Extended Data Fig. 5: Flow cytometry, scRNA-seq data analyses of HPCS lineage tracing, and immunofluorescence imaging examining traced HPCS cells and progression markers.

a-b, Quantification of HPCS in lineage-traced cells after tracing (n = 4 mice per time point; Mann-Whitney U test). c-d, mScarlet⁺/EpCAM⁺ (HPCS/all cancer cells, c) and GFP⁺/EpCAM⁺ (Traced cells/all cancer cells, d) at 3 days (3 d) or 14 days (14 d) of lineage tracing (6 to 8 weeks PTI). n = 4 mice per time point. Mann-Whitney U test. e-f, mScarlet⁺/EpCAM⁺ (HPCS/all cancer cells, e) and GFP⁺/EpCAM⁺ (Traced cells/all cancer cells, f) at 3 days (3 d) or 14 days (14 d) of lineage tracing (12 to 14 week). n = 4 mice per time point. Mann-Whitney U test. g, Cell states in combined tracing experiments from Fig. 2a. scRNA-seq data from HPCS lineage-traced cells are plotted at the indicated timepoints. h, Stacked bar graphs showing the distribution of cell states across individual mice from HPCS tracing experiments shown as stacked bar graphs as in Fig. 2c, e. Error bars are SEM. i, Distribution of Nkx2-1, Sftpc, Hopx, and Hmga2 expression from traced LUAD cells at 8 weeks (6 to 8 week tracing, left) or 14 weeks (12 to 14 week tracing, right) PTI. j, Percentage of HPCS (red) or other cell states (green) expressing the indicated genes in traced LUAD cells at 8 weeks (6 to 8 week tracing, left) or 14 weeks (12 to 14 week tracing, right) PTI. Fisher’s exact test. k, IF images showing co-staining of GFP (green) with either NKX2.1 (left, red), SPC (middle, red), or HMGA2 (right, red). Boxed areas are shown as individual insets below their respective images. Scale bar: 20 µm. l, Percentage of NKX2.1⁺ (left), SPC⁺ (middle) or HMGA2⁺ (right) cells in GFP⁺ LUAD cells quantified from IF co-staining of LUAD tissues harvested at 14 weeks PTI after 3 (3 d) and 14 (14 d) days post-tracing. NKX2.1: n = 21 tumours, 3 mice (3 d) and n = 111 tumours, 3 mice (14 d). SPC: n = 59 tumours, 3 mice (3 d) and n = 89 tumours, 3 mice (14 d). HMGA2: n = 84 tumours, 3 mice (3 d) and n = 35 tumours, 3 mice (14 d). Mann-Whitney U test. Error bars are SEM.