CCK8 analysis showed that verteporfin treatment reversed the pro-proliferative effect of CD155 overexpression in EC cells. G, H CCK-8 assay was performed to examine the effect of verteporfin (VP) in combination with irradiation on the proliferative capacity of esophageal cancer cell lines. I WB was performed to detect the expression levels of p-YAP and CD155 in esophageal cancer cells after irradiation. J WB for the effect of VP on CD155 and YAP
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