A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Abstract

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Rapid identification of mRNA processing defects with a novel single-cell yeast reporter

“…3, 4), quickly identifies potential pathways to explore further with orthogonal assays, such as the RT-qPCR we present in Figure 2. Additionally, multiplexing of reporters can also be a way to screen for effects on multiple reporters simultaneously (Chen et al 2012;Kulak and Lum 2013), as long as their signals do not overlap or disturb each other. But as with all other reporter technologies, it is still critical to ascertain the effect on the original or endogenous targets.…”

Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying

“…More advanced innovations toward multiplexing allow for the simultaneous detection of three luciferases with activities that can be spectrally distinguished using appropriate emission filters after the addition of one 18 or more substrates 43 . Alternatively, three luciferases that each use a unique substrate have been used to monitor three distinct cellular phenomena in vitro 44 or, sequentially, in vivo 45 . Thus, currently the maximum number of luciferases that can be measured simultaneously is only three.…”

Zebrafish small molecule screens: Taking the phenotypic plunge

“…At the complex end, Lum and colleagues have screened small molecules in HCT116 human colorectal cancer cells using multiplexed luciferase assays and dot blotting to monitor multiple pathways simultaneously [25] . By assessing multiple pathways in a quantitative manner, they were able to collapse the cellular phenotypes elicited by individual compounds into a “fingerprint.” Traditionally, determining mechanism of action (MOA) can be laborious, however; such an approach provides mechanistic insights by clustering compound induced “fingerprints” to those obtained from an siRNA library [24] . Cell based screens have also been conducted in an image based analytics paradigm.…”