Unraveling the complex code of tumor microenvironment and heterogeneity by comparing multiple primary lung adenocarcinomas and intrapulmonary metastasis: A multidimensional exploration.

e20542 Background: Current diagnostic criteria for multifocal lung adenocarcinoma rely primarily on histopathology and next-generation sequencing. However, distinguishing multiple primary lung adenocarcinomas (MPLC) from intrapulmonary metastasis (IM) is challenging due to similar histology. This study explores key differences in the tumor microenvironment (TME) and tumor heterogeneity between MPLC and IM. Methods: Eight patients with 16 lung nodules were selected for single-cell sequencing. Samples were categorized into three pairs for the MPLC group and five pairs (five primary and five metastatic) for the IM group. Differences in the TME and tumor cell heterogeneity were analyzed using multiplex immunofluorescence, spatial proteomics, and bioinformatics. Results: In the TME, IM group T cells showed stronger immunosuppressive features, while MPLC group had enriched CD8 effector and follicular helper T cells. IM group macrophages exhibited pro-inflammatory and pro-angiogenic traits, contrasting with MPLC group's lipid metabolism-linked macrophages. MPLC group endothelial cells expressed tumor-suppressor genes, whereas IM group fibroblasts showed genes related to stromal remodeling and tumor progression. Tumor cell heterogeneity revealed upregulated CRABP2, CEACAM5, and PLAT in IM group, associated with aggressive phenotype, while MPLC group showed higher extracellular matrix-related protein expression. Four genes (CXCL8, RNASE1, CTSH, AQP4) showed significant transcriptional differences. Conclusions: Our study identifies key TME differences between IM and MPLC, particularly in immune suppression and cellular composition. The distinct roles of macrophages, endothelial cells, and fibroblasts in IM suggest increased tumor aggressiveness. The identified interaction pairs and differential genes may serve as biomarkers for distinguishing MPLC from IM, aiding in diagnostic and therapeutic strategies.