Abstract
Although a large amount of screening data comprising target genes and/or drugs tested against cancer cell line panels are available, different assay conditions and readouts limit the integrated analysis and batch-to-batch comparison of these data. Here, we systematically produced and analyzed the anticancer effect of the druggable targetome to understand the varied phenotypic outcomes of diverse functional classes of target genes. A library of siRNAs targeting ~4,800 druggable genes was screened against cancer cell lines under 2D and/or 3D assay conditions. The anticancer effect was simultaneously measured by quantifying cell proliferation and/or viability. Hit rates varied significantly depending on assay conditions and/or phenotypic readouts. Functional classes of hit genes were correlated with the microenvironment difference between the 2D monolayer cell proliferation and 3D sphere formation assays. Furthermore, multiplexing of cell proliferation and viability measures enabled us to compare the sensitivity and resistance responses to the gene knockdown. Many target genes that inhibited cell proliferation increased the single-cell-level viability of surviving cells, leading to an increase in self-renewal potential. In this study, combinations of parallel 2D/3D assays and multiplexing of cell proliferation and viability measures provided functional insights into the varied phenotypic outcomes of the cancer targetome.
Highlights
Together with chemical and drug screening campaigns, functional screening of target genes using RNA interference (RNAi) technologies has been widely carried out to identify and evaluate the therapeutic potential of diverse target genes against cancer cells[1]
Two measures and two culture conditions were combined for the screening of the short interfering RNA (siRNA) library against 4,789 druggable genes in four different cancer cell lines (Table 1)
Among the three cell lines (A549, HT29 and U87) from different cancer lineages, the inhibitory hits identified by cell counting showed general consistency, while the consistency of hits inhibiting 3D sphere formation was relatively varied between cell lines (U87 and OVCAR8) (Fig. 1a)
Summary
Together with chemical and drug screening campaigns, functional screening of target genes using RNA interference (RNAi) technologies has been widely carried out to identify and evaluate the therapeutic potential of diverse target genes against cancer cells[1]. ATP-dependent luminescence (Cell Titer-Glo assay in the Cancer Cell Line Encyclopedia study), fluorescent DNA-binding dyes (SYTO 60 assay in the Cancer Genome Project study) and anionic general biomass stain (SRB assay in the National Cancer Institute study) are used to assess drug inhibition[11,12,13,14] These viability measures are assumed to be correlated with the mass change of cancer cells in the screen, representing the anticancer efficacy of treated RNAi. More recently, image-based direct cell counts in two-dimensional (2D) monolayer cultures or sphere counts in three-dimensional (3D) culture conditions have been multiplexed (or employed in parallel) with cell viability assays[15]. The multiplexing of cell count with the change in single cell viability will provide new insights for further dissecting functional classes of anticancer target genes. We report an analytical overview of the functional diversity of target genes displaying diverse outcomes in the multiplexed assays, together with the complete screening data
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