Recombinant noggin

The colonic fibroblast cell line CCD-18co was purchased from ATCC (American Type Culture Collection, Manassas, VA). 18co cells were cultured in DMEM/F12 GlutaMax (Gibco, Thermo Fisher Scientific, Bleiswijk, The Netherlands). Cell growth medium was supplemented with penicillin (100 U/ml, Gibco), streptomycin (100 µg/ml, Gibco), and 10% FCS (Gibco) unless stated otherwise. Fibroblasts were seeded in six well plates and stimulated for up to 96 h with 100 ng recombinant BMP2 (R&D Systems, Abingdon, UK), 200 nM LDN-193189 (Merck), or 100 ng recombinant Noggin (Peprotech, Rocky Hill, NJ, USA) in DMEM/F12. Normal intestinal mouse organoids were stimulated with 50 ng recombinant CXCL12 (Biolegend, Uithoorn, The Netherlands) and monitored for 5 consecutive days.

Patient-derived CRC organoids were cultured in droplets of Matrigel and the medium was exchanged every 48–72 h. The culture medium (ENA) contained basal medium (advanced DMEM/F12 (Life Technologies) with 1% v/v penicillin/streptomycin solution (Lif`e Technologies), 1% v/v HEPES buffer (Life Technologies) and 1% v/v Glutamax (Life Technologies), 100 ng/ml recombinant noggin (Peprotech), 1× B27 (Life Technologies), 1.25 mM n-acetyl-cysteine (Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 50 ng/ml EGF (Peprotech), 500 nM A83–01 (Tocris), 10 nM gastrin (Peprotech), 10 nM prostaglandin E2 (Santa Cruz Biotechnology), 10 µM Y-27632 dihydrochloride (Sigma) and 100 mg ml⁻¹ Primocin (Invivogen).

Fresh GC PDX tumor tissues were cut into pieces, washed with ice-cold PBS, and then digested with EDTA. Following Matrigel polymerization for 10 min at 37 °C, advanced DMEM/F12 was supplemented with penicillin/streptomycin, 2 mM GlutaMAX, 10 mM HEPES, 1× B27, 1× N2 (Life Technologies), 1mM N-acetylcysteine (Sigma), and 10 nM gastrin I (Biogems). The following factors were used: 50 ng/mL recombinant EGF, 500 nM A83-01 (Biogems), 100 ng/ mL recombinant Noggin (Peprotech), 10 μM Y-27632 (Abmole), 500ng/mL R-spondin-1(Peprotech), 10 mM nicotinamide (Sigma), and 10 μM SB202190 (Sigma). When organoids were formed, we extracted original medium and used medium without NAC in the latter experiments. They were randomly separated into scrambled group and shACTL6A group via lentivirus transduction. For NAC or Fer-1 rescue group, organoids were treated with 100 μM NAC or 5 μM Fer-1. After treatment with shRNA lentivirus and NAC or Fer-1, we chose 8 PDOs at similar sizes to observe. These organoids were pictured every 3 days, and the sizes were measured with Image J. All samples were collected with the patients’ written informed consent.

Murine organoids were isolated and cultured as described by Sato et al.^{31 (link)}. In brief, murine intestinal and colon crypts were isolated with EDTA treatment of washed tissue samples. Developing organoids were cultured in droplets of Matrigel (Fisher Scientific) and the medium exchanged every 48–72 h. The medium for colon organoids contained 30% basal medium (advanced DMEM/F12 (Life Technologies) with 1% v/v penicillin/streptomycin solution (Life Technologies), 1% v/v HEPES buffer (Life Technologies) and 1% v/v Glutamax (Life Technologies), 50% Wnt3A conditioned medium, 20% R-spondin conditioned medium, 100 ng/ml recombinant noggin (Peprotech), 1x B27 (Life Technologies), 1.25 mM n-acetyl-cysteine (Sigma-Aldrich), 10 mM nicotinamide (Sigma-Aldrich), 50 ng/ml EGF (Peprotech), 500 nM A83–01 (Tocris), 3 μM SB202190 (Biomol), 10 µM Y-27632 dihydrochloride (Sigma) and 100 µg/ml primocin (Invivogen). For APC-KRAS colon organoid lines, the Wnt and R-spondin conditioned medium was replaced by basal medium. For the intestinal organoid medium, the Wnt conditioned medium was replaced by basal medium and A83–01 and SB202190 were removed.