Ephrin receptor A2, the epithelial receptor for Epstein-Barr virus entry, is not available for efficient infection in human gastric organoids

Abstract

Epstein-Barr virus (EBV) is best known for infection of B cells, in which it usually establishes an asymptomatic lifelong infection, but is also associated with the development of multiple B cell lymphomas. EBV also infects epithelial cells and is associated with all cases of undifferentiated nasopharyngeal carcinoma (NPC). EBV is etiologically linked with at least 8% of gastric cancer (EBVaGC) that comprises a genetically and epigenetically distinct subset of GC. Although we have a very good understanding of B cell entry and lymphomagenesis, the sequence of events leading to EBVaGC remains poorly understood. Recently, ephrin receptor A2 (EPHA2) was proposed as the epithelial cell receptor on human cancer cell lines. Although we confirm some of these results, we demonstrate that EBV does not infect healthy adult stem cell-derived gastric organoids. In matched pairs of normal and cancer-derived organoids from the same patient, EBV only reproducibly infected the cancer organoids. While there was no clear pattern of differential expression between normal and cancer organoids for EPHA2 at the RNA and protein level, the subcellular location of the protein differed markedly. Confocal microscopy showed EPHA2 localization at the cell-cell junctions in primary cells, but not in cancer cell lines. Furthermore, histologic analysis of patient tissue revealed the absence of EBV in healthy epithelium and presence of EBV in epithelial cells from inflamed tissue. These data suggest that the EPHA2 receptor is not accessible to EBV on healthy gastric epithelial cells with intact cell-cell contacts, but either this or another, yet to be identified receptor may become accessible following cellular changes induced by inflammation or transformation, rendering changes in the cellular architecture an essential prerequisite to EBV infection.

Organoids as host models for infection biology – a review of methods

“…Microinjection with bacteria, such as Helicobacter pylori ( H. pylori) 13 , 19 – 22 , Clostridioides difficile ( C. difficile) 23 , 24 , Escherichia coli ( E. coli ) 25 , 26 , and Salmonella enterica serovar Typhimurium ( S . Typhimurium) 27 , 28 , as well as parasites, such as Cryptosporidium parvum ( C. parvum) 29 , 30 , viruses 31 – 33 , and toxins/drugs 32 , 34 , 35 has proven instrumental for understanding the initial steps of pathogenesis and establishing organoids as an advanced model for studying host–pathogen interactions. Microinjection was also used to experimentally test the hypothesis that pks-positive E. coli contribute to colon carcinogenesis; for this, long-term exposure to the bacteria was necessary.…”

“…The medium composition can also be modified to induce or inhibit cell differentiation, depending on the experimental approach. 2D monolayers have been used to model a range of infections, including H. pylori 56 , 59 , 60 , pathogenic E. coli 61 , norovirus 57 , 62 and, recently, Epstein Barr virus (EBV) infections 33 . In the case of EBV, infected B cells, the primary target cell of the virus, were added to the epithelial culture and transferred the virus to cells 33 , an infection method termed “transfer infection” that has been previously described for cell lines 63 .…”

“…Future challenges to the field relate to the incorporation of further complexity, namely, the presence of immune and nerve cells, vasculature, and even the microbiome 97 – 100 . Advances in these areas include the development of several approaches for coculturing immune cells with epithelial organoids 97 , e.g., the coculture of neutrophils with an RSV-infected airway organoid 31 , coculture with B cells for transfer infection with EBV 33 , or coculture with monocytes in an S . Typhimurium-infected tissue engineered intestinal model 76 .…”

Homeostasis and Cancer Initiation: Organoids as Models to Study the Initiation of Gastric Cancer

“…Co-infection with H. pylori is an increased risk factor, but it is unclear which pathogen initiates the process. A recent study that compared EBV infectivity for matched normal and GC organoids generated from the same patient made an interesting observation—that EBV was only able to infect GC organoids, not normal organoids [ 64 ]. The EBV receptor ephyrin receptor A2 (EPHA2) is expressed on carcinoma cell lines, including the gastric carcinoma line AGS [ 65 ] but was not present on normal organoids [ 64 ].…”

“…A recent study that compared EBV infectivity for matched normal and GC organoids generated from the same patient made an interesting observation—that EBV was only able to infect GC organoids, not normal organoids [ 64 ]. The EBV receptor ephyrin receptor A2 (EPHA2) is expressed on carcinoma cell lines, including the gastric carcinoma line AGS [ 65 ] but was not present on normal organoids [ 64 ]. This study raises questions about whether EBV can only infect epithelia after they have been transformed, or whether EPHA2 or another previously unidentified receptor may become expressed after chronic inflammatory activity.…”