Extended Data Fig. 6: Validation of individual sgRNAs targeting top hits with differential 3D/2D growth effects.
From: CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities
a, A schematic showing the competitive growth assay used to validate individual sgRNAs in 2D and 3D conditions. Cells expressing a gene-targeting sgRNA (mCherry) are mixed with cells expressing a control sgRNA (safe sgRNA, encoding GFP). Relative changes of mCherry to GFP ratios are monitored to compute growth phenotypes of gene-targeting sgRNAs. b, Genes within the CPD module and selected top hits with differential effects in 3D versus 2D growth were targeted with individual sgRNAs and subjected to competitive growth assays in both 2D and 3D culture. Relative 2D and 3D growth phenotypes of individual sgRNAs were measured by tracking changes in ratios of mCherry (gene-targeting sgRNAs) to GFP (control sgRNA) in the assays by automated fluorescence microscopy. (n = 3 wells in a 24-well plate, mean ± s.e.m.). c, Binary masks of H23 spheroids with the indicated gene knockouts. H23 knockout cell lines expressing sgRNAs against top hits from the 3D/2D phenotypes were seeded at equal density on ultra-low attachment plates. 3D spheroids generated from the knockout lines were imaged in a fluorescent microscope 72 h after seeding. For each knockout line, 48 images were taken from three wells in a 24-well plate using a 10× objective. Binary masks were then generated from mCherry signals of 3D spheroids. Forty-eight images were then stitched together to be shown as one large image for each knockout. d, Relative colony masses of H23 spheroids with gene knockouts are quantified and displayed in bar graphs (n = 3 wells in a 24-well plate, mean ± s.e.m.). e, Genes in the CPD module and KRAS were targeted with corresponding small-molecule inhibitors. Cells were seeded in 96-well plates in 2D (blue line) and 3D (red line) conditions, and grown in the presence of titrating doses of inhibitors for 72 h. Live cells were quantified with alamar blue assays. Relative growth of treated cells compared with the untreated samples are plotted in the drug titration curves. n = 3 wells in a 96-well plate for linsitinib and n = 4 for all other drugs; mean ± s.e.m.
