Single-Cell Profiling of Tumor Immune Microenvironment Reveals Immune Irresponsiveness in Gastric Signet-Ring Cell Carcinoma

Background & Aims: Gastric cancer (GC) is a major cancer type characterized by high heterogeneity in both tumor cells and the tumor immune microenvironment (TIME). One intractable GC subtype is gastric signet-ring cell carcinoma (GSRCC), which is associated with poor prognosis. However, it remains unclear what the GSRCC TIME characteristics are and how these characteristics may contribute to clinical outcomes. Methods: We enrolled 32 patients with advanced GC of diverse subtypes and profiled their TIME using an immune-targeted single-cell profiling strategy, including (1) immune-targeted single-cell RNA sequencing (n = 20 patients) and (2) protein expression profiling by a targeted antibody panel for mass cytometry (n = 12 patients). We also generated matched V(D)J (variable, diversity, and joining gene segments) sequencing of T and B cells along CD45⁺ immunocytes. Results: We found that compared to non-GSRCC, the GSRCC TIME appears to be quiescent, where both CD4⁺ and CD8⁺ T cells are difficult to be mobilized, which further impairs the proper functions of B cells. CXCL13, mainly produced by follicular helper T cells, T helper type 17, and exhausted CD8⁺ T cells, is a central coordinator of this transformation. We show that CXCL13 expression can predict the response to immune checkpoint blockade in GC patients, which may be related to its effects on tertiary lymphoid structures. Conclusions: Our study provides a comprehensive molecular portrait of immune cell compositions and cell states in advanced GC patients, highlighting adaptive immune irresponsiveness in GSRCC and a mediator role of CXCL13 in TIME. Our targeted single-cell transcriptomic and proteomic profiling represents a powerful approach for TIME-oriented translational research.