Dissecting transcriptional heterogeneity in primary gastric adenocarcinoma by single cell RNA sequencing

Abstract

ObjectiveTumour heterogeneity represents a major obstacle to accurate diagnosis and treatment in gastric adenocarcinoma (GA). Here, we report a systematic transcriptional atlas to delineate molecular and cellular heterogeneity in GA using single-cell RNA sequencing (scRNA-seq).DesignWe performed unbiased transcriptome-wide scRNA-seq analysis on 27 677 cells from 9 tumour and 3 non-tumour samples. Analysis results were validated using large-scale histological assays and bulk transcriptomic datasets.ResultsOur integrative analysis of tumour cells identified five cell subgroups with distinct expression profiles. A panel of differentiation-related genes reveals a high diversity of differentiation degrees within and between tumours. Low differentiation degrees can predict poor prognosis in GA. Among them, three subgroups exhibited different differentiation grade which corresponded well to histopathological features of Lauren’s subtypes. Interestingly, the other two subgroups displayed unique transcriptome features. One subgroup expressing chief-cell markers (eg, LIPF and PGC) and RNF43 with Wnt/β-catenin signalling pathway activated is consistent with the previously described entity fundic gland-type GA (chief cell-predominant, GA-FG-CCP). We further confirmed the presence of GA-FG-CCP in two public bulk datasets using transcriptomic profiles and histological images. The other subgroup specifically expressed immune-related signature genes (eg, LY6K and major histocompatibility complex class II) with the infection of Epstein-Barr virus. In addition, we also analysed non-malignant epithelium and provided molecular evidences for potential transition from gastric chief cells into MUC6+TFF2+ spasmolytic polypeptide expressing metaplasia.ConclusionAltogether, our study offers valuable resource for deciphering gastric tumour heterogeneity, which will provide assistance for precision diagnosis and prognosis.

High MHC-II expression in Epstein–Barr virus-associated gastric cancers suggests that tumor cells serve an important role in antigen presentation

“…7 . Our conclusions are supported by a recent single cell RNA sequencing analysis that conclusively shows much higher expression of HLA-DPA1 and HLA-DPB1 mRNAs in malignant epithelial cells isolated from an EBVaGC compared to the EBV-negative GCs they characterized 47 . Single cell RNA sequencing also demonstrated that the EBVaGC malignant epithelial cells exhibited a characteristic enrichment of genes fitting into the gene ontology terms “antigen processing and presentation of peptide antigen via MHC-II” and “interferon-gamma-mediated signaling processes” 47 , precisely mirroring our conclusions based on sequencing of bulk tumor mRNA.…”

“…This is supported by immunohistochemical analyses reporting expression of MHC-II molecules, particularly HLA-DR, by most GC epithelial cells 40 , including EBVaGCs 41 , 42 . A very recent single cell RNA sequencing study reported that malignant epithelial cells from an EBVaGC expressed higher levels of both HLA-DPA1 and HLA-DPB1 compared to the other GCs studied, definitively showing that these mRNAs originate from carcinoma cells, rather than professional APCs within the tumor 47 .

Figure 1 Expression of the DP MHC-II α- and β-chain genes in GC subtypes and normal gastric tissue.

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“…Importantly, the detection of high mRNA levels for all components of the MHC-II antigen presentation apparatus does not necessarily translate to a high level of expressed protein, correct protein localization, or function in EBVaGCs. However, immunohistochemical analyses of EBVaGCs observed high levels of at least HLA-DR and HLA-DP protein 41,42,47 , and the coordinated regulation of these genes suggests that the other MHC-II molecules will be similarly present. EBV-positive nasopharyngeal carcinomas frequently express high levels of HLA-DR protein as well 69 .…”

Potentiality of multiple modalities for single-cell analyses to evaluate the tumor microenvironment in clinical specimens

“…In this study, we evaluated the two distinct single-cell-based platforms, scRNA-seq and CyTOF, for single-cell level immune profiling using surgery-/biopsy-resected tumors and PBMC from gastric cancer patients. The number of previous studies focusing on gastric cancer biopsy in single-cell level is limited [31][32][33] . Our study revealed that the scRNA-seq analysis could cover a broader range of immune cells of TME in the endoscopically-resected small biopsy tumor samples, detecting even small subgroups of B cells or Treg cells in the tumors.…”

“…In addition, if the biopsy samples could be collected before and after the drug treatment in the same patients, the single-cell analysis with these pre-/ post-treatment specimens should provide a great advantage for a deep understanding of the mechanisms of actions and/or biomarker development for cancer therapeutic drugs [41][42][43] . In fact, the single-cell analyses in the biopsy samples, including the scRNA-seq analysis in gastric cancer, have received a lot of attention in recent years, while the number of reports is extremely limited [31][32][33] . Although several technical challenges still need to be cleared for scRNA-seq of the biopsy-resected tumors, especially the cell isolation steps to miss certain cell populations by "bottle-neck effects" 33,44 , this research platform of scRNA-seq should have potential to open the doors to the new generation of cancer biology, overcoming a number of difficulties that currently-used conventional methodologies are facing.…”