Single‐cell profiling reveals tumour cell heterogeneity accompanying a pre‐malignant and immunosuppressive microenvironment in gastric adenocarcinoma

To the Editor: Gastric adenocarcinoma (GAC) is a leading cause of cancer-related disease and death.1 Current phenotypical classifications, however, lack precision in guiding therapy decisions.2 The challenge lies in the molecular and morphologic heterogeneity within and across tumours. Herein, we performed single-cell RNA sequencing (scRNA-seq) and single-cell T-cell receptor sequencing (scTCR-seq) on nine treatment-naive GAC patients, including different Lauren's subtypes (Figure 1A and Figure S1A; Table S1). Six major cell lineages emerged, including epithelial cells, endothelial cells, fibroblasts, B cells, T cells, and myeloid cells, based on canonical markers (Figure 1B and Figure S1B–E; Table S2). Subpopulations were characterised by reclustering within each major cell type (Figure S1F,G). Malignant epithelial cells were identified using inferCNV (Figure S2A). Evaluation of pair-wise similarity of cancer cell transcriptomics revealed distinct levels of intratumour heterogeneity (ITH) across patients (Figure 1C and Figure S2B). Accordingly, we classified patients into higher ITH (ITH-H) and lower ITH (ITH-L) groups based on the median of ITH score (Supporting Information Methods), validated by the observation that ITH-H tumours spanned multiple epithelial clusters, which were scarce in ITH-L tumours (Figure 1D and Figure S2C,D). ITH-H cancer cells exhibited higher levels of copy number variation (CNV), proliferation and hypoxia compared with their ITH-L counterparts (Figure 1E–G), along with elevated VEGFA expression, indicating hypoxia-driven angiogenesis (Figure 1H). Differentially expressed genes (DEGs) analysis between ITH-H and ITH-L groups identified an ITH signature (PSCA, PPIB and CALR) dividing TCGA-STAD data into ITH-H and ITH-L groups based on the median ITH signature score calculated by ssGSEA (Figure 1I and Figure S2E). ITH-H TCGA-STADs displayed increased proliferation, hypoxia and angiogenesis compared with the ITH-L tumours (Figure S2F–H). Furthermore, ITH correlated with inferior disease-free survival (DFS) in TCGA-STADs, even after adjusting for confounding factors in multivariate COX regression analysis (Figure 1J,K), suggesting ITH as an independent prognostic factor for recurrence.3 Considering Lauren's subtype, intestinal-type epithelial cells exhibited higher levels of CNV (Figure S2I), consistent with previous reports.4 However, no association was observed between Lauren's subtype and ITH degree either in our cohort or in TCGA-STAD data (Figure S2J,K). In the stromal compartment, three endothelial cell types and two fibroblast types were identified. Notably, CXCL14+ fibroblasts, enriched in the ITH-H tumours, displayed oncogenic potential (Figures S3 and S4, Supporting Information). Immune cell analysis explored the immune tumour microenvironment (TME) in relation to ITH. Among myeloid cells, we identified dendritic cells, monocytes, mast cells and macrophages (Figure S5A–D). Mφ-SPP1, a transitional state between Mφ-FCN1 and Mφ-APOE, expressed mixed expression of SPP1 (a tumour-associated macrophage marker), ALOX5AP (a crucial immune-modulating lipid mediator associated with M2 macrophage polarization) and CD163 (an M2 macrophage marker)5, 6 (Table S3; Figure 2A–C). Distinct macrophage compositions were observed between different ITH statuses or among different Lauren's subtypes (Figure S5E). Unique transcriptional factors (TFs) regulatory network activity, particularly MYC(+), was observed in Mφ-SPP1 (Figure 2D). MYC was previously reported as a potent regulator in tumour-associated macrophages (TAMs).7, 8 Consistently, we detected high expression of MYC and its target genes specific to Mφ-SPP1 (Figure 2E,F and Figure S5F). Moreover, the targets of MYC were mainly upregulated in Mφ-SPP1 compared with the other two cell types (Figure 2G,H). In addition to the top targets inferred by pySCENIC, we observed upregulation of MRC1 and PARG in Mφ-SPP1, which were previously reported as the targets of MYC in TAMs (Figure 2H).8 The above findings confirm Mφ-SPP1 as a TAM subtype and suggest potential downstream genes involved in the TAM function. We also observed that macrophages in the ITH-H group were mainly located at the end stage of pseudotime and exhibited an enriched anti-inflammatory signature (Figure S5G,H, Supporting Information). T-cell analysis revealed fewer activated and more exhausted CD8 T cells in ITH-H tumours compared with ITH-L tumours (Figure 3A). Stratified analysis in ITH-H and ITH-L groups revealed more frequent state transitions between CD8 cells in the ITH-H group by scTCR-seq analysis (Figure 3B,C). The T-cell clones with state transition showed marked expression of immunosuppressive molecules (Figure 3D). These observations further indicate an immunosuppressive TME associated with ITH-H (Supporting Information). Finally, we compared the receptor–ligand pairs functioning as immune checkpoints between ITH-H and ITH-L groups (Supporting Information). Interestingly, we found no significant interactions between PDCD1 and its ligands when analysing the interaction between epithelial cells and other cell populations, suggesting that anti-PD1 therapy might not be suitable for some GACs. However, significant interaction of PVR and its receptors was only observed in ITH-H tumours (Figure 3E). We also observed enrichment of PVR+ cancer cells in the ITH-H group compared with ITH-L (Figure 3F). Considering the activated CD8 T cells and NK cells, along with their interactions with cancer cells through PVR–TIGIT and PVR–CD226 interactions, the ratio of PVR–TIGIT to PVR–CD226 in the ITH-H group exceeded that in the ITH-L group. This implies that the dominance of TIGIT over CD226 was more pronounced in the ITH-H tumours (Figure 3G), which might contribute to the immunosuppressive feature. Given that targeting PVR and TIGIT attracted increasing interest in the field of cancer immunotherapy,9 these findings suggest that patients with higher ITH might benefit from PVR/TIGIT inhibition treatment. In conclusion, using a novel scoring method based on cellular similarity of transcriptional profiles, we classified GACs into different ITH groups and comprehensively analysed the associated TME heterogeneity. The study suffered limitations of small sample size and potential bias in sample selection. The presence of only one mixed GAC among the cohort hindered the accurate comparison of Lauren's subtypes. And the study only provided a cross-sectional snapshot of the intertwining of ITH and TME, lacking over-time observations. Nevertheless, our research suggests that ITH can serve as a prognostic marker indicative of recurrence. Immunosuppressive features such as M2 macrophage polarization, T-cell exhaustion and interactions between certain immune checkpoint molecules are enriched in ITH-H tumours, offering valuable insights for immunotherapeutic strategies. Longitudinal studies involving larger cohorts are essential to unravel the dynamic interaction between ITH and TME with increased statistical power. ZH Chen, HL Liu and XF Xia conceived and designed the study. J Ge, HY Zhou, MM Tang, CH Huang, X Feng and T Liu collected samples as well as clinical information. J Ge, HY Zhou, MM Tang, CH Huang, X Feng and T Liu performed the experiments. CL Zhang, J Ge, XC Zou and X Xiao analyzed the data. X Xiao, CL Zhang and J Ge wrote the manuscript. J Ge, CL Zhang, J Bai, XF Xia, HL Liu, ZH Chen and X Xiao provided intellectual discussions and ideas regarding the content of manuscript. ZH Chen, HL Liu, XF Xia and J Bai supervised the study. All authors read and approved the final manuscript. The authors would like to thank the patients, their families and caregivers for their contributions to this study. The authors declare no potential conflicts of interest. This study was supported by the National Natural Science Foundation of China (8197103463) to Zihua Chen, Hunan Province Science and Technology Innovation Plan Project (2018SK52604) ‘Key Techniques for Prevention and Treatment of Postoperative Peritoneal Recurrence and Metastasis of Gastric Cancer Based on Minimally Invasive Surgery’ to Heli Liu and Guangdong Yiyang Healthcare Charity Foundation (JZ2022014) to Jie Ge. The data reported in this paper have been deposited in the OMIX, China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (https://ngdc.cncb.ac.cn/omix: accession no. OMIX004144). 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