Single cell genomic characterization reveals the cellular reprogramming of the gastric tumor microenvironment

Abstract

Purpose The tumor microenvironment (TME) consists of a heterogenous cellular milieu that can influence cancer cell behavior. The characteristics of the cellular TME have a dramatic impact on treatments such as immunotherapy. These features can be revealed with single-cell RNA sequencing (scRNA-seq). We hypothesized that single cell gene expression studies of gastric cancer (GC) together with paired normal tissue and peripheral blood mononuclear cells (PBMCs) would identify critical elements of cellular dysregulation not apparent with other approaches. Methods Single cell gene expression studies were conducted on seven patients with GC and one patient with intestinal metaplasia. We sequenced 56,167 cells comprising GC (32,407 cells), paired normal tissue (18,657 cells) and PBMCs (5,103 cells). Protein expression of genes of interest was validated by multiplex immunofluorescence. Results Tumor epithelium had copy number alterations and a distinct gene expression program compared to normal with intra-tumor heterogeneity. The GC TME was significantly enriched for stromal cells, macrophages, dendritic cells (DCs) and Tregs. TME-exclusive stromal cells expressed extracellular matrix components distinct from normal tissue. Macrophages were transcriptionally heterogenous and did not conform to a binary M1/M2 paradigm. Gene expression program of tumor DCs was unique from PBMC DCs. TME-specific cytotoxic T cells comprised of two exhausted heterogenous subsets. Helper, cytotoxic T, Treg and NK cells expressed multiple immune checkpoint or costimulatory molecules. Receptor-ligand analysis revealed TME-exclusive inter-cellular communication. Conclusions Single cell gene expression studies revealed widespread reprogramming across multiple cellular elements in the milieu of the GC TME. Cellular remodeling was delineated by changes in cell numbers, transcriptional states and inter-cellular interactions. This characterization facilitates understanding of tumor biology and enables the identification of novel molecular targets including for cancer immunotherapy. STATEMENT OF TRANSLATIONAL RELEVANCE We leveraged the power of single-cell genomics to characterize the heterogenous cell types and states in the tumor microenvironment (TME). By profiling thousands of single cells from surgical resections of gastric cancer together with paired normal mucosa and peripheral blood mononuclear cells (PBMCs), we determined the deviations in the TME from physiological conditions. Our analysis revealed a cellular reprogramming of the TME compared to normal mucosa in immune and stromal lineages. We detected transcriptional heterogeneity within macrophages and a TME-specific gene expression program in dendritic cells. Cytotoxic T cells in the TME had heterogenous profiles of exhaustion and expression of multiple immune checkpoint and costimulatory molecules. We constructed a receptor-ligand based inter-cellular communications network that was exclusive to tumor tissue. These discoveries provide information at a highly granular cel...

Integrated transcriptional analysis reveals macrophage heterogeneity and tumor-macrophage interactions in the progression of pancreatic ductal adenocarcinoma

“…Although macrophages divisions based on their polarization states could be into three types: un-activated macrophages (M0 macrophages), the classically activated type 1 (M1 macrophages), and the alternatively activated type 2 [20]. Recent research on the genetically engineered mouse model of PDACs, as well as human primary gastric cancer revealed no such classi cation of clusters in the single-cell level [14,21]. The in vitro programming of M0 macrophage to M1 is thought to enhance the cytotoxicity against tumor cells by pro-in ammatory factors.…”

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.…”

Review: Gastric cancer: Basic aspects

“…Overall, their results suggest the potential value of GPNMB blockade as a new immunotherapy against GC 23 . Using single‐cell gene expression analysis, Sathe et al 24 demonstrated that GC TME suffers from a series of cellular changes compared to matched stomach mucosa. They reported an increased number of Tregs, which contribute to an immunosuppressive microenvironment and identified transcriptional cell states unique to GC in dendritic cells and two subclasses of exhausted cytotoxic T lymphocytes.…”