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. 2022 Mar 18;23(6):3312.
doi: 10.3390/ijms23063312.

Barrett's Metaplasia Progression towards Esophageal Adenocarcinoma: An Attempt to Select a Panel of Molecular Sensors and to Reflect Clinical Alterations by Experimental Models

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Barrett's Metaplasia Progression towards Esophageal Adenocarcinoma: An Attempt to Select a Panel of Molecular Sensors and to Reflect Clinical Alterations by Experimental Models

Edyta Korbut et al. Int J Mol Sci. .

Abstract

The molecular processes that predispose the development of Barrett's esophagus (BE) towards esophageal adenocarcinoma (EAC) induced by gastrointestinal reflux disease (GERD) are still under investigation. In this study, based on a scientific literature screening and an analysis of clinical datasets, we selected a panel of 20 genes covering BE- and EAC-specific molecular markers (FZD5, IFNGR1, IL1A, IL1B, IL1R1, IL1RN, KRT4, KRT8, KRT15, KRT18, NFKBIL1, PTGS1, PTGS2, SOCS3, SOX4, SOX9, SOX15, TIMP1, TMEM2, TNFRSF10B). Furthermore, we aimed to reflect these alterations within an experimental and translational in vitro model of BE to EAC progression. We performed a comparison between expression profiles in GSE clinical databases with an in vitro model of GERD involving a BE cell line (BAR-T) and EAC cell lines (OE33 and OE19). Molecular responses of cells treated with acidified bile mixture (BM) at concentration of 100 and 250 μM for 30 min per day were evaluated. We also determined a basal mRNA expression within untreated, wild type cell lines on subsequent stages of BE and EAC development. We observed that an appropriately optimized in vitro model based on the combination of BAR-T, OE33 and OE19 cell lines reflects in 65% and more the clinical molecular alterations observed during BE and EAC development. We also confirmed previous observations that exposure to BM (GERD in vitro) activated carcinogenesis in non-dysplastic cells, inducing molecular alternations in the advanced stages of BE. We conclude that it is possible to induce, to a high extent, the molecular profile observed clinically within appropriately and carefully optimized experimental models, triggering EAC development. This experimental scheme and molecular marker panel might be implemented in further research, e.g., aiming to develop and evaluate novel compounds and prodrugs targeting GERD as well as BE and EAC prevention and treatment.

Keywords: Barrett’s esophagus; bile acids; cancer transformation; esophageal adenocarcinoma; gastroesophageal reflux disease; translational model.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Alterations in molecular profile of BAR-T, OE33 and OE19 cells representing subsequent stages of Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC) development. (AC): mRNA fold change for selected genes was normalized to the basal expression for EPC2 cell line; (D,E): results are normalized to the basal expression for BAR-T cells; (F): mRNA expressions in OE19 are normalized to the values observed in OE33 cells. Dotted lines indicate 1.5-fold up-/downregulation of mRNA expression compared to the reference cell line. Results shown as the mean ± SEM of 3 values per group for each gene. Statistically and biologically significant differences compared to reference cell line are indicated by asterisk (*) (p < 0.05).
Figure 2
Figure 2
The viability of cells in different developmental stages of Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC) exposed to various concentrations of bile mixture (BM) applied in pH 5.0 and pH 7.0. BAR-T (A), OE33 (B) and OE19 (C) were treated for 30 min with 50–1250 μM of BM. Results are shown as the mean ± SEM. Asterisk (*) indicates a statistically significant difference as compared with respective BM concentration applied in pH 5.0 (p < 0.05).
Figure 3
Figure 3
Alterations in PTGS1 (A), PTGS2 (B), IL1A (C), IL1B (D), IL1R1 (E), IL1RN (F), TIMP1 (G), TMEM2 (H), IFNGR1 (I), NFKBIL1 (J), SOCS3 (K), SOX4 (L), SOX9 (M), SOX15 (N), KRT4 (O), KRT8 (P), KRT15 (Q), KRT18 (R), FZD5 (S), TNFRSF10B (T) mRNA expression profiles for BAR-T, OE33 and OE19 cells treated for 6 days with acidified bile mixture (BM) to reflect clinical gastroesophageal reflux disease (GERD). Cell lines were exposed daily to 100 or 250 μM of BM applied in acidified medium. Vehicle indicates the cells cultured in regular medium without BM. Dotted lines indicate 1.5-fold up-/downregulation of mRNA expression compared to vehicle. Results are shown as the mean ± SEM of 3 values per experimental group. Statistically significant changes compared with a vehicle were marked with an asterisk (*) (p < 0.05).
Figure 3
Figure 3
Alterations in PTGS1 (A), PTGS2 (B), IL1A (C), IL1B (D), IL1R1 (E), IL1RN (F), TIMP1 (G), TMEM2 (H), IFNGR1 (I), NFKBIL1 (J), SOCS3 (K), SOX4 (L), SOX9 (M), SOX15 (N), KRT4 (O), KRT8 (P), KRT15 (Q), KRT18 (R), FZD5 (S), TNFRSF10B (T) mRNA expression profiles for BAR-T, OE33 and OE19 cells treated for 6 days with acidified bile mixture (BM) to reflect clinical gastroesophageal reflux disease (GERD). Cell lines were exposed daily to 100 or 250 μM of BM applied in acidified medium. Vehicle indicates the cells cultured in regular medium without BM. Dotted lines indicate 1.5-fold up-/downregulation of mRNA expression compared to vehicle. Results are shown as the mean ± SEM of 3 values per experimental group. Statistically significant changes compared with a vehicle were marked with an asterisk (*) (p < 0.05).
Figure 3
Figure 3
Alterations in PTGS1 (A), PTGS2 (B), IL1A (C), IL1B (D), IL1R1 (E), IL1RN (F), TIMP1 (G), TMEM2 (H), IFNGR1 (I), NFKBIL1 (J), SOCS3 (K), SOX4 (L), SOX9 (M), SOX15 (N), KRT4 (O), KRT8 (P), KRT15 (Q), KRT18 (R), FZD5 (S), TNFRSF10B (T) mRNA expression profiles for BAR-T, OE33 and OE19 cells treated for 6 days with acidified bile mixture (BM) to reflect clinical gastroesophageal reflux disease (GERD). Cell lines were exposed daily to 100 or 250 μM of BM applied in acidified medium. Vehicle indicates the cells cultured in regular medium without BM. Dotted lines indicate 1.5-fold up-/downregulation of mRNA expression compared to vehicle. Results are shown as the mean ± SEM of 3 values per experimental group. Statistically significant changes compared with a vehicle were marked with an asterisk (*) (p < 0.05).
Figure 3
Figure 3
Alterations in PTGS1 (A), PTGS2 (B), IL1A (C), IL1B (D), IL1R1 (E), IL1RN (F), TIMP1 (G), TMEM2 (H), IFNGR1 (I), NFKBIL1 (J), SOCS3 (K), SOX4 (L), SOX9 (M), SOX15 (N), KRT4 (O), KRT8 (P), KRT15 (Q), KRT18 (R), FZD5 (S), TNFRSF10B (T) mRNA expression profiles for BAR-T, OE33 and OE19 cells treated for 6 days with acidified bile mixture (BM) to reflect clinical gastroesophageal reflux disease (GERD). Cell lines were exposed daily to 100 or 250 μM of BM applied in acidified medium. Vehicle indicates the cells cultured in regular medium without BM. Dotted lines indicate 1.5-fold up-/downregulation of mRNA expression compared to vehicle. Results are shown as the mean ± SEM of 3 values per experimental group. Statistically significant changes compared with a vehicle were marked with an asterisk (*) (p < 0.05).
Figure 3
Figure 3
Alterations in PTGS1 (A), PTGS2 (B), IL1A (C), IL1B (D), IL1R1 (E), IL1RN (F), TIMP1 (G), TMEM2 (H), IFNGR1 (I), NFKBIL1 (J), SOCS3 (K), SOX4 (L), SOX9 (M), SOX15 (N), KRT4 (O), KRT8 (P), KRT15 (Q), KRT18 (R), FZD5 (S), TNFRSF10B (T) mRNA expression profiles for BAR-T, OE33 and OE19 cells treated for 6 days with acidified bile mixture (BM) to reflect clinical gastroesophageal reflux disease (GERD). Cell lines were exposed daily to 100 or 250 μM of BM applied in acidified medium. Vehicle indicates the cells cultured in regular medium without BM. Dotted lines indicate 1.5-fold up-/downregulation of mRNA expression compared to vehicle. Results are shown as the mean ± SEM of 3 values per experimental group. Statistically significant changes compared with a vehicle were marked with an asterisk (*) (p < 0.05).

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