293t cells

293T cells (DSMZ), GP2-293 cells (Takara Bio), and 293FT cells (Invitrogen) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine-penicillin-streptomycin solution (PSG; Sigma) at 37°C in a 5% CO₂ atmosphere. Jurkat cells (DSMZ), B-LCL cells (RIKEN Cell Bank), and NALM6 cells (ATCC) were cultured in RPMI-1640 (Sigma) supplemented with 10% FBS and 1% PSG at 37°C in a 5% CO₂ atmosphere.

Intestinal organoids were cultured in growth medium at 37 °C and 5% CO₂, as described above. In the first seven days after isolation and in the first five days after transduction, the concentration of the ROCK inhibitor Y-27632 (Tocris, Bristol, UK) was increased from 10 µM to 20 µM. Ten days after isolation, the colonoids were genetically modified via lentiviral transduction. The lentiviruses were produced via a third or second generation split packaging protocol in 293T cells (DSMZ, Braunschweig, Germany), as previously described [25 (link)]. One of the two transfer plasmids contained a sequence for the expression of a fusion protein from histone 2A and mCherry under the control of a CMV promotor. The other transfer plasmid was used to express a Wnt-sensitive promoter with seven TGP repeats that leads to fluorescence by EGFP synthesis [26 (link)]. 7TGP was a gift from Roel Nusse (Addgene plasmid #24305; http://n2t.net/addgene:24305 (accessed on 14 June 2021); RRID: Addgene_24305). For lentiviral transduction, 12 wells of 10-day-old colonoids from a 24-well plate were transduced with lentiviral particles produced from 4 × 10 cm dishes (5 × 10⁷ IU). The protocol for lentiviral transduction of intestinal organoids was described previously by van Lidth de Jeude [27 (link)].

Cells were maintained in a humidified atmosphere at 37 °C in 5% CO₂ in complete medium, consisting of DMEM (Sigma-Aldrich, MO, USA) or RPMI-1640 supplemented with 10% FBS, 1% penicillin/streptomycin (Sigma-Aldrich), and 1% L-glutamine (Sigma-Aldrich). 293T cells were purchased from DSMZ (Braunschweig, Germany). MDA-MB-231^Luc+ and NCI-H460 ^Luc+ cells, stably expressing luciferase, (onwards referred to as NTR^-) were kindly provided by Prof. James Lorens (University of Bergen). Generation of MDA-MB-231^Luc+GFP+NTR+ and NCI-H460^Luc+GFP+NTR+ cells, stably expressing luciferase, GFP and NfsB (onwards referred to as NTR⁺) has been previously described by McCormack et al. 20 (link). Characterisation of the cell lines by flow cytometry can be found in Figures S1 and S3. To avoid possible expression drift due to cell culturing, NTR⁺ cells were sorted prior to the experiments employing the flow cytometric methods described previously.