Methylcellulose

For mammosphere formation, single cell suspensions of HMLER^shCntrol and HMLER^shEcad cells were seeded at 1000 cells/cm² in six-well ultralow attachment plates (Corning Inc., New York, NY, USA). Mammosphere medium consisted of serum-free F12/DMEM containing 5 mg/mL insulin, 0.5 mg/mL hydrocortisone, 2% B27 supplement (Invitrogen Ltd., Carslbad, CA, USA), and 20 ng/mL epidermal growth factor (Sigma, Madrid, Spain). The medium was made semi-solid by the addition of 0.5% methylcellulose (R&D Systems, Minneapolis, MN, USA) to prevent cell aggregation. The tumorsphere-forming efficiency (TFE) was calculated after 7 days using the following equation: $$\mathrm{TFE}(\%)=\frac{\#\mathrm{of}\mathrm{tumorspheres}(\mathrm{large}\mathrm{diameter}>50\mathsf{\mu }\mathrm{m})\mathrm{per}\mathrm{well}}{\#\mathrm{of}\mathrm{cells}\mathrm{seeded}\mathrm{per}\mathrm{well}}\times 100$$

DCIS.com cells were plated at 250 per well in six‐well (Falcon F3046, Corning, NY) plates. After 7 days, plates were stained with 0.2% crystal violet dissolved in 20% methanol in PBS. Area was measured, and the percentage relative to number of cells plated was calculated.
For single-cell colony assays and single-cell mammosphere assays, cells were FACS sorted and a single living cell plated per well in a 96-well or ultra-low attachment plate respectively. After 14 days, plates were analysed in a Celigo cytometer (Nexcelom, Manchester, UK).
For mammosphere assays, 5000 DCIS cells/ml were plated in low‐attachment six‐well plates (Corning 3471) and incubated in medium supplemented with 2% NeuroCult SM1 without vitamin A (StemCell Technologies) and 0.65% methylcellulose (R and D Systems, Abingdon, UK). After 14 days, wells were scanned with a Celigo cytometer. Mammosphere‐forming efficiency was calculated by dividing the number of mammospheres by the number of cells plated per well.

The isolated tubular cells were cultured (2 × 10⁵ cells/well/500 µL) in methylcellulose (R&D Systems, Minneapolis, MN, USA) (42%) as a three-dimensional (3D) culture and in the presence of FCS (25%) and RPMI (33%) according to our previous studies [26 (link),27 (link),33 (link)]. Those cultures were growing in the absence (CT) or presence of recombinant IL-34 (recombinant mouse IL34, BioLegend; final dilution 1–10,000 pg/mL). The cells were incubated for four weeks in a CO₂ incubator at 37 °C. Every 10–14 days, 50 µL/well of fresh media containing the relevant concentration of IL-34 were added to the cell cultures. At the end of the incubation period in MCS, 0.5 mL of PBS was added to the culture wells that contained 0.5 mL MC, mixed by pipetting, and collected from the suspension in 15-mL tubes. The tubes were centrifuged at 1600 rpm for 10 min. Most of the volume was removed and the remainder of around 100 µL was collected from the bottom of the tubes. This volume, which contained the cells, was smeared on a slide for histological examination and/or collected and kept at −70 °C in a lysis solution to be used for RNA extraction.

U-CH1 cells were plated using standard protocols for tumour-sphere formation, at 1,500 cells/well in 24-well non-adherent plates (Nunc, Thermo Scientific) and grown for 10 days in serum-free DMEM/F12 (Life Technologies) supplemented with B27 (1∶50 dilution), bFGF (20 ng/mL), EGF (20 ng/mL) and 1% methylcellulose (R&D Systems). For rCCN2 experiments, the above media was supplemented with 100 ng/mL rCCN2. Cells were fed every other day and spheres (defined as ≥50 µm) were counted after 10 days. The efficiency of sphere formation (number of spheres per 1000 cells) was calculated, as previously described [30] (link).