HTG EdgeSeq analysis of parental and CDK4/6i-resistant (Ribociclib or Palbociclib) T47D breast cancer cell clones
HTG EdgeSeq analysis of parental and CDK4/6i-resistant (Ribociclib or Palbociclib) T47D breast cancer cell clones
1 x 106 cells were plated and cultivated for 48 hours. For cell harvesting and counting, adherent cells were treated with Trypsin-EDTA solution (2x) for 5 minutes at 37°C. The cells were resuspended in DMEM (with 10% FBS) and centrifuged. After washing with PBS, viable cell count was determined, and the appropriate number of cells (either 4 million or 2 million) was collected in a 1.5 mL Eppendorf tube.  T47D cells lines were obtained from the American Tissue Collection Center (ATCC) and grown in high-glucose DMEM supplemented with 10% fetal bovine serum, 100U ml-1 and 100 µg ml-1 Streptomycin at 37°C with 5% CO2. All cell lines were regularly tested and found to be negative for mycoplasma contamination. The metabolic drugs and molecular inhibitors were obtained from Sigma-Aldrich unless indicated otherwise and used at the following concentrations: Ribociclib (MedChemExpress) 0,125 – 32 µM, and Palbociclib (MedChemExpress) 0,125 – 32 µM. CDK4/6i-resistant T47D cells were generated using a dose escalation approach. Starting concentrations were set at 100-fold lower than the IC50 values for the respective cell line-inhibitor combinations. Parental T47D cells were treated with Ribociclib at concentrations ranging from 9,1 nM to 1 µM, and Palbociclib at concentrations from 23,2 nM to 1 µM. Parental MCF-7 cells were treated with Ribociclib from 0,73 nM to 1 µM, and Palbociclib from 2,8 nM to 1 µM. The resistant cell clones were selected and continuously cultured with 1 µM of the respective CDK4/6i. For the experiments, the cells were cultured in absence of the CDK4/6-inhibitors. Gene expression analysis was performed targeting 2549 genes using the HTG Oncology Biomarker Panel (OBP) and HTG's extraction-free nuclease protection assay (HTG Molecular Diagnostics, Tucson, USA). Cells were lysed and diluted to 3000 cells/35 µL of lysis buffer. 35 µL of the lysate was processed in the HTG EdgeSeq processor. 