title: Combining High-Content Imaging and Phenotypic Classification Analysis of Senescence-Associated Beta-Galactosidase Staining to Identify Regulators of Oncogene-Induced Senescence.
abstract: Hyperactivation of the PI3K/AKT/mTORC1 signaling pathway is a hallmark of the majority of sporadic human cancers. Paradoxically, chronic activation of this pathway in nontransformed cells promotes senescence, which acts as a significant barrier to malignant progression. Understanding how this oncogene-induced senescence is maintained in nontransformed cells and conversely how it is subverted in cancer cells will provide insight into cancer development and potentially identify novel therapeutic targets. High-throughput screening provides a powerful platform for target discovery. Here, we describe an approach to use RNAi transfection of a pre-established AKT-induced senescent cell population and subsequent high-content imaging to screen for senescence regulators. We have incorporated multiparametric readouts, including cell number, proliferation, and senescence-associated beta-galactosidase (SA-βGal) staining. Using machine learning and automated image analysis, we also describe methods to classify distinct phenotypes of cells with SA-βGal staining. These methods can be readily adaptable to high-throughput functional screens interrogating the mechanisms that maintain and prevent senescence in various contexts.
authors.affiliation: 1 Division of Cancer Research, Peter MacCallum Cancer Centre , Melbourne, Australia .
authors.forename: Keefe T
authors.initials: KT
authors.lastname: Chan
authors.affiliation: 2 Institute for Molecular Medicine Finland, University of Helsinki , Helsinki, Finland .
authors.forename: Lassi
authors.initials: L
authors.lastname: Paavolainen
authors.affiliation: 3 John Curtin School of Medical Research, Australian National University , Canberra, Australia .|4 Department of Biochemistry and Molecular Biology, University of Melbourne , Melbourne, Australia .
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Hannan
authors.affiliation: 3 John Curtin School of Medical Research, Australian National University , Canberra, Australia .|5 School of Biomedical Sciences, University of Queensland , Brisbane, Queensland, Australia .
authors.forename: Amee J
authors.initials: AJ
authors.lastname: George
authors.affiliation: 1 Division of Cancer Research, Peter MacCallum Cancer Centre , Melbourne, Australia .|3 John Curtin School of Medical Research, Australian National University , Canberra, Australia .|4 Department of Biochemistry and Molecular Biology, University of Melbourne , Melbourne, Australia .|5 School of Biomedical Sciences, University of Queensland , Brisbane, Queensland, Australia .|6 Sir Peter MacCallum Department of Oncology, University of Melbourne , Melbourne, Australia .|7 Department of Biochemistry and Molecular Biology, Monash University , Clayton, Australia .
authors.forename: Ross D
authors.initials: RD
authors.lastname: Hannan
authors.affiliation: 6 Sir Peter MacCallum Department of Oncology, University of Melbourne , Melbourne, Australia .|8 Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre , Melbourne, Australia .
authors.forename: Kaylene J
authors.initials: KJ
authors.lastname: Simpson
authors.affiliation: 2 Institute for Molecular Medicine Finland, University of Helsinki , Helsinki, Finland .|9 Synthetic and Systems Biology Unit, Hungarian Academy of Sciences , Szeged, Hungary .
authors.forename: Peter
authors.initials: P
authors.lastname: Horvath
authors.affiliation: 1 Division of Cancer Research, Peter MacCallum Cancer Centre , Melbourne, Australia .|4 Department of Biochemistry and Molecular Biology, University of Melbourne , Melbourne, Australia .|6 Sir Peter MacCallum Department of Oncology, University of Melbourne , Melbourne, Australia .|7 Department of Biochemistry and Molecular Biology, Monash University , Clayton, Australia .
authors.forename: Richard B
authors.initials: RB
authors.lastname: Pearson
mesh_terms: D002461:Cell Line, Transformed; D016922:Cellular Senescence / Q000502:physiology*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009857:Oncogenes / Q000502:physiology*; D010641:Phenotype; D013194:Staining and Labeling / Q000379:methods*; D001616:beta-Galactosidase / Q000032:analysis*
chemical_list: D001616:beta-Galactosidase