real-time-detection-vitro-tumor-cell-apoptosis-induced-cd8-t-cells-to

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8<sup>+</sup> T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

University of Edinburgh

The development of new 2D and 3D phenotypic screening assays combined with high-throughput genomic and proteomic technologies are well placed to advance a new era of molecular pathway informed Phenotypic Drug Discovery. We describe the application of Reverse Phase Protein Array (RPPA) technology to elucidate the mechanism-of-action of small molecules at the post-translational pathway level. We propose that profiling of phenotypic hits and lead molecules in increasingly more complex 3D in vitro and ex vivo models at the post-translational pathway network level represents an effective strategy to both triage and progress the preclinical development of phenotypic screening hits.

Reverse Phase Protein Arrays elucidate mechanisms-of-action and phenotypic response in 2D and 3D models.

Reverse Phase Protein Arrays (RPPA) represent a sensitive antibody-based proteomic approach, which enables simultaneous quantification of the abundance of multiple proteins and posttranslational modifications across multiple samples. Here, we provide protocols for RPPA performed on two distinct protein-binding substrates associated with two most commonly used RPPA platform technologies. We compare and contrast the respective advantages and limitations of each platform within the context of drug discovery applications.

Reverse Phase Protein Arrays and Drug Discovery.

Principal component analysis enables dimensional reduction of multivariate datasets that are typical in high-content screening. A common analysis utilizing principal components is a distance measurement between a perturbagen-such as small-molecule treatment or shRNA knockdown-and a negative control. This method works well to identify active perturbagens, though it cannot discern between distinct phenotypic responses. Here, we describe an extension of the principal component analysis approach to multivariate high-content screening data to enable quantification of differences in direction in principal component space. The theta comparative cell scoring method can identify and quantify differential phenotypic responses between panels of cell lines to small-molecule treatment to support in vitro pharmacogenomics and drug mechanism-of-action studies.

Article	Year
Reverse Phase Protein Arrays elucidate mechanisms-of-action and phenotypic response in 2D and 3D models. Drug discovery today. Technologies\| PubMed ID: 28647089	2017
Reverse Phase Protein Arrays and Drug Discovery. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 28809001	2017
High-Dimensional Profiling: The Theta Comparative Cell Scoring Method. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29736718	2018

Neil O. Carragher

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