3D drug screen identifies HDAC inhibitors as anti‐invasive and selectively cytotoxic toward TSC2−/− LAM cells. A) Representative maximum intensity projection image of TSC2−/− cells in hydrogel culture for 3 days ± 200 × 10−9m carfilzomib. Scale bars of 250 µm. B) Computational reconstruction of cellular spatial positions following 3 day hydrogel culture of TSC2−/− cells ± 40 × 10−9m dasatinib. Note that treated and untreated were in separate wells; cells were plotted in the same volume for ease of visualizing relative distances traveled. C) Highest development status reported for the 800 compounds contained in the curated kinase inhibitor and tool compound libraries. A 3D drug screen was conducted on WT and TSC2−/− cells following 3 day treatment with 5 × 10−6m compounds ± 20 × 10−9m rapamycin. D) Compound invasion modulation plotted against cytotoxicity, aggregating results across genotype and rapamycin treatment. Fixed invasion threshold determined by median invasion distance of untreated controls. Hexagonal plot employed to demonstrate compound densities. E) Waterfall plot of compound invasion z‐scores in ranked order; positive values indicate invasion potentiation, while negative values indicate invasion attenuation. Compounds conferring statistically significant invasion modulation highlighted in black. Data presented for TSC2−/−, no rapamycin treatment condition. F) Number of compounds significantly modulating invasion (potentiating or attenuating) for each genotype in the presence of absence of 20 × 10−9m rapamycin. Bubble area proportional to number of statistically significant targets. G) Overlap of compounds identified as anti‐invasive in each listed condition. H) Waterfall plots of compound selective toxicity z‐scores in ranked order; positive values indicate increased cytotoxicity toward TSC2−/− cells, negative values indicate increased cytotoxicity toward WT cells. Compounds conferring statistically significant selective cytotoxicity highlighted in black. I) Overlap of compounds identified to be selectivity cytotoxic toward TSC2−/− cells, with or without 20 × 10−9m rapamycin. J) Enrichment plot for compounds annotated to target HDACs, derived from an adapted implementation of GSEA. Hits (black vertical lines) in the red region indicate compounds with a favorable effect, hits in the blue region indicate compounds with an undesirable effect. K) Top 10 most statistically significant GO terms. Analysis performed using targets identified as statistically significantly enriched in screen data by Elion algorithm.
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