Single-cell dissection of prognostic architecture and immunotherapy response in Helicobacter pylori infection associated gastric cancer

Reviewer #1 (Public Review):

In this study, the authors conducted a single-cell RNA sequencing analysis of the cellular and transcriptional landscape of the gastric cancer tumor microenvironment, stratifying patients according to their H. pylori status into currently infected, previously infected, and non-infected patients. The authors comprehensively dissect various cellular compartments, including epithelial, stromal, and immune cells, and describe specific cell types and signatures to be associated with H. pylori infection, including i) inflammatory and EMT signatures in malignant epithelial cells, ii) inflammatory CAFs in stromal cells, iii) Angio-TAMs, TREM2+ TAMs, exhausted and suppressive T cells in immune cells. Looking at ligand-receptor interactions as well as correlations between cell type abundances, they suggest that iCAFs interact with immunosuppressive T cells via a NECTIN2-TIGIT axis, as well as Angio-TAMs through a VEGFA/B-VEGFR1 axis and thereby promote immune escape, tumor angiogenesis and resistance to immunotherapy.

The authors conduct a comprehensive and thorough analysis of the complex tumor microenvironment of gastric cancer, both single-cell RNA sequencing data as well as the analysis seem of high quality and according to best practices. The authors validate their findings using external datasets, and include some prognostic value of the identified signatures and cell types. However, most of their conclusions throughout the manuscript are based on the comparison between HPGC and healthy controls, which is not a valid comparison to determine which of the phenotypes are specifically driven by HP infection, e.g. Tregs are high in all GC types, independent of HP status. The same holds true for TREM+ TAMs and iCAFs, which are higher in GC in general. This makes it very difficult to assess the actual HP-driven signatures and cell types. Also, when looking at the correlation/transcriptional differences across different cell types and cellular interactions, the authors do not explicitly define if they are looking at the whole dataset (including healthy controls?) or only at certain patients (HPGC?), which again makes it difficult to interpret the results.

The authors aim to confirm some of their findings via immunofluorescence, which in principle is a great approach to validate their results. However, to be able to conclude that e.g. suppressive TIGIT+ T cells are located close to NECTIN2+ malignant epithelium and that this might facilitate immune escape in HPGC (Figure 4K), the authors should include stains that show that this is not the case in the other groups (nonHPGC, exHPGC and HC). The same holds true for Figure 5G.

In summary, this study provides a valuable resource on the cellular and transcriptional heterogeneity of the tumor microenvironment in gastric cancers, distinguishing between positive, negative, and previously positive HP-infected gastric cancer patients. Given that HP is the main risk factor for gastric cancer development, the study provides valuable insights into HP-driven transcriptional signatures and how these might contribute to this increased risk, however, the study would highly benefit from a clearer and more stringent comparison between HPGC and nonHPGC.

Reviewer #2 (Public Review):

Summary:

This study aims to describe the single-cell transcriptomes of H pylori-associated (Hp) gastric cancers and tumour microenvironment (TME), as a starting point to understand TME diversity stratified by Hp status.

RNAseq was performed for gastric cancers with current Hp+ (from N=9 people), ex-Hp+ (N=6), non-Hp (N=6), and healthy gastric tissue (N=6).

The study expands on previous single-cell transcriptomic studies of gastric cancers and was motivated by previous observations about the effect of H pylori status on therapeutic outcomes. The study includes a brief review of previous work and provides valuable context for this study.

Strengths:

The observations are supported by solid RNAseq study design and analysis. The authors describe correlations between Hp status and inferred molecular characteristics including cell lineages, enrichment for cell subclusters identified as tumour-infiltrating lyphocyte cell types, tumour-infiltrating myeloid cells, and cancer-associated fibroblasts.

The observed correlations between Hp status and enrichment of cell subclusters were broadly corroborated using comparisons to deconvolved bulk RNAseq from publicly available gastric cancer data, providing a convincing starting point for understanding the diversity of tumour microenvironment by Hp-status.

Weaknesses:

The authors acknowledge several limitations of this study.

The correlations with HP-status are based on a small number of participants per Hp category (N=9 with current Hp+; N=6 for ex-HP+ and non-HP), and would benefit from further validation to establish reproducibility in other cohorts.

The ligand-receptor cross-talk analysis and the suggestion that suppressive T cells could interact with the malignant epithelium through TIGIT-NECTIN2/PVR pairs, are preliminary findings based on transcriptomic analysis and immunostaining and will require further validation.