Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour

Silvia Blacher; Charlotte Erpicum; Bénédicte Lenoir; Jenny Paupert; Gustavo Moraes; Sandra Ormenese; Eric Bullinger; Agnès Noel

doi:10.1371/journal.pone.0097019

Cell invasion in the spheroid sprouting assay: a spatial organisation analysis adaptable to cell behaviour

PLoS One. 2014 May 7;9(5):e97019. doi: 10.1371/journal.pone.0097019. eCollection 2014.

Authors

Silvia Blacher¹, Charlotte Erpicum¹, Bénédicte Lenoir², Jenny Paupert¹, Gustavo Moraes³, Sandra Ormenese³, Eric Bullinger⁴, Agnès Noel¹

Affiliations

¹ Laboratory of tumor and developmental biology, GIGA-Cancer, University of Liège, Liège, Belgium.
² Laboratory of tumor and developmental biology, GIGA-Cancer, University of Liège, Liège, Belgium; Laboratory of cardiovascular research, CRP santé, Luxembourg, Luxembourg.
³ GIGA-Imaging and Flow Cytometry Platform, University of Liege, Liege, Belgium.
⁴ GIGA Systems Biology and Chemical Biology, University of Liege, Liege, Belgium.

Abstract

The endothelial cell spheroid assay provides a suitable in vitro model to study (lymph) angiogenesis and test pro- and anti-(lymph) angiogenic factors or drugs. Usually, the extent of cell invasion, observed through optical microscopy, is measured. The present study proposes the spatial distribution of migrated cells as a new descriptor of the (lymph) angiogenic response. The utility of this novel method rests with its capacity to locally characterise spheroid structure, allowing not only the investigation of single and collective cell invasion but also the evolution of the spheroid core itself. Moreover, the proposed method can be applied to 2D-projected spheroid images obtained by optical microscopy, as well as to 3D images acquired by confocal microscopy. To validate the proposed methodology, endothelial cell invasion was evaluated under different experimental conditions. The results were compared with widely used global parameters. The comparison shows that our method prevents local spheroid modifications from being overlooked and leading to the possible misinterpretation of results.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Angiogenesis Inhibitors / pharmacology
Cell Movement / drug effects
Cell Movement / genetics
Cell Tracking / methods*
Endothelial Cells / drug effects
Endothelial Cells / ultrastructure*
Humans
Imaging, Three-Dimensional
Microscopy, Confocal
Neovascularization, Pathologic / drug therapy
Neovascularization, Pathologic / genetics*
Spheroids, Cellular / drug effects
Spheroids, Cellular / ultrastructure*
Telomerase / chemistry
Telomerase / isolation & purification
Vascular Endothelial Growth Factor A / chemistry
Vascular Endothelial Growth Factor A / metabolism

Substances

Angiogenesis Inhibitors
Vascular Endothelial Growth Factor A
TERT protein, human
Telomerase

Grants and funding

This work was supported by Fonds de la recherche scientifique (FNRS-FRS) no. 3.4598.10; Fondation contre le Cancer (Fondation of public interest, Belgium) no. 2010-170; Centre anticancéreux près de l’Université de Liège; Fonds Léon Frédéricq (University of Liège); Action de Recherche Concertées (University of Liège) no. 11/16-02; and Interuniversity Attraction Pole: phase VII-P7/03 (IAP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.