Extended Data Fig. 1: Ageing suppresses lung tumorigenesis by degrading the stemness of the cell of origin.

(a) Survival of aged vs. young KP LUAD mice post-tumor initiation (n = 13 young and 7 aged mice) (Source Data). (b) Representative images of LUAD tumors in aged vs. young KPT (4 and 8 weeks) or KP (12 weeks) mice. At 4 and 8 weeks post-tumor initiation, LUAD cells were visualized by tdTomato immunohistochemistry. Scale bar: 200 µm. (c) Percentage of single tdTomato⁺ cells (singletons) per total number of tdTomato⁺ lesions at 2 weeks post-tumor initiation using adeno-SPC-Cre in aged vs. young KPT mice. N = 5 young and 3 aged mice. Representative images of early LUAD lesions, visualized by tdTomato fluorescence, in aged vs. young KPT mice is shown on the right. Arrowheads point to singletons. Scale bar: 50 µm. (d) Quantification of adeno-SPC-Cre transduction efficiency. Shown on the left is the total number of transduced AT2 cells, defined by co-expression of fluorescent reporter and the AT2 cell surface markers in Supplementary Fig. 2, and quantified by flow cytometry. Successful transduction is defined by the switch from tdTomato to GFP fluorescence in aged and young Rosa26^mTmG mice following intratracheal administration of adeno-SPC-Cre (n = 6 young and 5 aged mice). Shown on the right is the area density of tdTomato⁺ AT2 (SPC⁺) cells in tissue sections obtained from aged vs. young Rosa26^LSL-tdTomato mice 3 days after intratracheal administration of adeno-SPC-Cre (n = 4 mice per group). (e) Quantification of tumor burden in 3, 6, 9, 12, 18, and 24 months-old KP mice at 12 weeks following tumor initiation with adeno-SPC-Cre (n = 5, 6, 6, 5 and 5 mice per condition). (f) Schematic summary of the Eml4-Alk LUAD model (image representing chromosomal rearrangement is reproduced from Maddalo et al²⁶). Lungs of aged (104-130 weeks old) and young (12-16 weeks old) wild-type C57BL/6 mice were intratracheally transduced with adeno-sgEml4-sgAlk-Cas9 virus, which induces an Eml4-Alk gene fusion via an intra-chromosomal inversion in chromosome 17. (g-h) Tumor number (g, n = 19 young and 8 aged mice) and sizes (h, n = 231 and 184 tumors from young and aged mice, respectively) of Elm4-Alk fusion driven-LUAD in aged vs young C57BL6/J mice. (i) PCR assay used to detect Eml4-Alk inversion (top panel) and quantification of the inversion rate using the PCR assay in aged and young mice (bottom panel, n = 4 mice per condition). Note that the PCR primers specifically amplify the fusion gene. Raw gel image is shown in Supplementary Fig. 1. (j) Quantification of AT2 cells in aged and young mouse lungs. AT2 cells (red arrowheads) identified by surfactant protein-C (SPC) immunohistochemistry (n = 3 aged and 6 young mice). Scale bar; 50 µm. (k) FACS strategy for isolating mouse AT2 cells. AT2 cells are identified as DAPI^-/Lineage (CD45, CD11b, CD11c, F4/80 and Ter119)^-/EpCAM⁺/MHCII⁺ (AT2). Gating strategy of AT2 cells is shown in Supplementary Fig. 2. Validation of AT2 cell purity was performed by qPCR for the AT2 marker gene Sftpc. Note lack of detectable Sftpc expression in non-AT2 lineage-negative cells (EpCAM- and other EpCAM+) (n = 3 biological replicates). (l) Quantification of the percentage of AT2 cells in the total EpCAM⁺ epithelial cell pool in young (12-16 weeks old), middle-aged (52 weeks old), and aged (104-130 weeks old) wild-type C57BL/6 mice (n = 5 mice per condition). (m) Representative images of 6 young and 6 aged primary and 5 young and 4 aged secondary alveolar organoids. Scale bar: 100 µm. (n) Representative images (left) and quantification (right) of senescent cells identified by C₁₂RG fluorescence-based detection of senescence-associated β-galactosidase activity. Note that C₁₂RG positive senescent cells (arrowheads) do not express the AT2 cell marker SPC (n = 7 young and 6 aged mice). Scale bar: 50 µm. Y: young, A: aged. Mean with SD is shown in (c-e), (g), (i-l) and (n). Median and 25^th and 75^th percentiles are showed by dashed lines in (h). Log rank test was used in (a). Two-tailed Mann-Whitney test was used in (c), (i) and (j). Two-tailed Student’s t-test was used in (d), (g-h), (k) and (n). One-way ANOVA was used in (e) and (l).

Source Data