title: Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids.
abstract: Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.
authors.affiliation: The Centenary Institute; Sydney Medical School, University of Sydney; k.beaumont@centenary.org.au.
authors.forename: Kimberley A
authors.initials: KA
authors.lastname: Beaumont
authors.affiliation: The Centenary Institute; Sydney Medical School, University of Sydney.
authors.forename: Andrea
authors.initials: A
authors.lastname: Anfosso
authors.affiliation: The University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland.
authors.forename: Farzana
authors.initials: F
authors.lastname: Ahmed
authors.affiliation: The Centenary Institute; Department of Dermatology, Royal Prince Alfred Hospital; Discipline of Dermatology, University of Sydney.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Weninger
authors.affiliation: The Centenary Institute; The University of Queensland Diamantina Institute, Translational Research Institute, The University of Queensland; Discipline of Dermatology, University of Sydney.
authors.forename: Nikolas K
authors.initials: NK
authors.lastname: Haass
mesh_terms: D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D015687:Cell Hypoxia / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods*; D016193:G1 Phase / Q000502:physiology; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D018613:Microscopy, Confocal / Q000379:methods*; D018874:Spheroids, Cellular / Q000473:pathology; D014407:Tumor Cells, Cultured; D059016:Tumor Microenvironment / Q000187:drug effects; D054875:Ubiquitination