Psc833

The APC analogs CLR1404, CLR1501 and CLR1502 were provided by Cellectar Biosciences, Inc. (Madison, WI, USA). These APC analogs consist of a cancer-targeting alkylphosphocholine scaffold with an attached cancer imaging, visualization or therapeutic moiety (refs ^{17 , 18 (link), 25 (link), 28 (link)} and Figure 1): iodine isotopes for CLR1404; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (i.e. BODIPY, green fluorescence) for CLR1501; and 2-[2-[2-Chloro-3-[2-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)-ethylidene]-1-cyclohexen-1-yl]-ethenyl]-1,3,3-trimethyl-3H-indolium chloride (i.e. IR-775, near infrared fluorescence) for CLR1502. Pharmacological cellular efflux transporter inhibitors were purchased: PSC833 and Ko143 from Tocris Biosciences, and MK571 from Sigma-Aldrich (St. Louis, MO, USA). [¹⁴C]-sucrose and [³H]-diazepam were purchased from American Radiolabeled Chemicals (St Louis, MO, USA).

Tubuloids were disrupted using a P200 pipette and cultured overnight with 5 μM P-gp inhibitor PSC-833(4042, Tocris) in organoid maintenance medium or 0.2% DMSO in organoid maintenance medium as control. After washing in Hank’s buffer (14025050,Gibco) tubuloids were treated with 1 μM calcein-AM (C1430, Invitrogen) in Hank’s buffer supplemented with 5 μM P-gp inhibitor or 0.2% DMSO for 1 hour at 37 °C. After washing tubuloids were fixed in 4% PFA, and counterstained by 1 μg/ml DAPI for 15 minutes. P-pg transport image analyses were performed on a LSM 710 (Zeiss). For full details see Supplementary Methods.