Single cell biomass tracking allows identification and isolation of rare targeted therapy-resistant DLBCL cells within a mixed population

Abstract

Adaptive resistance is a major limitation in the use of targeted therapies for cancer. Using real time biomass tracking, we demonstrate the isolation and identification of rare (1% fraction) diffuse large B cell lymphoma cells resistant to the PI3K inhibitor idelalisib, from an otherwise sensitive population. This technique allows direct study of these rare, drug tolerant cells.

Fig. 1. (a) Schema of HSLCI. (1) Wide field phase detection camera, (2) illumination source, (3) micropipette system, (4) tubes for cell collection. (5) Dynamic focus control (b) examples of drug-tolerant and -sensitive DLBCL cells present in same culture. Left shows cell mass vs. time. Right shows HSLCI cellular mass density images of the two cells. Scale bars 10 μm.

Fig. 2. (a & b) Representative box-plots of SU-DHL-6 and SU-DHL-10 cells treated with idelalisib. Individual dots in the overlaying box plot represent the mass accumulation rates of single cells measured over the interval 24–36 h post-dosing. The carrot “^” indicates the maximum dose reached in the blood of human patients given the prescribed dose. (c & d) The EC₅₀ is calculated by sigmoidal fit to the median mass accumulation rates for SU-DHL-6 (N = 3) and SU-DHL-10 (N = 4). These responses indicate SU-DHL-6 is sensitive and SU-DHL-10 is resistant to idelalisib.

Fig. 3. (a) Representative example (n = 3) of the isolation of the top 1% of growing cells from 1 : 100 SU-DHL-10/SU-DHL-6 mixture. Cells were treated with 2.5 μM idelalisib for 24 hours and mass accumulation rates were measured from 24–40 hours post dosing at which point the top 1% of growing cells were isolated by micropipette. (b) Cells isolated in (a) were cultured for 20 days then rescreened at 2.5 μM idelalisib. The median growth rate at 2.5 μM idelalisib increased from −0.2% ± 0.02% per hour in the isolation to 4.1% ± 0.12% per hour after re-culture. (c). In an experiment identical to (a), cells in the bottom <95^th percentiles were isolated and cultured but no growth occurred. (d) PCR at the IGH-BCL2 loci for the cell line-specific breakpoints was performed on cells from stock SU-DHL-6 and -10 lines, and re-cultured cells indicating SU-DHL-10 cells were isolated from the 1 : 100 mixture.