e20532 Background: Malignant pleural effusion (MPE) is a poor prognostic factor in non-small cell lung cancer (NSCLC), which often develops resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs). Depicting the TKI-resistant MPE landscape is key for clarifying resistance mechanisms and finding therapeutic strategies. Methods: We used single-cell RNA sequencing and GO/KEGG/Reactome enrichment analysis to examine the immune microenvironment of fresh MPE samples (n=7), and compared the 3 rd -genration TKI-resistant subgroup (3 rd -GR, n=4) with the 1 st -genration TKI-resistant subgroup (1 st -GR, n=2). Results: 63,843 cells were isolated, with minimal epithelial cells (3.45%) and most being immune cells. T lymphocytes made up the largest proportion (49.72%), followed by macrophages (13.87%, mainly M1 phenotype), monocytes (8.27%), dendritic cells (7.23%), B cells (7.20%), neutrophils (3.01%), natural killer T (NKT) cells (3.00%), natural killer (NK) cells (2.30%), fibroblasts (0.88%), plasma cells (0.59%), mast cells (0.31%) and endothelial cells (0.16%). CD4+ T cells were dominated by Th1-type central memory T (TCM) cells, though few in low/recent activation states. Regulatory T cells (Tregs) were identified as natural central Tregs rather than peripherally induced Tregs. Cell proportions in 3 rd -GR MPE did not differ significantly from those in 1 st -GR MPE. However, cancer cells in 3 rd -GR MPE showed stronger aerobic metabolism and protein synthesis, with reduced adhesion and antigen presentation abilities, and more strongly inhibited apoptotic pathways. Functional differences in immune cells were also seen in 3 rd -GR MPE, showing a more markedly suppressed immune microenvironment. There was a trend toward higher Treg proportions, with significantly enhanced survival/ immunosuppressive capacity, and higher exhausted cytotoxic T lymphocyte (CTL) proportions, with more complete functional collapse. Despite enhanced protein synthesis and direct cytotoxic potential in mucosal-associated invariant T (MAIT) cells, NKT and NK cells, their aerobic metabolism was significantly reduced, insufficient to sustain their functions. Macrophages had weaker adhesion ability; M1 subtypes showed stronger aerobic metabolism and phagocytic/pro-inflammatory activities, but weaker antigen-presenting capacity, while M2 subtypes shifted from aerobic to lipid metabolism, with enhanced anti-inflammatory activity. These findings matched sequential MPE samples from a single patient after developing resistance to 1 st -G and then 3 rd -G TKIs. Conclusions: This study is the first to depict the immune microenvironment of EGFR-TKI resistant MPE. We found far more severe immune suppression in 3rd-GR MPE. These findings may offer a theoretical basis for creating local immunomodulatory strategies to overcome TKI-resistant MPE.
Gu et al. (Thu,) studied this question.