Determining combination treatment effects on the ratio of KRAS G13C/WT patient-derived BM CD34+ cells

From the BM aspirate obtained from patient 1, CD34⁺ cells were isolated by immunomagnetic separation using the CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer's instructions. Aliquots of 1 × 10⁵ cells were stored in liquid nitrogen using CELLBANKER® (Zenoaq) until use. After thawing, primary BM CD34⁺ cells were expanded in StemPro-34 (Thermo Fisher Scientific) supplemented with SCF, TPO, FLT3LG (all at 50 ng/ml), and IL-3 (10 ng/ml). Control cells were untreated, while treated groups were expanded in the presence of a single drug only (Navitoclax at 50 nM or Trametinib at 5 nM) or a combination of both. One week later, genomic DNA was extracted from expanded cells using the NucleoSpin Tissue extraction kit (Macherey–Nagel). Using custom probes for the detection of KRAS (WT or G13C; Bio-Rad), the ratio of G13C/WT alleles was estimated by ddPCR with the QX200 droplet digital PCR system (Bio-Rad) as previously described [28]. Allele ratios were finally converted to cell mixture ratios, considering the situation of a heteroallelic mutation existing in diploid genomes.