Highly multiplexed, single-cell spatial phenotyping of Epstein-Barr virus infected tissues.

Abstract

Abstract The Epstein-Barr virus (EBV), a group 1 carcinogen, is a causative factor in nine different cancers that cause ~165,000 deaths globally each year. Almost all adults carry EBV asymptomatically; the virus persisting primarily in rare memory B cells for the lifetime of the host. However, in some infected individuals, for reasons that are unknown, EBV may contribute to the development of cancer. The immune microenvironment in which infected cells reside may influence or trigger cancer development, but we know very little about how this occurs. We have begun to address these critical questions by deploying a spatial biology assay for comprehensive study of the immune microenvironment of EBV-infected tissues. Using AKOYA’s immunofluorescence based Phenocycler assay, we developed an antibody panel that can detect 40 or more different biomarkers in situ at single cell resolution. The panel contains antibodies directed at EBV infected cells at various stages of viral infection, as well as biomarkers for different immune cell lineages, immune cell activation states and tissue structures. This design allows simultaneous identification of infected immune cells within their microenvironment, while, crucially, maintaining spatial relationships between them. We have now set out to produce unbiased and comprehensive spatial phenotypic maps of normal and EBV infected human FFPE tissues. Our data will provide an initial understanding of how the immune microenvironment of virus-infected cells influences tissue pathology and, ultimately, the development of cancer.