Extended Data Fig. 8: Histopathological and flow cytometry analyses of HPCS ablation in early neoplasias and in adenocarcinomas.

a, Number of tumours normalized by lung area in saline vs. DT treated groups. n = 10 lung areas, 3 mice per group. Mann-Whitney U test. b, Tumour burden (total tumour area / total lung area) in saline (6 mice) vs. DT (8 mice) treated groups. Mann-Whitney U test. c, Representative H&E stained lung sections showing tumour boundaries (dashed lines) in saline control vs. HPCS-ablated (DT) mice. Scale bar: 100 µm (low magnification) and 40 µm (high magnification). d, Distribution of tumour numbers across tumour grades in saline vs. DT treated groups. n = 6 mice (saline) and 8 mice (DT). Two-way ANOVA with Šídák’s multiple comparisons test. e, HNF4α (red) and DNA (blue) in lung sections in saline control vs. HPCS-ablated (DT) mice. Scale bar: 100 µm. f, Predicted end cell states (EMT, Proximal ciliated-like) and the HPCS projected on the LUAD progression PHATE⁸² map from Marjanovic et.⁴. g, Hnf4a expression. h, Gene expression trajectories for Hnf4a plotted in Palantir⁸⁰ pseudotime space, with estimated time spent in the HPCS shaded in red. i-j, Experimental design to test the effect of ablating Slc4a11⁺ HPCS on tumour growth in autochthonous KPfrt; Hipp11^GGCB/+; Slc4a11^MCD/+ LUAD-bearing mice as determined by longitudinal G-Luc activity monitoring. Experimental schema of LUAD bearing mice 14 weeks PTI were administered saline or DT (50 μg/kg, daily) and analysed at 7 days as indicated. k, G-Luc activity (secreted by GG⁺ tumour cells) shown as fold change at indicated time points normalized to day 0 post-DT treatment. n = 3 mice per group. Welch’s t test. l, Representative flow cytometry plot of saline- and DT-treated (7 day) tumours. Shaded regions indicate gated populations analysed. m, Total tumour burden (GFP⁺ cells, left) or HPCS (mScarlet⁺/GFP⁺ cells, right) as a percentage of live, non-immune cells [CD45⁻/CD31⁻/CD11b⁻/CD11c⁻/F480⁻/TER-119⁻/Helix NP NIR⁻(Live)⁺]. n = 5 (Saline) and 7 (DT) mice. Mann-Whitney U test. n, Heatmap of scaled mean gene expression for DTR (Slc4a11^MCD/+ allele) and native Slc4a11 in saline vs. HPCS-ablated (DT) GFP⁺ cancer cells. Error bars are SEM. The diagrams in i and j were created using BioRender. Tammela, T. (2025) https://BioRender.com/0lgfrw5.