Rank fc

All cell lines used in this study were previously isolated from female BC patients, and include: MCF-7GFP+Luc+, herein designated by MCF-7, and T47D (provided by Dr. Phyllippe Clézardin, INSERM, Lyon, France). Their derivatives MCF-7 RANK OE (MCF-7^OE) and T47D RANK OE (T47D^OE) were previously generated by our group.²⁰ (link) MCF-7 and T47D parental or derived cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 1% (v/v) Penicillin/Streptomycin (Pen/Strep, 10,000 U/mL Penicillin, 10,000 μg/mL Streptomycin, Gibco), and 0.01 mg/mL insulin (Gibco). PalbR cells were derived from MCF-7 or T47D cells by continuous exposure to increasing concentrations of palbociclib (10 nM - 2.5 μM), with or without 100 ng/mL OPG-Fc (Amgen, Inc.) or 100 ng/mL RANK-Fc (R&D Systems) for 3 months. Cells were maintained at 37°C with 5% CO₂, used at low passage number, and regularly tested for Mycoplasma contamination by qPCR (Eurofins Genomics). In all studies related to acquired resistance, parental cells were cultured in parallel, for the same period of time under the indicated experimental conditions (untreated, Palbo-treated or Palbo+RANKLi-treated).

To measure cell proliferation, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and 1:10 (v/v) Alamar blue (Invitrogen) was added to each well. Fluorescence (excitation 560nm; emission 590nm) was measured 2h after adding Alamar blue and at indicated time points in an Infinite M200 microplate reader (Tecan). To quantify cell viability, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well, with or without fulvestrant (Selleck Chemicals), palbociclib (Sigma-Aldrich), ribociclib (Santa Cruz Biotechnology), abemaciclib (Selleck Chemicals), OPG-Fc (Amgen Inc), RANK-Fc (R&D Systems), 3-ATA (Santa Cruz Biotechnology), seliciclib (Focus Biomolecules), and/or fludarabine (Santa Cruz Biotechnology). Complete medium with drugs was replaced every 2 days. After 8 days, viability was measured by Alamar blue assay as described above. For clonogenic assays, cells were seeded in 6-well plates at a density of 2 × 10⁴ - 4 × 10⁴ cells/well for 7 days and allowed to recover for additional 7 days in drug-free medium. Cells were washed and fixed with 4% PFA (Merck) for 10min and stained with 2% crystal violet (Sigma-Aldrich) for 10 min. crystal violet was solubilized with 1% SDS (Merck) for 15–30 min and absorbance at 570 nM was measured in an Infinite M200 microplate reader (Tecan).