Integrated single-cell transcriptomic analysis of brain and lymph node metastases in non-small cell lung cancer

Figure 1 Single-cell transcriptomic atlas of primary and metastatic non-small cell lung cancer. A: UMAP visualization of 174643 cells integrated from adult human non-small cell lung cancer primary tumors (lung cancer), lymph node metastases (mLN), normal lymph nodes (nLN), brain metastases (mBrain), and normal brain tissues (nBrain). Cells are color coded by annotated cell types: T cells, B cells, plasma cells, myeloid cells, mast cells, microglia, epithelial cells, endothelial cells, myofibroblasts (Myo_fibro), neuroendocrine cells, oligodendrocytes, and smooth muscle cells; B: Dot plot showing expression levels of selected canonical marker genes used for celltype identification; C: UMAP plot colored by tissue origin (nLN, nBrain, Lung Cancer, mBrain, and mLN); D: UMAP plot showing cell type distribution across the five tissue groups; E: Stacked bar plot showing cell proportion per tissue group; F and G: Bar plots highlighting changes in cell type composition between nBrain and mBrain (up) and between nLN and mLN (down). nLN: Normal lymph nodes; mLN: Lymph node metastases; mBrain: Brain metastases; nBrain: Normal brain tissues; ECs: Epithelial cells; SMC: Smooth muscle cells.

Figure 2 Different distribution of tumor epithelial subclusters in lymph node and brain metastases of non-small cell lung cancer. A: UMAP visualization of two epithelial subclusters and 14 tumor subclusters identified by subclustering analysis of 26587 epithelial/tumor cells derived from non-small cell lung cancer primary tumors (lung cancer), lymph node metastases (mLN), normal lymph nodes (nLN), brain metastases (mBrain), and normal brain tissues (nBrain). Each subcluster is annotated by its top marker genes; B: The UMAP shows the cell distribution of two epithelial subpopulations and 14 tumor subpopulations in the five tissue groups; C: Dot plot showing the expression levels of selected canonical marker genes used for cell type identification of the two epithelial subclusters and 14 tumor subclusters; D: Stacked bar plot showing the proportion of each tumor subcluster within different tissue types; E: UMAP plot showing the cell distribution of tumor and epithelial tissues in the five tissue groups; F: UMAP plot colored by tumor and epithelial tissues; G: UMAP plot colored by tissue origin (nLN, nBrain, Lung Cancer, mBrain, and mLN). nLN: Normal lymph nodes; mLN: Lymph node metastases; mBrain: Brain metastases; nBrain: Normal brain tissues.

Figure 3 Upregulation of unfolded protein response and associated metabolic pathways in brain and lymph node metastases of non-small cell lung cancer. A: Volcano plot showing differentially expressed genes (DEGs) between brain metastases (mBrain) and primary lung tumors. Red dots (UP) indicate significantly upregulated genes, blue dots (DOWN) indicate downregulated genes, and gray dots (NOT) represent non-significant changes; B: Gene Ontology (GO) enrichment analysis of DEGs in mBrain compared to primary tumors; C: KEGG pathway enrichment analysis of DEGs in mBrain compared to primary tumors; D: Volcano plot showing DEGs between mLN and primary lung tumors. Red dots (UP) indicate significantly upregulated genes, blue dots (DOWN) indicate downregulated genes, and gray dots (NOT) represent non-significant changes; E: GO enrichment analysis of DEGs in mLN compared to primary tumors; F: KEGG pathway enrichment analysis of DEGs in mLN compared to primary tumors.

Figure 4 T cell landscape in lymph node and brain metastases of non-small cell lung cancer. A: UMAP visualization of 59450 T cells extracted from non-small cell lung cancer (NSCLC) primary tumors, normal lymph nodes (nLN), lymph node metastases (mLN), normal brain tissues, and brain metastases (mBrain). Major immune lineages are annotated, including CD4⁺ T cells, CD8⁺ T cells, and γδT cells; B: Distribution of CD4⁺ T cells, CD8⁺ T cells, and γδT cells across the five tissue groups; C: UMAP plot of T cell subclusters (generated by subclustering of all T cells), identifying multiple CD4⁺ and CD8⁺ subsets as well as γδT cells; D: UMAP plot showing the cell distribution of the 11 T cell subclusters across the five tissue groups; E: Dot plot showing the expression levels of selected canonical marker genes used for cell type identification of the 11 T cell subclusters; F: Stacked bar plot showing the proportion of each cell subset among the five tissue sample groups; G: Representative graph showing the proportional distribution of T cell subclusters across different tissue samples. Adjusted P values for between-group comparisons are shown at the top; H and I: Dot plot showing the elevated expression of exhaustion markers, including PD-1 (PDCD1), TIM-3 (HAVCR2), CTLA4, and LAG3, T cells of metastatic sites (mBrain and mLN) compared to normal tissues. nLN: Normal lymph nodes; mLN: Lymph node metastases; mBrain: Brain metastases; nBrain: Normal brain tissues.

Figure 5 Myeloid cell landscape in brain and lymph node metastases of non-small cell lung cancer. A: UMAP visualization of 15916 myeloid lineage cells, including macrophages, monocytes, dendritic cells (DC), and neutrophils, derived from non-small cell lung cancer (NSCLC) primary tumors, normal tissues, and metastatic lesions [lymph node metastases (mLN) and brain metastases (mBrain)]; B: UMAP visualization of macrophages, monocytes, DCs, and neutrophils across the five tissue groups; C: UMAP plot of myeloid cell subclusters (generated by subclustering of all myeloid cells), identifying multiple macrophage, monocyte, DC, and neutrophil subsets; D: UMAP plot showing the cell distribution of the 14 myeloid cell subclusters across the five tissue groups; E: Dot plot showing the expression levels of selected canonical marker genes used for cell type identification of the 14 myeloid cell subclusters; F: Stacked bar plot showing the proportion of each cell subset among the five tissue groups; G: Representative graph showing the proportional distribution of myeloid cell subclusters across different tissue samples. Adjusted P values for between-group comparisons are shown at the top; H-I: Dot plot showing the expression levels of key immunosuppressive in myeloid cells of metastatic sites (mBrain and mLN) compared to normal tissues. nLN: Normal lymph nodes; mLN: Lymph node metastases; mBrain: Brain metastases; nBrain: Normal brain tissues.

Figure 6 Multiplex immunohistochemistry validation and prognostic value of tumor subpopulations. A: Multivariate Cox regression analysis of prognostic factors. Forest plot showing hazard ratio and P values for overall survival of lung adenocarcinoma patients from The Cancer Genome Atlas; B: Representative multiplex immunohistochemistry images showing the expression and spatial distribution of CD68⁺/CD163⁺, PD-1⁺/ CD8⁺, XBP1⁺/ATF6⁺/ATF4⁺ makers in Mets0 and Mets1 non-small cell lung cancer tissues. Scale bars: 50 μm; C: Comparison of positive cell density between Mets0 and Mets1 cancer regions; D: Comparison of positive cell rates between Mets0 and Mets1 cancer regions. Mets0 (n = 11) and Mets1 (n = 12); statistical significance was determined using Mann-Whitney U test; NS indicates not significant.