Single-cell and spatial transcriptomic analysis reveals PAX5's role and regulatory mechanisms in gastric adenocarcinoma lymph node metastasis.

BACKGROUND

Lymph node metastasis (LMN) is a key factor in the poor clinical prognosis of patients with gastric adenocarcinoma (GAC), but the underlying cellular regulatory networks and molecular mechanisms are not fully understood.

METHODS

A combined approach of single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) was employed to characterize the cellular landscape of the GAC lymph node metastasis microenvironment. Critical regulatory genes were screened. Their roles and underlying molecular mechanisms were then confirmed via in vitro and in vivo functional assays.

RESULTS

Eight core cell types were identified by scRNA-seq, with proliferative cell clusters further subdivided into six proliferative subpopulations, including proliferative B cells. ST revealed the presence of tertiary lymphoid structures (TLS) formed by T/B cell aggregates within tumor regions of each lesion. Proliferative B cells exhibited dynamic functional transitions. During the primary lesion (PL) stage, their function was primarily associated with regulating apoptosis. In the metastatic primary lesion (MPL) stage, this shifted to participating in T cell/NK cell activation. In the metastatic lymph node (MLN) stage, an immune evasion network was constructed through pathways such as mediating Th2-biased immune responses and uncontrolled somatic diversification of immune receptors. Lasso regression combined with a random forest model confirmed PAX5 as a highly significant regulatory gene for predicting the functional characteristics of proliferative B cells. Its expression levels progressively increased with tumor metastasis (MLN > MPL > PL, p < 0.0001). In vivo and in vitro studies confirmed PAX5’s pro-tumorigenic role, promoting proliferation by activating PI3K/AKT1 signaling and upregulating VEGF-C.

CONCLUSIONS

The systematic characterization of proliferative B cell functional dynamics, from anti-tumor immunity to immune evasion, was achieved. PAX5 was confirmed as a critical regulator of proliferative B cell function, mediating tumor progression through the VEGF-C/PI3K/AKT1 pathway.