Fig 2: The therapeutic potential of STAT6 inhibition in EoE. (A) IHC staining for phosphorylated STAT6 was performed, with representative images shown. Scale bars: 100 μm. (B) The positive rate of phosphorylated STAT6 in esophageal epithelium are compared. The data are shown as means ± SD (n = 5). (C) Relative expression of CD73 gene in EPC2-hTERT organoids treated with STAT6 inhibitor (AS151749, 400 nmol/L), IL4 (10 ng/mL), or IL13 (10 ng/mL). (D) The frequencies of CD73+CD104+ cells in organoids treated with AS151749 or dimethyl sulfoxide (DMSO) under the presence of Th2 cytokines. STAT6 silencing efficiency was evaluated by (E) qRT-PCR and (F) Western blot. (G) CD73 gene expression in EPC2-hTERT organoids treated with STAT6 siRNA or nonsilencing (scramble) siRNA with or without IL13. The data are shown as means ± SD (n = 3). Analysis of variance was performed with multiple comparisons. ∗∗P < .01, ∗∗∗∗P < .0001.

Fig 3: CD73+cells mediate epithelial renewal in human esophageal epithelium. (A–D) PDOs or EPC2-hTERT organoids were harvested on day 11 (1° organoids) and dissociated for FACS. The cells were sorted based on CD73 and CD104 expression and resuspended in Matrigel for organoid establishment and growth. OFR and organoid size (mean area) of 2° organoids were (B and C) quantified on a Celigo image cytometer and (D) harvested on day 11 and again dissociated for flow cytometry. (A) Representative phase-contrast images of organoids formed by corresponding cell populations. (B) OFR and (C) mean size were compared among organoids from the 3 populations. (D) The scheme of this experiment and the representative flow cytometry plots of 2° organoids are shown. The percentage of CD73+ cells in EPC2-hTERT were compared. The data are shown as means ± SD (n = 3). (E–H) EPC2-hTERT cells were treated for 48 hours with CD73 siRNA or nonsilencing (scramble) siRNA in 2D culture, followed by seeding into organoid cultures. (E) CD73 silencing efficiency was evaluated by flow cytometry. (F) Representative phase-contrast images of organoids formed by corresponding cells. (G) OFR and (H) mean size of organoids from EPC2-hTERT cells transfected with CD73 or scramble siRNA. The data are shown as means ± SD (n = 10). Two-tailed Student t test or analysis of variance were performed for single or multiple comparisons, respectively. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001.

Fig 4: CD73+CD104+cells represent a less-differentiated and slow-cycling cell population within the basal zone. (A and B) EPC2-hTERT organoids were harvested on day 11 and dissociated for FACS. (A) The cells in EPC2-hTERT organoids were sorted based on CD73 and CD104 expression. (B) CD73, CD104, IVL, and FLG relative gene expression in CD73+CD104+ and CD73+CD104- populations was validated by qRT-PCR. (C) EPC2-hTERT cells in 2-dimensional culture (organoid day 0) and EPC2-hTERT organoid (day 7 and day 11) were harvested for flow cytometry and the representative flow cytometry plots for CD73 and CD104 expression are shown. (D) Representative IHC images for CD73 staining in EPC2-hTERT organoids harvested on day 5, day 7, day 9, and day 11. Scale bars: 100 μm. (E–G) EPC2-hTERT organoids were harvested on day 11 after incubation in culture medium with EdU for 2 hours, and dissociated for flow cytometry. (E) A representative FACS dot plot of cells from EPC2-hTERT organoids subjected to EdU, with the frequencies of each population according to CD73 and CD104 expression, is shown. (F) Representative histograms of EdU staining in each cell population. The percentage of EdU+ cells in each population is shown. (G) Percentages of EdU+ cells in each population. All data are shown as means ± SD (n = 3). A 2-tailed Student t test or analysis of variance were performed for comparing 2 or multiple comparisons, respectively. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001.

Fig 5: The CD73+CD104+population is depleted in the esophagus of active EoE patients and a murine model of EoE. (A) Immunofluorescence staining for CD73 and CD104 on biopsy specimens from non-EoE controls and patients with active EoE. Representative images are shown. Dotted line represents the basement membrane. Scale bars: 100 μm. (B–E) Biopsy specimens from non-EoE controls (n = 10), and patients with active (n = 10) or inactive EoE (n = 10) were dissociated for flow cytometry. The representative flow cytometry plots are shown in panel B. (C) The percentage of CD73+CD104+ or CD73-CD104+ cells in non-EoE controls (n = 10), and patients with active (n = 10) or inactive EoE (n = 10). (D and E) Pearson correlation coefficient analyses comparing the percentage of CD73+CD104+ cells detected by flow cytometry and the number of eosinophils (eos/hpf) in (D) H&E-stained biopsy specimens or (E) CD45+ cells detected by flow cytometry. (F and G) After BALB/c mice were treated with the EoE protocol, their esophagi were resected for histologic sections or flow cytometry. (F) Histologic sections (H&E staining) of the esophagus from control (nontreated [NT]) or EoE protocol–treated mice. Arrowheads identify tissue-infiltrating eosinophils. (G) The percentage of CD73+ cells in esophageal epithelium of EoE mice (n = 3), compared with NT mice (n = 3). The data are shown as means ± SD. A 2-tailed Student t test or analysis of variance was performed for comparing 2 or multiple comparisons, respectively. ∗P < .05.