THE EMBRYOID BODY AS A NOVEL IN-VITRO ASSAY SYSTEM FOR ANTIANGIOGENICAGENTS

Tumor progression necessitates the induction of blood vessels that con verge upon the tumor and enhance the diffusibility of oxygen and nutri ents. Approaches to treat cancer by antiangiogenic therapy are therefo re straightforward, and there is a great need for suitable in vitro sy stems to test antiangiogenic agents. In the present study, embryoid bo dies (EBs) differentiated from totipotent mouse embryonic stem (ES) ce lls and cultivated using the spinner flask technique are introduced as an in vitro system for antiangiogenesis research. ES cells effectivel y differentiated endothelial cells within the three-dimensional tissue of EBs. The total area of capillary-like structures, which were posit ive for CD31 (platelet endothelial cell adhesion molecule, PECAM-1), w as assessed by confocal laser scanning microscopy and image analysis o f a series of optical sections. Endothelial differentiation occurred b etween Day 4-5 and Day 8 of EB development. Within 7 days, 100% of EBs contained capillary-like structures. Suramin, tamoxifen, tetrahydroco rtisol, and a combination of tetrahydrocortisol and heparin were teste d for their antiangiogenic capacity in the EB system and were found to efficiently inhibit endothelial differentiation. Diffusion studies of a 10-kd -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-de xtran and the fluorescent, amphiphilic agent doxorubicin in avascular and vascularized EBs revealed that the endothelial structures formed f unctional vessels that facilitated diffusion. The diffusion coefficien t D for doxorubicin was 296.10(-9) cm(2) s(-1) in vascularized 8-day-o ld EBs, ie, about 10-fold larger than in avascular 3-day-old EBs (18.1 0(-9) cm(2) s(-1)) and EBs treated with suramin (14.10(-9) cm(2) s(-1) ), tamoxifen (13.5.10(-9) cm(2) s(-1)), and tetrahydrocortisol/heparin (18.5.10(-9) cm(2) s(-1)). Consequently, avascular EBs treated with a ntiangiogenic agents developed central necrosis, which was absent in v ascularized EBs. Our findings indicate that EBs are a suitable in vitr o model system to study the effects of antiangiogenic agents in a thre e-dimensional tissue context. Furthermore, EBs provide a unique model to investigate the diffusion of anticancer agents in a tissue in both the avascular and vascularized states.