Mouse organoid cultures

Wt organoid medium: The medium for wt, KrasG12D/+, KrasG12D/+:p53R270H/+ organoids contained 50% of L-WRN (CRL-3276™, ATCC) conditioned medium, 20% of fetal bovine serum (Biosera), penicillin/streptomycin (ThermoFisher, 1/100), GlutaMax (ThermoFisher, 1/100), Y-27632 (final 10 nM), A83-01 (final 5 nM), SB203580 (final 10 μM), Gastrin (final 10 nM), N-Acetyl-L Cysteine (final 1 μM) and CHIR9902 (final 5 nM) in advanced DMEM-F12.

mTNFα (final 40 ng/ml, Wako), mIL6 (final 100 ng/ml, Wako), mIL-1β (final 10 ng/ml, Wako) and mIFNγ (final 1 ng/ml, Wako) were added to the organoid culture medium.

To activate CreERT2-mediated recombination, we treated organoids with 1 μM 4-hydroxytamoxifen (SIGMA) dissolved in the wt organoid medium for 6 days. The organoid genome was purified by LaboPass Blood Mini (COSMO GENENTECH), and recombination efficiency was determined by a standard PCR protocol.

To knock out genes by CRISPR-Cas9 in wt or KrasG12D/+ organoids, we cloned gRNA into plentiCRISPR_v2 (Addgene, #52961) and generated lentivirus. Sequences for gRNAs were described in Supplementary Table ³. The lentivirus dissolved in Transdux and Enhancer (System Bioscience) was mixed with organoids and placed onto the Matrigel which was polymerized in wells in advance. To confirm whether mutations were introduced by CRISPR-Cas9, we PCR amplified the loci, cloned into pCR™4-TOPO® TA vector (ThermoFisher #450030), and performed Sanger sequencing by M13F or M13R universal primers.

The medium for mouse tumor organoids contained 10 mM HEPES, Penicillin/Streptomycin, N2 supplement (1/100, ThermoFisher), B27 supplement (1/50, ThermoFisher), 50 ng/ml of mEGF (Wako), 5 nM of A83-01, 1 mM of N-Acetyl-L Cysteine in Advanced DMEM-F12 (ThermoFisher), in addition, Y-27632 (final 10 nM) and CHIR9902 (final 5 nM) are added at the time of passage. To pass organoids, TrypLE (ThermoFisher) or Cell recovery solution (Corning) was used, following the manufacturer’s protocol. AK organoids were obtained from Dr. Oshima⁵³. The establishment of AK-Cas9 organoids was described in our previous report.

To generate knockout in AK organoids, gRNAs⁵⁴ were cloned into the BbsI-digested pKLV2-U6gRNA5(Bbsl)-PGKpuro2ABFP-W (Addgene, #67974) lenthi-vector. Lentivirus was generated by transfection of plasmids (pLP1, pLP2, pVSVG, and gRNA-cloned lentivector) into 293FT. Lentiviruses were concentrated by Lenti-X™ Concentrator (TAKARA) and then transduced into AK-Cas9 organoids to knockout genes. Organoids carrying gRNAs were selected by puromycin. Sequences for gRNA were described in the Supplementary Table ³.