Deciphering the Immune Complexity in Esophageal Adenocarcinoma and Pre-Cancerous Lesions With Sequential Multiplex Immunohistochemistry and Sparse Subspace Clustering Approach

Abstract

Esophageal adenocarcinoma (EAC) develops from a chronic inflammatory environment across four stages: intestinal metaplasia, known as Barrett’s esophagus, low- and high-grade dysplasia, and adenocarcinoma. Although the genomic characteristics of this progression have been well defined via large-scale DNA sequencing, the dynamics of various immune cell subsets and their spatial interactions in their tumor microenvironment remain unclear. Here, we applied a sequential multiplex immunohistochemistry (mIHC) platform with computational image analysis pipelines that allow for the detection of 10 biomarkers in one formalin-fixed paraffin-embedded (FFPE) tissue section. Using this platform and quantitative image analytics, we studied changes in the immune landscape during disease progression based on 40 normal and diseased areas from endoscopic mucosal resection specimens of chemotherapy treatment- naïve patients, including normal esophagus, metaplasia, low- and high-grade dysplasia, and adenocarcinoma. The results revealed a steady increase of FOXP3+ T regulatory cells and a CD163+ myelomonocytic cell subset. In parallel to the manual gating strategy applied for cell phenotyping, we also adopted a sparse subspace clustering (SSC) algorithm allowing the automated cell phenotyping of mIHC-based single-cell data. The algorithm successfully identified comparable cell types, along with significantly enriched FOXP3 T regulatory cells and CD163+ myelomonocytic cells as found in manual gating. In addition, SCC identified a new CSF1R+CD1C+ myeloid lineage, which not only was previously unknown in this disease but also increases with advancing disease stages. This study revealed immune dynamics in EAC progression and highlighted the potential application of a new multiplex imaging platform, combined with computational image analysis on routine clinical FFPE sections, to investigate complex immune populations in tumor ecosystems.

Construction of the Interaction Network of Hub Genes in the Progression of Barrett’s Esophagus to Esophageal Adenocarcinoma

“…In BE is the only recognized precancerous lesion of EAC, and almost all EAC originates from BE. 28 However, majority of EAC patients are unaware of their history of BE. In this study, we screened RNA-seq data of 94 NE epithelial tissues, 113 BE tissues and 147 EAC tissues in the GEO database, and analyzed the DEGs between normal and BE, normal and EAC, BE and EAC by bioinformatics methods, respectively.…”

Dual-modality imaging of immunofluorescence and imaging mass cytometry for whole slide imaging with accurate single-cell segmentation

“…Surgical pathologists independently assessed the pathology as described in our previous study 23 . The H&E slides were scanned and printed out.…”

“… IF staining. Dewax and antigen retrieval were performed on formalin-fixed paraffin-embedded (FFPE) EMR tissues section as described 23 . The slides were blocked by 5% BSA in PBS for 45 minutes at room temperature, followed by incubation of IF primary antibodies of CD45 (#13917, Cell Signaling Technology, 1:100) and E-Cadherin (#14472, Cell Signaling Technology, 1:100) that diluted in PBS-TB (PBS supplemented with 0.05% Tween 20 and .05% BSA) overnight at 4C.…”

Dual-modality imaging of immunofluorescence and imaging mass cytometry for whole-slide imaging and accurate segmentation

“…Surgical pathologists independently assessed the pathology as described in our previous study. 26 The H&E slides were scanned and printed out. Two experienced gastrointestinal pathologists (M.T.…”