design: RNA sequencing of luminal breast cancer patient-derived xenografts (PDX) library to capture tumor heterogeneity and explore the mechanisms of resistance to CDK4/6 inhibitors
library_construction_protocol: Tumor samples were collected from CDK4/6 inhibitor sensitive and resistant PDX at necroscopy. Tumors were processed in ice and fragments flash frozen for subsequent nucleic acid extraction. Stable third-passage PDX derived from luminal BCRO#28 were implanted in NSG mice (10 mice/PDX). When tumor volume reached about 60 mm3, mice were randomly divided in two cohorts and continuously treated via oral gavage with vehicle or Palbociclib (PD-0332991 hydrochloride, Clinisciences, Nanterre, France) resuspended in ringer lactate (100 mg/kg, 5 days/week). Treatments were prolonged up to 8 months with the same dosing regimen.  After emergence of acquired-resistance tumors (volume >400mm3), mice were sacrificed, tumors and distant organs collected and stored for further molecular analysis. To confirm the resistant phenotype, tumor pieces derived from excised masses were implanted in 10 recipient NSG mice. As described above, when the tumors reached 60 mm3, mice were separated into two different groups (vehicle- and Palbociclib-treated) and the treatments followed as described. In all experiments, tumor dimensions were recorded weekly with a caliper and tumor volume was calculated based on the formula: length x width2/2. Total RNA was isolated from fresh solid tumor samples using TRIzol solution (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer protocol. GentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to disrupt tumors, and lysates were passed at least five times through a 23‐gauge needle fitted to an RNase‐free syringe. RNA quantification was performed using NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) and Tape Station RNA Screen Tape kit (Agilent Technologies, Inc., Santa Clara, CA, USA) according to manufacturer protocol. An input of 500 ng of total RNA per sample was used for subsequent RNA Sequencing analysis. RNA-Seq libraries were generated using the Illumina Stranded total RNA prep library kit (Illumina, San Diego, CA, USA) according to protocol's instructions. Pools of 28 purified libraries were quantified using High Sensitivity D1000 Screen Tape System (Agilent Technologies, Inc., Santa Clara, CA, USA) and normalized to 2nΜ.