Upregulated genes in the cell cycle are responsible for the high cancer stemness of EAC. (A) Venny analysis of upregulated genes in the cancer stemness of EA01 and EA02. A total of 131 genes were upregulated in the high-stemness single cancer cells of EA01 and EA02. Among these, 16 genes were identified with P<0.01. Previously reported stemness-associated genes were marked in red; references were listed in Table SI. (B) Database for Annotation, Visualization and Integrated Discovery-Kyoto Encyclopedia of Genes and Genomes analysis of upregulated genes in EAC. The signaling pathway of cell cycle and metabolic pathways were significantly enriched in the cancer stemness of EAC (P<0.05). (C) Dynamic expression of EAC cancer stemness-associated cell cycle genes in TCGA data as assessed with cBioPortal. E2F3, CHEK1, CDC20, BUB3, SMC3 and TFDP1 (EAC cancer stemness-associated cell cycle genes) were significantly upregulated in some patients with EAC (percentage is given), which correlated with poor prognosis in patients (median disease-free: 15.67 vs. 24.05 months). EAC, esophageal adenocarcinoma; SI, stemness index; TCGA, The Cancer Genome Atlas; E2F3, E2F transcription factor 3; CHEK1, checkpoint kinase 1; CDC20, cell division cycle 20; SMC3, structural maintenance of chromosomes protein 3; TRDP1, transcription factor Dp-1; HTLV-1, human T-lymphotropic virus type 1; ACVR1B, activin A receptor type 1B; ALDH1A1, aldehyde dehydrogenase 1 family member A1; AURKA, aurora kinase A; RIF1, replication timing regulatory factor 1; STMN1, stathmin 1; TRIM59, tripartite motif containing 59; CENPJ, centromere protein J; FAM168A, family with sequence similarity 168 member A; GPR89B, G protein-coupled receptor 89B; HMGB2, high mobility group box 2; LANCL2, lanthionine synthetase C-like 2; LONP2, lon peptidase 2, peroxisomal; PPP1R8, protein phosphatase 1 regulatory subunit 8; PTDSS1, phosphatidylserine synthase 1; SRSF8, serine and arginine rich splicing factor 8; ZNF107, zinc finger protein 107.