Mice
Previously published genetically engineered mouse strains were used in this study: KrasLSL-G12D/+ (ref. 19), Trp53flox/flox (ref. 62), KrasFSF-G12D/+ (ref. 21), Trp53frt/frt (ref. 26), Rosa26mTmG/+ (ref. 36); and Hipp11FSF-GGCB, Hipp11FSF-BG, Slc4a11FSF-MCD and HopxFSF-MACD reporters were generated in this study as described in detail below. All mice bearing autochthonous KP lung tumours were maintained in a C57BL/6 × Sv129 mixed background. NOD.Cg-Prkdcscid;Il2rgtm1Wjl/SzJ(NSG)63 mice (The Jackson Laboratory, 005557) were used as recipients in all allotransplant studies. All mice were monitored by the investigators and veterinary staff at the Research Animal Resource Center at Memorial Sloan Kettering Cancer Center (MSKCC) and housed under a 12 h–12 h light–dark cycle at 20–25 °C and 30–70% humidity with food and water provided ad libitum.
Autochthonous and transplantation models of lung cancer
Autochthonous LUAD tumours were induced in KrasLSL-G12D/+;Trp53flox/flox or KrasFSF-G12D/+;Trp53frt/frt (KPfrt) mice with 1 × 108–1 × 109 plaque-forming units (PFU) of AdSPC-Cre, AdSPC-FlpO (Iowa Viral Vector Core), or lentiviral FlpO at 3 × 105 or 6 × 105 transforming units, as previously described64, in mice that were aged between 8 and 12 weeks. Immunocompromised NSG mice were used as recipients for either subcutaneous, orthotopic or intravenous transplantation of KP LUAD cell line allografts. For subcutaneous transplantation, cells were resuspended in S-MEM (Gibco, 11380-037) and mixed with Matrigel (Thermo Fisher Scientific, CB-40230C) at a 1:1 ratio. Then, 250,000 cells were implanted subcutaneously into both flanks of NSG mice. For orthotopic transplantation, sorted cells were resuspended in PBS (Gibco, 10010-023) and intratracheally administrated to NSG mice. For intravenous transplantation, 200,000 cells were resuspended in S-MEM and injected into NSG mice through the tail vein. All cell lines were continuously monitored for mycoplasma contamination. Approximately equal numbers of male and female mice were included in all experimental groups in all mouse experiments. Mice were treated in accordance with all relevant institutional and national guidelines and regulations, and mice were euthanized by CO 2 asphyxiation, followed by intracardiac perfusion with S-MEM to clear tissues of blood when appropriate. A complete list of mice along with age, sex and age of tumour used in experiments is available (Supplementary Table 4). All animal studies were approved by the MSKCC Institutional Animal Care and Use Committee (protocol 17-11-008). Sample sizes were determined based on our previous experience with similar models rather than statistical methods. We found this sufficient to detect biologically meaningful differences while minimizing animal use; experiments were randomized when feasible. Blinding was not possible as treatment effects on tumour volume were readily distinguishable between groups. Tumour burden limit was defined as a single tumour >2 cm in diameter, tumour volume >10% of body mass or multiple tumours with a cumulative volume >3,000 mm3. These limits were not exceeded in any of our experiments.
Generation of donor vectors for embryonic stem cell targeting
For the generation of the Slc4a11-FSF-MCD donor vector, homology arms of around 1,200 bp in length 5′ and 3′ to the end of Slc4a11 exon 21 (Extended Data Fig. 1b) were amplified from genomic DNA of C57BL/6 mES cells using high-fidelity PCR (NEB, M0494). A homology-directed repair template donor vector was constructed by flanking the frt-bGlobinpA-(PGK-Hygromycin-pA)i-frt-P2A-mScarlet-T2A-CreERT2-P2A-DTR-WPRE-bGHpA cassette with the 5′ and 3′ homology arms and cloned into the pUC19 plasmid backbone (Takara Bio, 638949) using Gibson assembly (NEB, E2611).
For the generation of the Hipp11-FSF-GGCB donor vector, homology arms of around 5,000 bp in length 5′ and 3′ to the safe harbour of Hipp11 intergenic region (positioned between the Eif4enif1 and Drg1 genes; Extended Data Fig. 1e) were amplified from genomic DNA of C57BL/6 mES cells using high-fidelity PCR. A homology-directed repair template donor vector was constructed by flanking the CAG-loxP-frt-Neomycin-PGKpA-SV40pA-frt-G-Luc-P2A-meGFP-bGlobinpA-loxP-C-Luc-E2A-TagBFP-3xFlag-WPRE-bGHpA (GGCB) cassette with the 5′ and 3′ homology arms and cloned into the pUC19 plasmid backbone using Gibson assembly.
For the generation of Hopx-FSF-MACD donor vector, homology arms of around 1,500 bp in length 5′ and 3′ to the end of Hopx exon 3 (Extended Data Fig. 6a) were amplified from genomic DNA of C57BL/6 mES cells using high-fidelity PCR. A homology-directed repair template donor vector was constructed by flanking the frt-bGlobinpA-(PGK-Hygromycin-pA)i-frt-P2A-mScarlet-AkaLuc-T2A-CreERT2-P2A-DTR-WPRE-bGHpA cassette with the 5′ and 3′ homology arms and cloned into the pUC19 plasmid backbone using Gibson assembly.
Validation of the Hipp11 GGCBreporter
To validate the functionality of the GGCB cassette, we performed ex vivo transformation of AT2 cells isolated from a KPfrt;Hipp11FSF-GGCB/+ chimeric mouse using lentiviral vectors encoding either codon-optimized Flp recombinase (flpO) alone or flpO linked to creERT2. In these experiments, FlpO activates oncogenic KRAS(G12D), deletes Trp53, and initiates expression of the GG cassette, whereas subsequent activation of CreERT2 with 4-hydroxytamoxifen (4-OHT) results in a switch from GG to CB (Extended Data Fig. 1h–k). G-Luc activity was increased 13 days after transformation in all conditions (Extended Data Fig. 1i (top)), whereas C-Luc activity was observed only after 4-OHT stimulation in organoids transduced with the vector encoding both flpO and creERT2 (Extended Data Fig. 1i (bottom)). Moreover, we performed flow cytometry and fluorescence imaging analyses on organoids under these four conditions. We found that eGFP was expressed at the baseline following flpO and a switch to TagBFP was observed only after 4-OHT exposure in the organoids transduced with both flpO and creERT2 (Extended Data Fig. 1j,k). Similar results were obtained in subcutaneous transplants (Extended Data Fig. 2a–c) and autochthonous lung tumours (Extended Data Fig. 2d–g) in vivo, both by detection of G-Luc and C-Luc from repeated blood samples and by fluorescence imaging of tumours at the end point.
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