Network-based single-cell analysis identifies a proliferative GZMB⁺ plasma cell subset linked to Hedgehog signaling...

Lung squamous cell carcinoma (LUSC) is a heterogeneous cancer characterized by complex cellular interactions. Plasma cells, a critical immune population, exhibit functional diversity in LUSC. The aim of this study was to characterize plasma cell heterogeneity and their interactions with tumor cells using an integrative single-cell framework.

We constructed a high-resolution single-cell atlas of LUSC using single-cell RNA-sequencing data to identify plasma cell subsets. Plasma cell heterogeneity was assessed through clustering and differential expression analysis, and differentiation trajectories were plotted. Cell-cell communication was analyzed using the "CellChat" package. A prognostic nine-gene signature was derived using LASSO-Cox regression and further validated across independent GEO cohorts. Upstream regulatory mechanisms of prognostic genes were investigated through integrative transcriptomic analysis. The role of ZBTB20 in tumor progression was validated through functional assays.

Three plasma cell subsets were identified, including a proliferative GZMB⁺ (C2) subset characterized by higher G2M/S activity and stemness-associated features. Trajectory analysis revealed a differentiation axis from IGLC2⁺ to IGKC⁺ to GZMB⁺ plasma cells. Network-based communication analysis demonstrated that C2 plasma cells interact with LUSC tumor cells via the SHH-PTCH1 signaling axis. A nine-gene prognostic signature robustly stratified patients, and was associated with immune signaling, cytokine interactions, apoptosis regulation, and tumor microenvironment remodeling. Integrative regulatory analysis identified UBB as a central prognostic gene, with ZBTB20 and PPARG as potential upstream transcriptional regulators. The prognostic significance of the C2-related gene signature was validated in independent GEO cohorts. ZBTB20 was further identified as a key regulator of tumor-related signaling and cellular proliferation, and its oncogenic role was verified through functional experiments.

The proliferative C2 plasma cell subset has prognostic and functional significance in LUSC. ZBTB20 regulates key tumor-related pathways in LUSC cells through UBB-centered transcriptional control. These findings provide a systems-level framework for understanding plasma cell dynamics and tumor-immune interactions in LUSC progression for future development of targeted therapies.