Extended Data Fig. 1: Construction of the Slc4a11^FSF-MCD/+ and Hipp11^FSF-GGCB/+ reporter alleles and validation of the Hipp11^FSF-GGCB/+ allele in vitro.

a, Slc4a11 mRNA in KP LUAD at 20 weeks post-tumour initiation (PTI). Scale bar: 50 µm. b, Components of the Slc4a11^FSF-MCD/+ reporter and targeting strategy by CRISPR/Cas9 mediated homology dependent repair (HDR). To restrict the expression of the MCD cassette to Slc4a11⁺ cancer cells, we introduced a frt-stop-frt (FSF) cassette²¹ upstream of the MCD. Upon removal of the FSF cassette, MCD is linked to the last coding exon of Slc4a11 by a 2 A peptide sequence⁸². This approach preserves activity of the endogenous gene and links the MCD reporter tightly to native gene-regulatory elements, increasing the fidelity of the reporter. The strategy for generating genetically engineered Slc4a11^MCD/+ knock-in (KI) reporter enabling lineage tracing and ablation of Slc4a11⁺ HPCS in KP LUAD was to knock in the frt-stop-frt-P2A-mScarlet-T2A-CreERT2-P2A-DTR-WPRE (FSF-MCD) reporter construct in frame into the stop codon of exon 21 of Slc4a11 gene in the presence of CRISPR/Cas9 gene editing. c, Experimental outline to generate Kras^FSF-G12D/+; Trp53^frt/frt; Slc4a11^FSF-MCD/+ reporter mouse. The FSF-MCD reporter construct was knocked into a mouse embryonic stem cell (mESC) harbouring the KRAS(G12D) (Kras^{frt-stop-frt(FSF)-G12D/+})²¹ and p53 loss-of-function (Trp53^frt/frt)²⁶ alterations (KPfrt) and the correctly targeted mESC clones were microinjected into 8-cell stage embryos to obtain mESC-derived chimeras. d, Gel electrophoresis of the PCR products from a correctly targeted mESC clone (pos ctrl), parental KPfrt mESC, or mESC-derived chimeras using primer pairs detecting either wild-type (WT, 464 bp, top) or knock-in (KI, 259 bp, bottom) alleles, respectively. e, Vector map of Hipp11^FSF-GGCB/+ targeting vector and strategy for the generation of Hipp11^FSF-GGCB/+ reporter by CRISPR/Cas9 mediated dependent HDR. CAG-LoxP-frt-stop-frt-G-Luc-P2A-EGFP-pA-LoxP-C-Luc-E2A-TagBFP-pA-WPRE (FSF-GGCB) reporter construct was knocked into the Hipp11 intergenic region safe harbour (positioned between Eif4enif1 and Drg1 genes) by CRISPR/Cas9 gene editing. FlpO-mediated recombination removes the FSF cassette and activates the G-Luc-P2A-EGFP (GG) element, whereas Cre-mediated recombination removes the loxP-stop-loxP (LSL) cassette and activates the C-Luc-E2A-TagBFP (CB) element. G-Luc: Gaussia luciferase; C-Luc: Cypridia luciferase. f, Experimental outline to generate the Kras^FSF-G12D/+; Trp53^frt/frt; Hipp11^FSF-GGCB/+ reporter mouse. CAG-FSF-GGCB reporter construct described in panel (e) was knocked into KPfrt mESCs, the correctly targeted mESC clones were microinjected into 8-cell stage embryos to obtain mESC-derived chimeras. g, Gel electrophoresis of the PCR products from either a parental mESC (KPfrt mESC), a correctly targeted mESC clone (4D12), or mESC-derived chimeras using primer pairs detecting either wild-type (WT, 773 bp, top) or knock-in (KI, 545 bp, bottom) alleles. h, Experimental outline to validate the Hipp11^FSF-GGCB reporter in an alveolar type 2 (AT2) cell ex vivo transformation assay. AT2 cells isolated from the chimeras were transduced with lentivirus expressing either FlpO or FlpO-P2A-CreERT2 to remove the FSF cassette and activate oncogenic Kras^G12D/+ and delete Trp53, resulting in the generation of Kras^G12D/+ mutant and Trp53 deficient LUAD organoids. The transformed organoids were exposed to 4-OHT and analysed with the indicated approaches. i, Longitudinal monitoring of G-Luc (top) and C-Luc (bottom) activities depicted as fold change over day 0 from ex vivo transformed LUAD organoids as in (h). Organoids were exposed to 4-OHT (1 µM) at day 10. Media was refreshed every 3 days before supernatant was collected for luciferase activity measurement. n = 6 for each group. Error bars are SEM. Two-way ANOVA with Dunnett's multiple comparison test. j, Flow cytometry analysis of EGFP vs. TagBFP expression from PGK-FlpO (left) or PGK-FlpO-P2A-CreERT2 (right) transformed organoids 6 days after treatment with either vehicle control or 4-OHT. TagBFP is only induced in organoids harbouring CreER^T2 and exposed to 4-OHT. k, Fluorescent images of EGFP and TagBFP from LUAD organoids transformed with indicated lentivirus 6 days after treatment with either vehicle control or 4-OHT. Consistent with (j), TagBFP is only present in organoids harbouring CreER^T2 and exposed to 4-OHT. Scale bar: 200 µm. The diagrams in c, f and h were created using BioRender. Tammela, T. (2025) https://BioRender.com/0lgfrw5.