Objective To screen antigen targets for immunotherapy by analyzing over-expressed genes, and to identify significant pathways and molecular mechanisms in esophageal cancer by using bioinformatic methods such as enrichment analysis, protein-protein interaction (PPI) network, and survival analysis based on the Gene Expression Omnibus (GEO) database.Methods By screening with highly expressed genes, we mainly analyzed proteins MUC13 and EPCAM with transmembrane domain and antigen epitope from TMHMM and IEDB websites. Significant genes and pathways associated with the pathogenesis of esophageal cancer were identified using enrichment analysis, PPI network, and survival analysis. Several software and platforms including Prism 8, R language, Cytoscape, DAVID, STRING, and GEPIA platform were used in the search and/or figure creation.Results Genes MUC13 and EPCAM were over-expressed with several antigen epitopes in esophageal squamous cell carcinoma (ESCC) tissue. Enrichment analysis revealed that the process of keratinization was focused and a series of genes were related with the development of esophageal cancer. Four genes including ALDH3A1, C2, SLC6A1,and ZBTB7C were screened with significant P value of survival curve.Conclusions Genes MUC13 and EPCAM may be promising antigen targets or biomarkers for esophageal cancer. Keratinization may greatly impact the pathogenesis of esophageal cancer. Genes ALDH3A1, C2, SLC6A1,and ZBTB7C may play important roles in the development of esophageal cancer.
Humans ; Esophageal Neoplasms/metabolism* ; Esophageal Squamous Cell Carcinoma/metabolism* ; Epithelial Cell Adhesion Molecule/metabolism* ; Gene Expression Profiling/methods* ; Gene Regulatory Networks ; Gene Expression ; Gene Expression Regulation, Neoplastic ; Intracellular Signaling Peptides and Proteins

Objective To investigate the effects of natural killer (NK)-cell-derived miR-30e-3p-containing exosomes (Exo) on esophageal squamous cell carcinoma (ESCC) cell proliferation, apoptosis and invasion. Methods NK cells were isolated and amplified from the peripheral blood of healthy donors, and NK cell-derived Exo was isolated and identified, which were further co-cultured with NEC cells and were randomly grouped into Exo1 and Exo2 groups. Transmission electron microscopy (TEM) was used to observe the morphology and size of exosomes. Western blot analysis was used to detect the expression levels of exosome markers apoptosis related gene 2- interacting protein X(ALIX), tumor susceptibility gene 101(TSG101), CD81 and calnexin. The NC plasmids, mimics and inhibitors of miR030e-3p were respectively delivered into the NK cells, and the corresponding NK cells-derived Exo were co-cultured with NEC cells, which were divided into NC, Exo, mimic and inhibitor groups. CCK-8 assay was used to evaluate cell proliferation, flow cytometry was conducted to determine cell cycle, annexin V-FITC/PI double staining was employed to detect cell apoptosis, and Transwell^TM assay was performed to detect cell invasion abilities. Real-time quantitative PCR was used to detect the expression of miR-23b, miR-422a, miR-133b, miR-124, miR-30e-3p and miR-99a in NCE cells and exosomes. Results The percentages of CD56⁺CD3⁺ cells and CD56⁺CD16⁺ cells in NK cells were (0.071±0.008)% and (90.6±10.6)%, respectively. Exosome isolated from NK cells ranged from 30 nm to 150 nm, and was positive for ALIX, TSG101 and CD81, while negative for calnexin. NK cell-derived Exos inhibited the proliferation, reduced the proportion of S-phase cells and the number of invaded cells of NEC cells, and promoted the apoptosis and the proportion of G1 phase cells. Overexpression of miR-30E-3p in NK cell-derived exosome inhibited the proliferation and invasion of NEC cells, and blocked cell cycle and promoted apoptosis, while knockdown miR-30e-3p in NK cell-derived exosomes did the opposite. Conclusion miR-30e-3p in NK cell-derived exosomes can inhibit the proliferation and invasion of ESCC cells, block their cell cycle and induce their apoptosis.
Humans ; Esophageal Squamous Cell Carcinoma/genetics* ; Esophageal Neoplasms/genetics* ; Exosomes/metabolism* ; Calnexin/metabolism* ; Cell Movement/genetics* ; MicroRNAs/metabolism* ; Cell Proliferation/genetics* ; Killer Cells, Natural ; Cell Line, Tumor ; Apoptosis/genetics*

Objective To clarify the effect and mechanism of tumor antigen-loaded dendritic cells (Ag-DCs) combined with cytokine-induced killers (CIKs) on the killing of esophageal cancer tumor cells. Methods Peripheral blood DCs and CIKs were induced and cultured, and the DCs were loaded with tumor antigen to obtain Ag-DCs, and Ag-DCs were co-cultured with CIKs. The experiment was divided into CIK group, DC combined with CIK group, Ag-DC combined with CIK group. Flow cytometry was used to detect the phenotype of cells. MTT assay was employed to determine the killing activity against EC9706 cells. Annexin V-FITC/PI double staining was used to detect the apoptosis rate of cells, immunofluorescence staining to detect the expression of phosphorylated apoptotic signal-regulated kinase 1 (p-ASK1) and Western blot analysis to detect the expression of ASK1 pathway related proteins. A nude mouse model of esophageal cancer transplantation tumor was constructed and divided into control group, DC combined with CIK group and Ag-DC combined with CIK group. The corresponding immune cells were injected into the tail vein for treatment and the tumor volume was measured every 2 days. After 21 days, all nude mice were sacrificed with the tumors taken out. HE staining was used to observe the tumor pathological changes and immunohistochemical staining was performed to detect the expression of ki67 and ASK1 in the tumor tissue. Results Comparedwith the CIK group alone and the DC combined with CIK group, the ratio of CD3⁺ CD8⁺ and CD3⁺ CD56⁺ in the cells significantly increased after Ag-DCs and CIKs co-culture, along with the increased killing rate of EC9706 cells, increased apoptosis rate of EC9706 cells, and the improved activation level of ASK1. Compared with the CIK group and the DC combined with CIK group, the growth of the transplanted tumor in nude mice treated with Ag-DCs combined with CIKs was significantly inhibited, and after 21 days, it was observed that the tumor tissue mass in this group was relatively smaller, with sparsely arranged cells in the tumor tissue and a decline in the positive rate of ki67 in tumor tissue, while the positive rate of ASK1 was significantly increased. Conclusion Co-cultivation of tumor antigen-loaded DCs with CIKs can significantly increase the killing activity of esophageal cancer tumor cells. The mechanism of action may be related to the activation of the ASK1 pathway.
Animals ; Humans ; Mice ; Antigens, Neoplasm ; Cytokine-Induced Killer Cells ; Cytokines/metabolism* ; Cytotoxicity, Immunologic ; Dendritic Cells ; Esophageal Neoplasms/therapy* ; Ki-67 Antigen ; Mice, Nude

In recent years, immunotherapy represented by immune checkpoint inhibitors programmed death 1 (PD-1) has made great progress in the treatment of esophageal cancer and is rewriting the global paradigm for the treatment of esophageal cancer. According to current data, only a small number of patients with esophageal cancer could benefit from immunotherapy. Therefore, it is a challenge to screen the potential beneficiaries of PD-1 inhibitors. Studies have shown that the expression level of programmed death-ligand 1 (PD-L1) in esophageal cancer is closely associated with the efficacy of PD-1 inhibitors, and PD-L1 is the most important predictive biomarker of the efficacy of PD-1 inhibitors. With the clinical application of different PD-1 inhibitors and PD-L1 protein expression detection platforms, clarifying the clinical significance and timing of detection of PD-L1 protein expression in esophageal cancer, and establishing a standardized PD-L1 testing procedure, are of great significance to improve the accuracy of detection and reduce the difference between laboratories, so as to maximize the therapeutic benefits for patients. This consensus was finally reached, based on the combination of literature, expert experience, and internal discussion and voting of committee members, to provide an accurate and reliable evidence for clinicians to make decisions.
Humans ; B7-H1 Antigen/metabolism* ; Immune Checkpoint Inhibitors/therapeutic use* ; Consensus ; Esophageal Neoplasms/drug therapy* ; Immunotherapy/methods* ; Lung Neoplasms/pathology*

Objective: To investigate the effect of acetyl-CoA carboxylase 1 (ACC1) knockdown on the migration of esophageal squamous cell carcinoma (ESCC) KYSE-450 cell and underlying mechanism. Methods: Lentiviral transfection was conducted to establish sh-NC control cell and ACC1 knocking down cell (sh-ACC1). Human siRNA HSP27 and control were transfected by Lipo2000 to get si-HSP27 and si-NC. The selective acetyltransferase P300/CBP inhibitor C646 was used to inhibit histone acetylation and DMSO was used as vehicle control. Transwell assay was performed to detect cell migration. The expression of HSP27 mRNA was examined by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and the expressions of ACC1, H3K9ac, HSP27 and epithelial-mesenchymal transition-related proteins E-cadherin and Vimentin were detected by western blot. Results: The expression level of ACC1 in sh-NC group was higher than that in sh-ACC1 group (P<0.01). The number of cell migration in sh-NC group was (159.00±24.38), lower than (361.80±26.81) in sh-ACC1 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC group were statistically significant compared with sh-AAC1 group (P<0.05). The migrated cell number in sh-NC+ si-NC group was (189.20±16.02), lower than (371.60±38.40) in sh-ACC1+ si-NC group (P<0.01). The migrated cell number in sh-NC+ si-NC group was higher than that in sh-NC+ si-HSP27 group (152.40±24.30, P<0.01), and the migrated cell number in sh-ACC1+ si-NC group was higher than that in sh-ACC1+ si-HSP27 group (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-NC+ si-NC group were significantly different from those in sh-ACC1+ si-NC and sh-NC+ si-HSP27 groups (P<0.01). The protein expression levels of E-cadherin and Vimentin in sh-ACC1+ si-NC group were significantly different from those in sh-ACC1+ si-HSP27 group (P<0.01). After 24 h treatment with C646 at 20 μmmo/L, the migrated cell number in sh-NC+ DMSO group was (190.80±11.95), lower than (395.80±17.10) in sh-ACC1+ DMSO group (P<0.01). The migrated cell number in sh-NC+ DMSO group was lower than that in sh-NC+ C646 group (256.20±23.32, P<0.01). The migrated cell number in sh-ACC1+ DMSO group was higher than that in sh-ACC1+ C646 group (87.80±11.23, P<0.01). The protein expressions of H3K9ac, HSP27, E-cadherin and Vimentin in sh-NC+ DMSO group were significantly different from those in sh-ACC1+ DMSO group and sh-NC+ C646 group (P<0.01). The protein expression levels of H3K9ac, HSP27, E-cadherin and Vimentin in sh-ACC1+ DMSO group were significantly different from those in sh-ACC1+ C646 group (P<0.01). Conclusion: Knockdown of ACC1 promotes the migration of KYSE-450 cell by up-regulating HSP27 and increasing histone acetylation.
Humans ; Esophageal Neoplasms/pathology* ; Esophageal Squamous Cell Carcinoma/genetics* ; Vimentin/metabolism* ; Dimethyl Sulfoxide ; HSP27 Heat-Shock Proteins/metabolism* ; Histones/metabolism* ; Cadherins/metabolism* ; Cell Movement ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Gene Expression Regulation, Neoplastic

Treating patients with esophageal squamous cell carcinoma (ESCC) is challenging due to the high chemoresistance. Growth differentiation factor 15 (GDF15) is crucial in the development of various types of tumors and negatively related to the prognosis of ESCC patients according to our previous research. In this study, the link between GDF15 and chemotherapy resistance in ESCC was further explored. The relationship between GDF15 and the chemotherapy response was investigated through in vitro and in vivo studies. ESCC patients with high levels of GDF15 expression showed an inferior chemotherapeutic response. GDF15 improved the tolerance of ESCC cell lines to low-dose cisplatin by regulating AKT phosphorylation via TGFBR2. Through an in vivo study, we further validated that the anti-GDF15 antibody improved the tumor inhibition effect of cisplatin. Metabolomics showed that GDF15 could alter cellular metabolism and enhance the expression of UGT1A. AKT and TGFBR2 inhibition resulted in the reversal of the GDF15-induced expression of UGT1A, indicating that TGFBR2-AKT pathway-dependent metabolic pathways were involved in the resistance of ESCC cells to cisplatin. The present investigation suggests that a high level of GDF15 expression leads to ESCC chemoresistance and that GDF15 can be targeted during chemotherapy, resulting in beneficial therapeutic outcomes.
Humans ; Esophageal Squamous Cell Carcinoma/drug therapy* ; Cisplatin/metabolism* ; Esophageal Neoplasms/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Carcinoma, Squamous Cell/genetics* ; Growth Differentiation Factor 15/therapeutic use* ; Receptor, Transforming Growth Factor-beta Type II/therapeutic use* ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic

Objective: To investigate the clinicopathological characteristics of esophageal carcinoma with gland duct differentiation. Methods: The clinical, morphologic and immunohistochemical (IHC) features of eight cases of esophageal carcinoma with gland duct differentiation diagnosed from 2012 to 2022 at the First Affiliated Hospital of Soochow University were summarized. Results: There were four males and four females, with a mean age of 68.5 (range 59-82) years. Two tumors were located in middle esophagus, five in the lower esophagus, and one in the cardia. The mean diameter was 2.4 cm (range 0.6-4.5 cm). The tumor had a bilayer epithelial structure, including the inner luminal epithelium and the outer basal epithelium. Immunohistochemistry showed that CK7 (8/8) and CK18 (8/8) were positive in the inner epithelium. p40 (8/8), p63 (8/8) and CK5/6 (8/8) were positive in the outer epithelium. SMA, calponin and CD117 were all negative. p53 mutants were found in all eight cases (strong and diffuse positivity in 6/8; complete loss of expression in 2/8). No columnar metaplasia, intestinal metaplasia and ectopic gastric mucosa were observed in the surface squamous epithelium in the cases. The mean follow-up time was 21.5 months (range 5-51 months). Seven patients survived and one patient died 31 months after surgery due to recurrence and liver metastasis. Conclusion: Esophageal carcinoma with esophageal gland duct differentiation is a rare tumor with unique histologic and IHC characteristics.
Male ; Female ; Humans ; Middle Aged ; Aged ; Aged, 80 and over ; Esophageal Neoplasms/pathology* ; Epithelium/pathology* ; Metaplasia/metabolism* ; Carcinoma/pathology*

Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Esophageal Neoplasms/pathology* ; Esophageal Squamous Cell Carcinoma/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; MicroRNAs/metabolism* ; RNA, Long Noncoding/metabolism* ; Wnt Signaling Pathway/genetics*

Objective: To explore the function and mechanism of long non-coding RNA MIR503HG in esophageal squamous cell carcinoma (ESCC). Methods: The MIR503HG expression data in 60, 119 and 23 cases of ESCC and their paired adjacent tissues were chosen from three ESCC datasets GSE53622, GSE53624 and GSE130078, respectively. The expression data of MIR503HG in 81 ESCC tissues and 271 unpaired normal esophageal tissues were screened from the combined dataset of Cancer Genome Atlas and Genotype-Tissue Expression Database (TCGA+ GTEx). The MIR503HG knockdown plasmid was constructed, packaged into lentivirus. The lentivirus was used to infect with esophageal squamous cell carcinoma cell lines KYSE30 and KYSE510 to screen out the stable MIR503HG knockdown cell lines. ESCC cell line KYSE30 was transiently transfected with miRNA mimics to overexpress hsa-miR-503-3p and hsa-miR-503-5p.The expression levels of MIR503HG, hsa-miR-503-3p and hsa-miR-503-5p were detected by quantitative real-time polymerase chain reaction. The proliferation ability of the cells was detected by cell counting kit 8 and clone formation assay. The invasion and migration ability of the cells were detected by Transwell assay. Cell cycle was detected by flow cytometry. The effect of MIR503HG on the proliferation of ESCC was detected by xenograft experiment in BALB/c-nu/nu mice. Results: Both GEO and TCGA+ GTEx databases showed that the expression of MIR503HG in ESCC tissues was higher than that in adjacent tissues and normal esophageal tissues (P<0.01). Compared with shNC group, the proliferation rates of KYSE30 and KYSE510 cells after knockdown of MIR503HGwere significantly inhibited (P<0.001). The colony formation numbers of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were (2.00±1.41) and (1.33±0.47), respectively, significantly lower than that of the shNC group (P=0.002). The clone formation numbers of KYSE510 cells in shMIR503HG1 group and shMIR503HG2 group were (174.67±15.97) and (80.33±6.34), respectively, significantly lower than that of the shNC group (P<0.001). The invasive numbers of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were 75.33±6.02 and 45.67±7.59, significantly lower than that of the shNC group(P<0.001). The migrating number of KYSE30 cells in shMIR503HG1 group and shMIR503HG2 group were 244.00±10.23 and 210.67±13.52, significantly lower than that of the shNC group(P<0.001), and the cell cycle was arrested in G(0)/G(1) phase. The xenograft experiment showed that the subcutaneous tumor in shMIR503HG group was significantly smaller than that in shNC group, and the tumor weight in shMIR503HG group was (0.097±0.026) g, which was lower than (0.166±0.021) g in shNC group (P<0.001). After knockdown of MIR503HG, the relative expression levels of hsa-miR-503-3p in KYSE30 cells of shMIR503HG1 group and shMIR503HG2 group were 0.66±0.02 and 0.58±0.00, respectively, the relative expression levels of hsa-miR-503-5p were 0.64±0.00 and 0.68±0.03, respectively, which were all lower than those in shNC group (P<0.01). After knockdown of MIR503HG, overexpression of hsa-miR-503-3p and hsa-miR-503-5p attenuated the inhibitory effects of knockdown of MIR503HG on proliferation (P<0.001), invasion (P<0.01) and migration (P<0.001) of KYSE30 cells. Conclusions: MIR503HG promotes the proliferation, invasion and migration of ESCC cells by regulating hsa-miR-503 pathway and can be used as a new potential target for targeted therapy of ESCC.
Animals ; Humans ; Mice ; Cell Line, Tumor ; Cell Movement/genetics* ; Cell Proliferation/genetics* ; Esophageal Neoplasms/pathology* ; Esophageal Squamous Cell Carcinoma/pathology* ; Gene Expression Regulation, Neoplastic ; Mice, Nude ; MicroRNAs/metabolism*

BACKGROUND: Gene promoter methylation is a major epigenetic change in cancers, which plays critical roles in carcinogenesis. As a crucial regulator in the early stages of B-cell differentiation and embryonic neurodevelopment, the paired box 5 (PAX5) gene is downregulated by methylation in several kinds of tumors and the role of this downregulation in esophageal squamous cell carcinoma (ESCC) pathogenesis remains unclear. METHODS: To elucidate the role of PAX5 in ESCC, eight ESCC cell lines, 51 primary ESCC tissue samples, and eight normal esophageal mucosa samples were studied and The Cancer Genome Atlas (TCGA) was queried. PAX5 expression was examined by reverse transcription-polymerase chain reaction and western blotting. Cell apoptosis, proliferation, and chemosensitivity were detected by flow cytometry, colony formation assays, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays in ESCC cell lines with PAX5 overexpression or silencing. Tumor xenograft models were established for in vivo verification. RESULTS: PAX5 methylation was found in 37.3% (19/51) of primary ESCC samples, which was significantly associated with age (P = 0.007) and tumor-node-metastasis stage (P = 0.014). TCGA data analysis indicated that PAX5 expression was inversely correlated with promoter region methylation (r = -0.189, P = 0.011 for cg00464519 and r = -0.228, P = 0.002 for cg02538199). Restoration of PAX5 expression suppressed cell proliferation, promoted apoptosis, and inhibited tumor growth of ESCC cell lines, which was verified in xenografted mice. Ectopic PAX5 expression significantly increased p53 reporter luciferase activity and increased p53 messenger RNA and protein levels. A direct interaction of PAX5 with the p53 promoter region was confirmed by chromatin immunoprecipitation assays. Re-expression of PAX5 sensitized ESCC cell lines KYSE150 and KYSE30 to fluorouracil and docetaxel. Silencing of PAX5 induced resistance of KYSE450 cells to these drugs. CONCLUSIONS As a tumor suppressor gene regulated by promoter region methylation in human ESCC, PAX5 inhibits proliferation, promotes apoptosis, and induces activation of p53 signaling. PAX5 may serve as a chemosensitive marker of ESCC.
Animals ; Carcinoma, Squamous Cell/genetics* ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Epithelial Cells/metabolism* ; Esophageal Neoplasms/genetics* ; Esophageal Squamous Cell Carcinoma/genetics* ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; PAX5 Transcription Factor/genetics* ; Tumor Suppressor Protein p53/genetics* ; Xenograft Model Antitumor Assays