Introduction

Avascular tumors in vivo or tumor spheroids in vitro will not grow beyond a size of a few millimeters, at which size passive diffusion can no longer provide nutrients for the cells in the depth of the tissue or waste products adequately diffuse out of the tumor tissue. The gradients in oxygen, pH, nutrients, and the absence of sufficient detoxification then result in growth arrest, development of central

From: Methods in Molecular Biology, vol. 331: Human Embryonic Stem Cell Protocols Edited by: K. Turksen © Humana Press Inc., Totowa, NJ

necrosis, and induction of a multidrug resistance (MDR) phenotype, which is correlated with the increased expression of the MDR transporter P-glycoprotein (1,2). Such tumors usually remain in this state of equilibrium for decades unless they get access to the host circulation in the process of tumor-induced angiogene-sis (3). Compelling evidence shows that vascularization of the tumor by the host vasculature is a prerequisite for rapid tumor growth as well as for metastasis. Tumor-induced angiogenesis is mediated by tumor-secreted proangiogenic growth factors that interact with their surface receptors, which are expressed on the surface of endothelial cells. The most commonly found angiogenic factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, bind to tyrosine kinase receptors on endothelial cell membranes which results in dimerization of the receptors and activation of autophosphorylation of tyrosines on the receptor surface thereby initiating several signalling proteins (4). Binding of the SH-2 regions of these proteins to the phosphotyrosines on the receptor tyrosine kinases activates several pathways, which are crucial for triggering the cell cycle machinery. However, upregulation of an angiogenic factor is not sufficient in itself for a tumor to become angiogenic. Indeed certain negative regulators of blood vessel growth may need to be downregulated (5,6). If there is a preponderance of proangiogenic factors in the local milieu, the neovasculature may persist. However, if the levels of angiostatic factors increase over the level of proangio-genic factors, the new tumor blood vessels may regress. Hence the switch to the angiogenic phenotype is regulated by a change in the local equilibrium between positive and negative regulators of the growth of microvessels.

Vasculogenesis, the in situ assembly of capillaries from undifferentiated endothelial precursor cells as well as angiogenesis, the sprouting of capillaries from preexisting blood vessels has been extensively studied in embryonic stem (ES) cells of mouse and human (7,8) origin (Fig. 1). It has been discussed that the vasculogenic potential of embryonic stem cells could be specifically of use in tissue engineering for the induction of tissue vascularization (8). It has been conclusively demonstrated that ES cell-derived embryoid bodies represent a suitable in vitro model to study molecular events involved in vascular development. ES cells differentiate in vitro to endothelial cells through successive maturation steps with sequential expression of cell lineage-specific markers: platelet endothelial cell adhesion molecule (PECAM), Flk-1, tie-1, tie-2, vascular endothelial cad-herin, MECA-32, and MEC-14.7 (9). These endothelial cells differentiated from ES cells form functional capillary structures, which facilitate diffusion within the tissue and dissipate oxygen gradients within the tissue. We have recently introduced the embryoid body as a model system for in vitro testing of antiangiogenic agents (10). Several agents that were already proven to be effective in clinical patient treatment were applied to embryoid bodies and displayed antiangiogenic capacity (10). Furthermore, it was shown that the teratogenic agent thalidomide exerted antiangiogenic effects, which was related to generation of hydroxyl

Fig. 1. Capillaries differentiated from human embryonic stem cells. Embryoid bodies were outgrown on cover slips. Endothelial cells were visualized by the use of an antibody directed against PECAM-1. The bar represents 50 |im. (Please see the companion CD for the color version of this figure.)

radicals by this compound (11). In a further study, we elaborated confrontation cultures consisting of embryoid bodies and multicellular tumor spheroids to study tumor-induced angiogenesis (12). Multicellular tumor spheroids are three-dimensional cell systems that have been used for more than 30 yr as model systems for avascular micrometastases or avascular microregions of solid tumors

(13). By the use of the multicellular tumor spheroid model studies on mechanisms of cell cycle regulation, the tumor tissue micromilieu and the action of anticancer agents have been performed (13). Recently, we and others have demonstrated that upregulation of the MDR transporter P-glycoprotein in hypoxic regions of multicellular tumor spheroids is regulated by hypoxia-inducible factor (HIF)-la

(14). For generation of confrontation cultures of multicellular tumor spheroids and embryoid bodies, cells are initially cultured separately by a spinner flask technique and subsequently merged in hanging drops (Fig. 2). Invasion of endothelial cells into the tumor tissue was monitored by immunolabeling of cells

Fig. 2. Scheme of confrontation culture generation. Multicellular tumor spheroids are grown in spinner flasks whereas embryoid bodies are grown in liquid overlay culture. At a size of 350-450 |im single tumor spheroids and embryoid bodies are placed in hanging drops on the lid of a Petri dish. After 24 h, the tissues coalesce and form confrontation cultures, which allow to investigate the process of tumor-induced angiogenesis. (Please see the companion CD for the color version of this figure.)

Fig. 2. Scheme of confrontation culture generation. Multicellular tumor spheroids are grown in spinner flasks whereas embryoid bodies are grown in liquid overlay culture. At a size of 350-450 |im single tumor spheroids and embryoid bodies are placed in hanging drops on the lid of a Petri dish. After 24 h, the tissues coalesce and form confrontation cultures, which allow to investigate the process of tumor-induced angiogenesis. (Please see the companion CD for the color version of this figure.)

with the endothelial cell-specific antibody PECAM-1. After 24-48 h in confrontation culture, first PECAM-1-positive cells appeared in the contact region between the embryoid body and the tumor spheroids. Within 5 d of confrontation cultures, endothelial cells migrated toward the tumor tissue, which resulted in vascularization of the tumor spheroid (Fig. 3). During confrontation culture, changes in the expression of genes involved in angiogenesis (e.g., HIF-1a, VEGF) as well as multidrug resistance can be monitored. We observed that during tumor-induced angiogenesis, matrix metalloproteinases (MMPs) are upregulated. MMPs degrade the extracellular matrix and basal membrane thereby allowing the invasion of endothelial cells into the tumor tissue. This process involved the generation of reactive oxygen species, which are known to act as signalling molecules in a variety of signaling cascades including VEGF-signaling and MMP expression (15). Recently confrontation cultures consisting of multi-cellular tumor spheroids and human embryonic stem cells were established (Fig. 4). It was shown that capillary structures derived from embryonic stem cells readily invaded the tumor tissue. Hence this model may be ideally suited to study signal transduction events occurring during tumor-induced angiogenesis and may be useful in antiangiogenesis research.

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