Single-cell and spatial dissection of necroptosis spatiotemporal evolution driving lymph node metastasis in gastric...

Lymph node metastasis is a common metastatic route of gastric cancer. However, the heterogeneity of tumor cells and tumor microenvironment between primary gastric cancer and metastatic lymph nodes, as well as the driving mechanisms of metastasis, remain not fully understood. In this study, single-cell RNA sequencing and spatial transcriptomics analysis were performed on 4 gastric cancer patients (four primary tumors and two paired metastatic lymph nodes). Additionally, the TCGA database was used as an external validation dataset to validate the unfavorable clinical outcomes of necroptosis genes and the correlation between MDK-NCL and immune infiltration. Our single-cell analysis revealed significant heterogeneity in the tumor microenvironment between primary and metastatic lymph nodes of gastric cancer. Pseudotime analysis indicated that gastric cancer cells may follow two potential differentiation trajectories during lymph node metastasis. One type of cells possess the ability to migrate to lymph nodes, while the other type remain in the primary site during tumor progression. We observed that the expression of necroptosis-related gene CHMP3 was significantly associated with lymph node metastasis. Immunofluorescence further suggested upregulated CHMP3 protein expression in metastatic lymph node. Furthermore, we found that MDK-NCL interaction was active in metastatic lymph nodes. External validation using the TCGA cohort indicated that high MDK-NCL expression correlated with an immunosuppressive microenvironment. Finally, through integrated single-cell and spatial analysis, we observed that gastric cancer cells may remodel the tumor microenvironment via the MDK-NCL signaling pathway. In summary, our study revealed the dynamics of tumor cells in lymph node metastasis in GC and identified a subtype of GC cells with potential metastatic capability. Our data suggest that necroptosis and the MDK-NCL signaling pathway may be involved in facilitating lymph node metastasis, providing new insights into the mechanisms of GC progression and potential therapeutic targets.