The development of double barcoded shRNA screen for assessment of tumor heterogeneity

Introduction.Tumor heterogeneity (TH) is supposed to cause drug resistance and is associated with a poor prognosis. One of the efficient methods to study TH is shRNA screen, which allows not only to distinguish subclones, but also to find drug sensitivity genes. The present methods have a low level of specificity because of false positive cases. Here we propose the new shRNA screen using a double barcoded shRNA system to study TH response in osimertinib treatment.

Methods.A double barcoded library contained a unique barcode, a common sequence, and a random sequence. H1975 cells were infected with libraries with MOI 10–20%. Cells were treated with 20 µM osimertinib or with vehicle control. After a period of recovery, the DNA extraction was performed. Analysis of barcodes was done with multiplexing nested PCR. Barcodes were sequenced using Ion Torrent and Illumina platforms.

Results.The deviation of the reads number in the standard library group was wider than in double barcoded, which represents the specificity of the method. The number of reads representing each barcode 2 within a population of cells was proportional to the number of cells in the corresponding clone. Osimertinib equally suppressed the growth of cells derived from different clones.

Conclusion. We developed a double barcoded shRNA screen, which allowed the measurement of drug effects in different tumor clones more precisely in comparison with the standard cell-barcoding. There was little clonal variability in sensitivity to osimertinib in cells that formed primary tumor, which represents a low level of TH in response to the drug.