Fig 2: TSTA3 overexpression increased in vivo ESCC metastasis. (A) Representative images of metastatic tumor nodules in the lung of nude mice intravenously injected with KYSE150 cells stably overexpressing TSTA3 (TSTA3-WT) or control vector (NC). (B) Number of metastatic tumor nodules in the lung were compared between nude mice injected with TSTA3-WT and control cells and statistically analyzed. All data are presented as the mean ± standard deviation. (C) Representative images of H&E staining in metastatic tumor nodules in the lung section of nude mice. (D) Representative images transverse (upper) and coronal (bottom) sections of CT images of nude mice. White arrows show water-density nodule lung in CT.

Fig 3: TSTA3 silencing reduced ESCC cells invasion and fucosylation level. (A) Relative TSTA3 mRNA levels in KYSE510 (left) and KYSE180 (right) cells treated with the lentivirus expressing TSTA3-shRNA (sh1 or sh2) or sh-NC (negative control) were assessed by qPCR. (B) Relative expression of TSTA3 protein in KYSE510 (left) and KYSE180 (right) cells treated with the lentivirus expressing TSTA3-shRNA (sh1 or sh2) or sh-NC were assessed by western blot. (C) Transwell invasion assays on KYSE510 (upper) and KYSE180 (bottom) with TSTA3 knockdown and control group. Right is a bar chart for quantitative analysis. (D) UEA-I fluorescence of KYSE180 (upper) and KYSE510 (bottom) cells was analyzed by FACS. The right panel shows quantitation of UEA-I fluorescence. All data are presented as the mean ± standard deviation and three independent experiments. (E) Representative immunohistochemistry images of TSTA3 expression in tumor tissues, matched normal tissues and metastatic lymph node from paraffin-embedded formalin-fixed 11 ESCC tissues. Right: TSTA3 expression in metastatic lymph node tissue showed significantly higher expression than that of matched tumor and normal tissues.

Fig 4: Copy number amplification and expression of TSTA3 in ESCC. (A) The significant focal SCNA filtered by GISTC across 663 ESCC genomes. (B) Heatmap of CNA log2 ratio of read coverage across 225 ESCC individuals in 8q24.3 covering TSTA3 regions and detected significant amplification of TSTA3. (C) Kaplan-Meier's survival analysis between ESCC patients with TSTA3-WT (TSTA3-wild type) and TSTA3-Gain (TSTA3-copy number amplification). The horizontal axis is the survival time and the vertical axis is the percentage of survival. Every cross on the survival curves stands for censored data. Log-rank test P-value, Cox regression P-value, Hazard Ratio and 95% CI are displayed on the graph. (D) Scatter plots of positively correlating TSTA3 amplification and RNA expression. RNA expression values and DNA copy number values are plotted against each other for the 155 cases where WGS and RNA sequencing were performed. (E) Representative images of TSTA3 protein expression from FFPE tissue microarrays containing 104 tumors and 60 adjacent normal esophageal tissues by IHC. (F) Comparison of the TSTA3 protein expression in non-paired (upper) and paired (bottom) ESCC tumor tissues and normal tissues using t-test.

Fig 5: N-glycoproteomics analysis identified glycoproteins mediating the effects of TSTA3 on ESCC metastasis. (A) Schematic illustration of our systems biology approach to identify N-glycoproteins mediating the effects of TSTA3 on ESCC metastasis. (B) Motif analysis of glycosylation sites identified in N-glycoproteomics analysis. (C) KEGG pathways analysis of the differentially expressed glycoproteins in KYSE150 NC group and TSTA3-WT group. (D) The classification of differentially expressed glycoproteins in cell components, molecular function and biological process (N = 3 biological triplicates).