Unraveling tumoral heterogeneity and angiogenesis-associated mechanisms of PD-1 and LAG-3 dual inhibition in lung cancers by single-cell RNA sequencing.

Background: Lymphocyte activation gene 3 (LAG-3) is a promising immune checkpoint for combination immunotherapy. This study aims to elucidate the exact synergistic anti-tumor mechanism of programmed death 1 (PD-1) and LAG-3 dual inhibition in lung cancer. Methods: Multiple patient-derived xenograft (PDX) models of lung cancer were constructed and analyzed by single-cell RNA sequencing (scRNA-seq). Clustering of all human-derived cells, identification of biomarker genes of three cell types, trajectory analysis, and calculation of tumor heterogeneity scores were performed. Differentially expressed genes (DEGs) were identified and functional enrichment analyses of cancer-associated genes were conducted. The functional significance of DEGs in the immune system was evaluated using the Reactome online server. Major histocompatibility complex (MHC) pathways and angiogenesis-associated pathways were analyzed. The Cancer Genome Atlas (TCGA) was used for further verification. Results: PD-1 and LAG-3 dual inhibition achieved synergistic tumor inhibition in squamous cell carcinoma (SCC) PDX models, but not in adenocarcinoma and small cell lung cancer PDX models. A total of 8127 cells, including 2699 basal, 4109 malignant, and 1319 epithelial cells, were identified by scRNA-seq. Malignant cells evolved from basal and epithelial cells in the trajectory analysis. The responders to the combination therapy of PD-1 and LAG-3 inhibitors had lower heterogeneity scores than non-responders. Compared with anti-PD-1 monotherapy, the combination group exhibited higher levels of neutrophil degranulation. The DEGs were correlated with disease, metabolism, and programmed cell death-associated pathways. The MHC class I-associated pathways and pericyte pathways were upregulated, whereas the vascular endothelial growth factor pathway was downregulated in the combination group. Conclusion: We discovered the superior efficacy of PD-1 and LAG-3 dual inhibition in SCC PDX models, and showed that it may be associated with low tumor heterogeneity scores, upregulation of the MHC class I pathway, and normalization of tumor angiogenesis.