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Experimental proceduresOrganoid culture and maintenanceThepatient-derived organoids used during this research were previously characterisedand established 21.

Colorectalcancer organoids were cultured according to these guidelines. P18T organoidswere cultured in small drops of Basement Membrane Extract (BME, Amsbio) andprovided with Colorectal Cancer medium (CRC medium). CRC medium consists of +++medium: Advanced Dulbecco’s Modified Eagle Medium/Ham’s F-12 (AdvancedDMEM/F12, Invitrogen) supplied with 1% Penicillin/Streptomycin (Pen/Strep, Lonza),1% Glutamax (Invitrogen) and 1% Hepes buffer (Invitrogen).In addition to the +++ medium, 10% R-spondin conditioned medium, 10% Nogginconditioned medium, 1x B27 (Invitrogen), 10 mM Nicotinamide (Sigma-Aldrich),1.25 mM N-Acetyl Cysteine (Sigma-Aldrich), 10 mlSB202190 (ApexBio), 500 nM A83-01 (Tocris), 50 ng/mLEGF (Invitrogen) and – to recently splitted cultures – 10 mM Y-27632 dihydrochloride, were added tocreate CRC medium.

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Mediaof the organoid lines were refreshed every other day. Organoids were splittedby shearing the BME drops and using Trypsin/EDTA (Sigma-Aldrich).Flipase and FACS-sortingPuromycin cassettes were deleted usingflipase-GTP. Organoids were trypsinised into single cells, before FACS-sortingthe GFP positive cells.

To ascertain all puromycin cassettes were flipped out,the sorted organoids were treated with 100 mM ganciclovir, which leads to toxicity in cellsstill containing the DTK promoter. PCRproducts of the KRAS exon 1 in surviving cells were sequenced, using wt KRASprimers.RNA isolation, preparation of cDNA and qRT-PCROrganoids were harvested incold PBS (Lonza) and resuspended in RLT buffer. RNA was isolated using theQiagen RNeasy kit.25qRT-PCR was performed using IQ CYBR green mix (Bio-Rad) 25. The??Ct method was used to calculate results. All lines were grown on both CRCmedium and CRC medium + 200 nM afatinib. Primers are shown in Table 2.

MicroscopyOrganoids were trypsinisedand grown on either CRC medium or CRC medium + 200 nM afatinib. Five days aftertrypsinisation, organoids were plated in BME on a NuncTM Lab-TekTM II ChamberedCoverglass (ThermoFischer). 1 µM afatinib was added to some of the samples. Theorganoids were imaged on a Leica SP8X. Organoids were imaged on day 1, 3 and 7.The amount of live and dead cells was shown by adding respectively Hoechst andDRAQ7 (Cell signalling) 90 minutes before imaging on day 7.Western BlotSamples were lysed using RIPA buffer (50 mMTRIS-HCl pH 8.0, 150 mM NaCl, 0,1% SDS, 0,5% Na-Deoxycholate, 1% NP-40(IGEPAL), 1 µM Leupeptin, 0,1 µM Aprotinin and Sodium Orthovanadate).

Theprotein content of each sample was determined using standard Bradford assay(Roche). Equal amounts of protein were loaded on 10% SDS-page gels andtransferred to PVDF membranes (Millipore). Membranes were blocked and probedfor pERK ½ (Cell Signaling Technology), tERK (Cell Signaling Technology) andGAPDH (Millipore).

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