Endoxifen

Fetal bovine serum and RPMI 1640 medium were obtained from Gibco (Grand Island, NE, USA). Modified Lowry protein assay kits were procured from Pierce (Rockford, IL, USA). Tamoxifen, 4-hydroxyTamoxifen, endoxifen, phenacetin, paracetamol, dextromethorphan, and dextrorphan were purchased from Sigma (St. Louis, MO, USA). Midazolam and 1-hydroxymidazolam were obtained from Gentest (Woburn, MA, USA). HPLC-grade formic acid and acetonitrile were obtained from Fisher (Dayton, OH, USA). Deionized water was purified with a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals, unless otherwise indicated, were procured from Sigma-Aldrich (St. Louis, MO, USA).

CD44-Cre/Cdh1^fl/fl/tdTomato and CD44-Cre/tdTomato organoids were cultured for 24 h, then Cre recombinase activity was induced with 1 µM endoxifen (Sigma-Aldrich, St Louis, MO, USA). After an additional four days of culture, organoids were passaged and resuspended in 1 mL of filter-sterilised fluorescence-activated cell sorting buffer comprising 2 mM EDTA and 1% fetal bovine serum (Scharlau, Barcelona, Spain) in PBS, pH-adjusted to 7.2. A total of 12.5 µL of Matrigel (Corning, Corning, NY, USA) was dispensed into each well of a 96-well, black-walled, clear-bottom tissue culture plate (Corning, Corning, NY, USA) on ice. Fluorescence-activated cell sorting was performed on a BD FACSAria™ Fusion Cell Sorter (BD Biosciences, San Jose, CA, USA) to sort and dispense 20 individual tdTomato-positive cells into each well of the 96-well plate. Organoids were cultured from single cells for a period of 11 days and monitored via brightfield microscopy on a Nikon Eclipse Ti inverted microscope (Nikon, New York City, NY, USA), with images captured by a DS-QiMc camera (Nikon, New York City, NY, USA).

The kinase inhibitors dasatinib (#AOB87355), imatinib mesylate (#AOB6752), nilotinib (#AOB87155) and ponatinib (#AOB87302), were purchased from AOBious (Gloucester, MA, USA) and the powders reconstituted in sterile DMSO, aliquoted and stored at −20 °C until use. Endoxifen (E8285) was purchased from Sigma. Information regarding the components utilised for organoid media are detailed in Table S1.

All SERMs were obtained from the following commercial sources. tamoxifen and Y-134 were purchased from Tocris Bioscience (Bristol, United Kingdom). N-desmethyl tamoxifen, 4-hydroxy tamoxifen, endoxifen, SO₄-tamoxifen, toremifene, 4-hydroxy toremifene, OSP, RAL, lasofoxifene, NAF, and BAZ were all obtained from Sigma Aldrich (St. Louis, MO, USA). Acolbifene was procured from AdooQ Bioscience (Irvine, CA, USA).
AM-630, AM-251, DAMGO, and WIN-55,212-2 were purchased from Tocris Bioscience. CP-55,940 was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). [³⁵S]GTPγS (1250 Ci/mmol) was procured from American Radiolabeled Chemicals (St. Louis, MO, USA) and [³H]CP-55,940 (131.4 Ci/mmol) was purchased from PerkinElmer (Waltham, MA, USA).
All other reagents were purchased from Fisher Scientific Inc. (Pittsburgh, PA, USA). All compounds were dissolved in 100% DMSO to produce a stock concentration of 10 mM.

For PC ablation experiments in larvae (4–6 days post-fertilization), 4-OHT (cis/trans-4-hydroxy-tamoxifen, Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 8 µM was added to the rearing medium for 18–20 hr. Since EtOH was used as solvent for 4-OHT, 0.4% EtOH was applied to the rearing medium of a control group for the same time period. On the next day, the medium was exchanged three times and the ablation rate of PCs was analyzed under a confocal laser scanning microscope (LSM). Ordinary ablation rates higher than 85–90% at day 2 post-treatment (dpt) compared to the control group were considered successful to use these larvae for further experiments.
To deplete PCs in adults, 5 months old zebrafish were incubated overnight in fish facility water supplemented with 4 µM Endoxifen (Sigma-Aldrich, St. Louis, MO, USA), an active metabolite of 4-OHT, which proved more efficient in adult PC ablations. Three consecutive treatments were performed each overnight at 28°C in the dark with resting and feeding periods during the day. Since dimethyl sulfoxide (DMSO) is used as solvent for Endoxifen, control groups were treated with 0.13% DMSO. Afterwards, the chemicals were washed out at least three times with fresh fish facility water and specimens were returned to the fish facility.