EpCAM Mutant Enteroids Show Alterations in Differentiation and Permeability
Authors: Das Barun , Okamoto Kevin , Rabalais John , Das Soumita , Sivagnanam Mamata 
Published: June 2021

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein which is expressed in the basolateral cell surface of most of the epithelial tissues with the highest expression in the gut. Apart from its role in homotypic cellular adhesion and tight junctions, EpCAM is known to be involved in cellular signaling that promotes proliferation, migration, and maintenance of the undifferentiated state of pluripotent stem cells. Several tumors and carcinomas have been linked with EpCAM overexpression, while the mutations in EpCAM is directly associated with a severe diarrheal disease of infancy known as Congenital Tufting Enteropathy (CTE). Alterations in intestinal cell types and barrier permeability have been observed in the patient and the murine model with CTE. We hypothesize that EpCAM plays a pivotal role in intestinal cell type differentiation and intestinal barrier function. In the current study, we evaluate whether the mutation of EpCAM has an effect on the differentiation and permeability of the intestinal epithelial cells (IECs).We have developed an inducible EpCAM mutant enteroid model based on mutations found in CTE patients (deletion of EpCAM exon 4) to allow for ex vivo study of the consequences of EpCAM mutation on all types of IECs. We first established the mutant enteroid model by confirming the deletion of EpCAM exon 4. Immuno‐fluorescence study demonstrates the mislocalization of mutant EpCAM expression away from the cell surface. The different IECs markers were studied by RNA and protein. The gene expression of different transcription factors that are involved in the secretory pathway of intestinal cell differentiation were analyzed. We observe that mutant enteroids have significantly decreased (n=3; p<0.0001) gene expression of the differentiated goblet and paneth cells markers (Muc2, Lysozyme respectively) than the control enteroids. The expression of other cell types and differentiation markers (enteroendocrine cell marker ChgA, intestinal stem cell marker Lgr5, intestinal differentiation marker Krt20) are also decreased in the mutant model (n=3) as shown by qPCR. Moreover, the gene expression of Hnf1b and ATOH1, two early transcription factors for secretory pathway differentiation, are significantly decreased (n=3; p< 0.05) in mutant enteroids compared with controls. The enteroids were polarized onto monolayers to check the trans‐epithelial resistance and barrier integrity with FITC‐dextran flux assay. Trans‐epithelial resistance values remained intact between the control and mutant enteroid derived monolayer, while the epithelial permeability was significantly impaired in the EpCAM mutant enteroid model compared to the control (n=3; p<0.0001).In this study, we develop the first EpCAM mutant enteroid model. With this model, we note the role of EpCAM in the differentiation of IECs from intestinal crypt progenitor/stem cells. The involvement of EpCAM in barrier permeability is also confirmed. This study further establishes a new facet of EpCAM biology which will help in understanding the pathophysiology CTE and other EpCAM associated diseases.Support or Funding InformationNIH R01 DK107764This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.