Characterizing the Immune Microenvironment in Esophageal Adenocarcinoma

Abstract

Background: In recent years, rates of esophageal adenocarcinoma (EAC) in the United States and other Western countries has markedly increased, but EAC patients have remarkably poor prognosis. Growing evidence suggests that many types of cancers co-opt immune checkpoint pathways, including the programmed cell death protein 1 (PD-1) pathway, to escape anti-tumor immune responses, and antibodies that block these pathways are emerging as promising therapies. Recently, epithelial PD-L2 up-regulation has been found in approximately 50% of EAC tumors, suggesting that immunotherapy may serve as a novel therapeutic strategy for EAC. Further investigation of the mechanism by which EAC up-regulate PD-L2 and further characterization of the tumor microenvironment carries great potential to facilitate the development of novel therapies. Methods: EAC cell lines were treated with IL-4, IL-13, IFNγ or STAT6 siRNA constructs, and PD-L1 and PD-L2 expression were determined by qRT-PCR or immunoblotting. Immunohistochemistry using antibodies specific to different immune cell populations, CD4, CD8, CD20, CD45, CD68, FOXP3, PD-1, and tBET, was performed on formalin-fixed paraffin-embedded EAC tissue samples. RNA was extracted from formalin-fixed paraffin-embedded EAC tissue samples and analyzed using Nanostring Technologies. Results: We found that IL-4 and IL-13 specifically up-regulated PD-L2, while IFNγ induced PD-L1 and PD-L2 expression in EAC cell lines. While STAT6 was activated by IL-4 and IL-13, STAT6 knockdown by siRNA did not affect basal PD-L2 expression in an EAC cell line. Furthermore, EAC tumor microenvironments were heterogeneous with varying types and quantity of immune cell populations in each tumor, but distinct subsets could be identified with low or high amounts of T-cell infiltration. Immune cell populations were predominantly located in the invasive margins of EAC tumors. Finally, immune profiling of EAC tumors further verified subsets of EAC tumors with low or high immune cell infiltration, and identified a subset of EAC tumors with an IFNγ gene signature, which has been shown to predict response to anti-PD-1 therapy in melanoma. Conclusions: Taken together, our data demonstrates that TH2 cytokines, IL-4 and IL-13, specifically induces PD-L2 in EAC cell lines; however, loss of STAT6 is not sufficient to alter basal PD-L2 expression. EAC tumors have heterogeneous immune cell infiltration, but can be grouped by low or high immune cell infiltration and a subset of tumors shares a specific immune-related gene signature that may be of prognostic value in EAC. These findings suggest that immunotherapies, such as PD-1 inhibitors, may be a novel treatment strategy in EAC.