Abstract 6774: Establishment and optimization of an air liquid interface co-culture system to evaluate phenotypic changes in primary human Barrett's esophagus cells upon different environmental exposures

Abstract

Abstract Esophageal adenocarcinoma (EAC) is a deadly cancer with an increasing incidence which arises out of it's precancerous precursor, termed Barrett’s esophagus (BE). A better understanding of the factors that influence BE progression is essential for early detection and novel therapeutics. 3D Air Liquid Interface (ALI) cultures provide an edge over traditional 2D cultures as they better mimic the in vivo conditions of the esophagus. The aim of the study was to establish conditions for a BE ALI model and to evaluate the phenotypic and molecular changes in primary human BE cells upon interaction with different types of esophageal fibroblasts (EF) and exposure to bile acids(BA) (a major component of gastroesophageal reflux). ALI conditions that allowed growth of both primary BE and fibroblast for at least 30 days were optimized. Non dysplastic BE cells from two patients were utilized with one having EAC (P) and the other with no history of progression (NP). The BE cells were cultured in our ALI model with 3 types of EFs: normal esophageal fibroblasts(NFs), BE associated fibroblasts(BFs) and EAC associated fibroblasts(CFs). To determine the effect of BA exposure, ALI cultures underwent daily 1 hour exposure to bile salts. To determine if BE cells that lacked p53 responded differently to BA exposure, CRISPR Cas9 was utilized to knockout TP53. ALI cultures were conducted for 28 days. Phenotypic differences were determined by H&E staining and immunofluorescence (IF). Changes in cell proliferation was evaluated by Ki67 IF. Successful ALI cultures were validated by positive expression of laminin, pan-cytokeratin, Muc2 and Muc5ac by IF. Upon co-culture, we observed opposing histologic changes in the BE cells from the P and NP patients. The NP BE cells had a less complex structure upon culture with the BFs and CFs compared to NFs. The P BE cells showed the development of a complex morphology upon culture with the CFs compared to the BFs and the NFs. In both patients, we observed an increase in Ki67 positivity with fibroblast co culture; NFs (NP: 6.91%, p=0.070, P: 10.6%, p=0.001), BFs (NP: 12.49%, p=0.0001, P: 11.196%, p=0.003) and CFs (NP: 19.88%, p<0.0001, P:14.63 %, p=0.0001) compared to no fibroblasts (NP = 5.32%, P= 7.25% Ki67+). Co-culture with CFs increased Ki67 staining compared to either NFs or BFs (p=0.007,0.014). Exposure to BA showed a thinning but intact BE epithelial surface in both patients with and without TP53 knockout. Overall, primary BE cells and fibroblasts can be maintained long term in an ALI culture and co-culture with fibroblasts drive increased proliferation in BE cells. The histology changes observed in NP and P cell could be an indication of possible activation and interaction of differential signaling pathways. Ongoing studies aim to identify these pathways and determine transcriptional changes due to co-culture and BA exposure. Citation Format: Annesha Chatterjee, Jordana Maria Azevedo-Martins, Chira Chen Tanyolac, Qurat-Ul Ain, Hui Yu, Sophie Camilleri-Broet, Lorenzo Ferri, Philippe Gascard, Thea D. Tlsty, Matthew D. Stachler. Establishment and optimization of an air liquid interface co-culture system to evaluate phenotypic changes in primary human Barrett's esophagus cells upon different environmental exposures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6774.