Fig. 1: Study design and scRNA-seq analysis of aged lung immune microenvironment.

a Representative images and quantitative results of lung metastases in young and aged mice. Each mouse was injected with 5 × 10⁵ B16F10 cells via the tail vein. Each group contains six mice. Young mice are 2 months old, aged mice are 20 months old. Data expressed as mean ± SD. P = 5.0E−05. Significance was determined using unpaired two-tailed Student’s t test. ****P < 0.0001. YM young model, AM aged model. Scale bars: 200 mm. b Representative images and quantitative results of liver metastases (arrow) after intraocular tumor establishment in young and aged mice. Each mouse was injected with 2.5 × 10⁵ B16F10 cells via subretina. Young mice are 2 months old, aged mice are 20 months old. Each group contains eight mice. Data expressed as mean ± SD. P = 1.7E−04. Significance was determined using an unpaired two-tailed Student’s t test. ***P < 0.001. Scale bars: 100 um, 200 um. c Schematic of the experimental design for scRNA-seq. YM and AM groups were respectively injected with B16F10 cells via the tail vein at day 0, and then the lung immune cells of YC, AC, YM, and AM groups were sorted for scRNA-seq at day 21. YC young control, AC aged control. Each image was drawn by PaintTool SAI (version 1.2) and Adobe illustrator 2023 (version 27.0). d Uniform manifold approximation and projection (UMAP) plot showing the cluster of cell types, pie charts showing the percentages of the immune cells in AC and YC groups. MoMøDC macrophages, monocytes and dendritic cells, NK natural killer cells. e Volcano plot showing upregulated and downregulated DEGs of all immune cells (as a whole) between AC and YC mice. Significance was determined using “FindMarkers” functions of Seurat package with Wilcoxon Rank Sum test and adjusted by Bonferroni correction. Representative GO terms and KEGG pathways enriched in upregulated DEGs (f) and downregulated DEGs (g) of all immune cells (as a whole) in the AC/YC comparison. Significance was calculated based on the accumulative hypergeometric distribution by Metascape webtool. h Kernel density estimation plot showing the shift of senescence-associated secretory phenotype (SASP) gene set score with age in total immune cells from mouse lung, calculated based on gene expression. The x-axis shows the range of scores, the y-axis indicates the calculated density, representing the likelihood of data falling within each range on the x-axis SASP: Senescence-associated secretory phenotype. i MFI of SA-β-Gal in all immune cells and neutrophils in lungs from YC/AC groups, which were measured by flow cytometry, calculated on CD45+ cells or CD45+CD11B+LY6G+ cells. Each group contains six mice. Data expressed as MFI of SA-β-Gal (mean ± SD). All immune cell (P = 1.2E−03), neutrophil (P = 2.0E−05). Significance was determined using unpaired two-tailed Student’s t test. SA-β-Gal senescence-associated β-galactosidase, MFI mean fluorescence intensity. ****P < 0.0001, **P < 0.01. Source data are provided as a Source Data file.