Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC235
Quantification of tumour cell adhesion on endothelial cell monolayers
A. Abdullah1, F. I. Arrigoni1, A. K. Snabaitis1, N. S. Freestone1
1. School of Pharmacy and Chemistry, Kingston University London, Kingston Upon thames, United Kingdom.
Introduction: Adhesion of tumour cells (TCs) to the endothelial cells (ECs) that line the circulatory system plays an important role in the process of metastasis [1-2]. A fluorescent activated cell sorting (FACS) technique has previously been used to determine the number of tumour cells that adhere to endothelial cells [3, 4]. In this study we have developed a simple assay to quantify the number of tumour cells that adhere to an endothelial cell monolayer. Haddad and colleagues (2010) have shown in their FACS analysis that after washing, about 50% of the total added TCs adhered to the EC monolayer which represented a 1 TC for 2 ECs ratio . In the current study, a different but simpler approach was used to determine this ratio. Aims and objectives: The primary aim of this study was to develop an alternative fluorescence-based assay to quantify the adhesion of tumour cells (PC3, DU-145 and MCF7) to human vascular umbilical vein endothelial cells (HUVECs). Methods: Monolayers of human vascular endothelial cells (HUVEC) were activated by treating them with 10ng/mL of TNF-α. Different numbers of PC3, DU-145 and MCF7 cells (10,000-160, 000) in three different experiments, labelled with cell-tracker greenTM (Invitrogen, UK) were then added to HUVEC monolayers. Phase contrast and fluorescent microscopy was performed to observe tumour adhesion onto endothelial cell surfaces. Tumour cell fluorescence, as an index of cell adherence, was detected using a Fluostar Optima plate reader (BMG Labtech) with the excitation wavelength set at 485 nm and emission at 520nm. Statistical analysis was done using a one tailed t-test. Results: The attachment of TCs to activated HUVEC monolayers was increased in proportion to the number of TCs added. We have also found that there is a specific ratio of TCs adhering to HUVEC monolayers which depend on the number of tumour cells added. Conclusion: We conclude that the number of tumour cells that adhere to HUVECs is proportionally related to the number of tumour cells used in the assay and this adherence is increased by the pre-activation of HUVECs by TNF-α.
Where applicable, experiments conform with Society ethical requirements