Extended Data Fig. 2: Ageing suppresses the proliferation of LUAD cells and delays LUAD progression.

(a) Airspace size of aged and young mice before and after hyperoxia-induced lung injury measured by mean cord length (MCL) measurement (n = 4 mice per group). Shown on the right are representative images of alveoli from young and aged animals pre- and post-hyperoxia injury (28 days). Scale bar: 50 µm. (b) Transformation efficiency of aged and young KP-Cas9 AT2 cells, calculated as the ratio of GFP⁺ transformed organoids/non-transformed alveolar organoids from the same animal (n = 8 biological replicates). (c) Quantification of lentiviral transduction efficiency of aged and young AT2 cells in the ex vivo transformation assay. AT2 cells were isolated from both aged and young KP mice and transduced with lenti-PGK-GFP. Transduction efficiency was defined as the percentage of GFP⁺ AT2 cells over total number of AT2 cells (n = 3 biological replicates). (d) Quantification of lentiviral transduction efficiency of aged and young AT2 cells in vivo. Aged and young Rosa26^mTmG mice were transduced by Lenti-PGK-Cre and transduction efficiency was defined by the total number of GFP⁺ AT2 cells (GFP⁺/MHCII⁺/EpCAM⁺/lineage^-/DAPI^-), where Cre recombinase converts the tdTomato fluorescence to GFP fluorescence (n = 3 mice). (e) Quantification of KP LUAD tumor size in aged vs. young mice at 4, 8, 12, and 17 weeks post-tumor initiation. Number of cancer cells per tumor nodule was quantified in KPT mice using tdTomato to visualize cancer cells at the 4- and 8-week time points; tumor size was quantified in KP mice from HE stained sections for at the 12- and 17-week time points. N = 109, 60, 371, and 358 tumors from young mice and n = 95, 66, 121, and 144 tumors from aged mice at 4, 8, 12, and 17 weeks. (f) Quantification of proliferating LUAD cells at different stages of tumor development. The proportion of Ki67 positive cells of total lung cancer cells (identified by endogenous reporter alleles or SPC immunofluorescence) was calculated and normalized to the mean of young tumors at the corresponding time point. N = 55, 62, 127, and 306 tumors from young mice and n = 32, 62, 25, and 237 tumors from aged mice at 4, 8, 12, and 17 weeks. (g-i) Quantification of senescent cancer cells in young and aged KP LUAD tumors at 12 weeks post-tumor initiation. The senescent cells were identified by p16 (g-h) (n = 8 young and 6 aged mice) or C₁₂RG staining (i) (n = 11 and 9 tumors from young and aged KP tumor-bearing mice, respectively). Representative images of p16 staining are shown in (g). Scale bar: 50 µm. (j) Quantification of cleaved caspase-3 (c-Casp3) positive LUAD tumors, defined as tumors with ≥ 1 c-Casp3⁺ cell (n = 4 mice). (k-l) Histopathological grading of KP LUAD tumors in aged vs. young mice at 12 and 17 weeks post-tumor initiation. Representative images of the grading by an automated deep neural network (Aiforia Technologies) are shown (k). Proportion of G1-G4 tumor area per total tumor area. Proportion in each mouse was calculated individually and mean is shown in (l, n = 10 young and 5 aged mice for 12 week time point and n = 10 young and 10 aged mice for 17 week time point, respectively). Scale bar: 1 mm. Y: young, A: aged. Mean with SEM is shown in (e) and mean with SD is shown in (a-c) and (h-j). Median and 25^th and 75^th percentiles are shown by dashed lines in (f). One-way ANOVA was used in (a). Two-tailed Student’s t test was used in (b), (e-f), (h-j) and (l). Two-tailed Mann-Whitney test was used in (c-d).

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