Single-cell and spatial transcriptomic profiling reveal CST6 + epithelial-SPP1+ macrophage crosstalk driving lung adenocarcinoma metastasis.

Metastasis remains the primary cause of mortality in lung adenocarcinoma (LUAD), yet the microenvironmental drivers of this progression are not fully elucidated. In this study, we performed single-cell transcriptomic profiling on primary LUAD and multi-site metastatic lesions (lymph nodes, pleura, and brain), complemented by spatial transcriptomics and multiplex immunohistochemistry (mIHC) to map the tumor microenvironment (TME). Single-cell analysis identified a conserved subset of CST6 + epithelial cells and SPP1 + macrophages significantly enriched at metastatic sites. Integrated spatial analysis and mIHC verification revealed that the physical proximity of these two subsets establishes a pro-metastatic microenvironment. In vitro co-culture experiments and secretome analysis further demonstrated that the CST6-SPP1 interaction triggers the secretion of pro-metastatic factors, such as TGF-beta and MMP9, which significantly enhances tumor cell invasion and migration. Furthermore, bioinformatic analyses suggest that this coordinated infiltration is associated with the formation of an immunosuppressive microenvironment, potentially leading to reduced lymphocyte infiltration and decreased TCR richness. In conclusion, our findings provide novel mechanistic insights into how the interaction between CST6+ epithelial cells and SPP1 + macrophages drives LUAD metastasis. The integration of spatial and functional evidence highlights this cellular axis as a potential therapeutic vulnerability, suggesting that targeting these key interactions could provide significant clinical benefits for patients at high metastatic risk.