Tumor cell adhesion: Dynamic adhesion of RAW117 lymphoma to organ-specific endothelial cells and the development of an invasion assay
Dombrowski, John Joseph
McIntire, Larry V.
Doctor of Philosophy
The role of cell adhesion molecules was explored in a model of hematogenous metastasis. RAW117 lymphoma cells were perfused over hepatic sinusoidal endothelial (HSE) and murine microvascular lung endothelial (MLE) monolayers in a parallel plate flow chamber. Using video microscopy, cell adherence was examined at various wall shear stresses. Perfused cells were observed to rapidly adhere without rolling at or below shear stresses of 0.5 dynes/cm2 (dyn/cm2). During initial adherence of H10 and L17 cells to HSE, VCAM-1 and E-selectin epitopes helped to establish adhesive interactions more significantly than for P cells. Rolling velocity studies illustrated higher levels of interaction with human E-selectin for H10 than L17 and P cells. MLE did not illustrate any direct role for E-selectin or VCAM-1. Upon neuraminidase treatment, only L17 cells showed greater adhesion to MLE over control. This unique adhesion of L17 cells may indicate a sequential role for each organ vasculature. Squamous cell carcinomas (UMSCC-17A, UMSCC-17B, UMSCC-22A, and UMSCC-22B), prostate epithelial cells (normal and LNCAP), melanomas (B16-Br15 and B16-F1), and normal human fibroblasts (MRC-5) were tested for their invasive abilities. Transwell invasion chambers (8 m m pore) demonstrated that the diaphragm was a selective barrier for some cell types and restricted invasion to single cells. A novel approach to evaluation of cell invasion was performed in 96 well plates. Cell suspensions were seeded over coatings and layers of gelatin, collagen, matrigel, and/or agarose inside each well. At the end of a period of time, layers of substrate were removed and quantities of invasive cells at lower levels were evaluated through calcein and a fluorescent plate reader. Fluid-like gels permitted cells to invade as individuals and aggregates without distinction and separation from surface cells. The assay was modified in several variations to isolate invasion from cell aggregation, reduce the selectivity of invasion due to substrate adhesion through the incorporation of agarose at various concentrations and mixtures, and reduce matrix, cell, and chemical usage. Each invasion assay could explore the role of cell adhesion for invasion, but quantity of fastidious results and accurate portrayals of invasion needed to be considered.
Cell biology; Biomedical engineering; Biophysics; Oncology