Ellipsoid Segmentation Model for Analyzing Light-Attenuated 3D Confocal Image Stacks of Fluorescent Multi-Cellular Spheroids.
In oncology, two-dimensional in-vitro culture models are the standard test beds for the discovery and development of cancer treatments, but in the last decades, evidence emerged that such models have low predictive value for clinical efficacy. Therefore they are increasingly complemented by more physiologically relevant 3D models, such as spheroid micro-tumor cultures. If suitable fluorescent labels are applied, confocal 3D image stacks can characterize the structure of such volumetric cultures and, for example, cell proliferation. However, several issues hamper accurate analysis. In particular, signal attenuation within the tissue of the spheroids prevents the acquisition of a complete image for spheroids over 100 micrometers in diameter. And quantitative analysis of large 3D image data sets is challenging, creating a need for methods which can be applied to large-scale experiments and account for impeding factors. We present a robust, computationally inexpensive 2.5D method for the segmentation of spheroid cultures and for counting proliferating cells within them. The spheroids are assumed to be approximately ellipsoid in shape. They are identified from information present in the Maximum Intensity Projection (MIP) and the corresponding height view, also known as Z-buffer. It alerts the user when potential bias-introducing factors cannot be compensated for and includes a compensation for signal attenuation.
Discovery Sciences, Janssen Pharmaceutical companies of Johnson & Johnson, Beerse, Belgium.
Michaël
M
Barbier
Discovery Sciences, Janssen Pharmaceutical companies of Johnson & Johnson, Beerse, Belgium.
Steffen
S
Jaensch
Pharma R&D IT, Janssen Pharmaceutical companies of Johnson & Johnson, Beerse, Belgium.
Frans
F
Cornelissen
Discovery Sciences, Janssen Pharmaceutical companies of Johnson & Johnson, Beerse, Belgium.|Department of Urology, Erasmus MC Rotterdam, Rotterdam, The Netherlands.|Faculty of Mathematics, Natural Sciences and Information Technologies, University of Primorska, Koper, Slovenia.
Suzana
S
Vidic
Discovery Sciences, Janssen Pharmaceutical companies of Johnson & Johnson, Beerse, Belgium.|Department of Urology, Erasmus MC Rotterdam, Rotterdam, The Netherlands.
Kjersti
K
Gjerde
Discovery Sciences, Janssen Pharmaceutical companies of Johnson & Johnson, Beerse, Belgium.
Ronald
R
de Hoogt
Translational Medicine and Clinical Pharmacology, Boehringer Ingelheim, Ingelheim am Rhein, Germany.
Ralph
R
Graeser
Discovery Sciences, Janssen Pharmaceutical companies of Johnson & Johnson, Beerse, Belgium.
Emmanuel
E
Gustin
Discovery Sciences, Janssen Pharmaceutical companies of Johnson & Johnson, Beerse, Belgium.
Yolanda T
YT
Chong
D000465:Algorithms; D000072645:Cancer-Associated Fibroblasts / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D003198:Computer Simulation; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008027:Light; D018613:Microscopy, Confocal / Q000379:methods*; D008954:Models, Biological; D015203:Reproducibility of Results; D018874:Spheroids, Cellular / Q000166:cytology*; D059016:Tumor Microenvironment