Thomas Korff

Single endothelial cells (EC) seeded in suspension culture rapidly undergo apoptosis. Addition of survival factors, such as VEGF and FGF-2, does not prevent apoptosis of suspended EC. However, when cells are allowed to establish cell-cell contacts, they become responsive to the activities of survival factors. These observations have led to the development of a three-dimensional spheroid model of EC differentiation. EC spheroids remodel over time to establish a differentiated surface layer of EC and a center of unorganized EC that subsequently undergo apoptosis. Surface EC become quiescent, establish firm cell-cell contacts, and can be induced to express differentiation antigens (e.g., induction of CD34 expression by VEGF). In contrast, the unorganized center spheroid cells undergo apoptosis if they are not rescued by survival factors. The responsiveness to the survival factor activities of VEGF and FGF-2 was not dependent on cell shape changes since it was retained after cytochalasin D treatment. Taken together, these findings characterize survival factor requirements of unorganized EC and indicate that polarized surface EC differentiate to become independent of exogenous survival factors. Furthermore, they demonstrate that spheroid cell culture systems are useful not just for the study of tumor cells and embryonic stem cells but also for the analysis of differentiated functions of nontransformed cells.

During angiogenesis, anastomosing capillary sprouts align to form complex three-dimensional networks of new blood vessels. Using an endothelial cell spheroid model that was developed to study endothelial cell differentiation processes, we have devised a novel collagen gel-based three-dimensional in vitro angiogenesis assay. In this assay, cell number-defined, gel-embedded endothelial cell spheroids act as a cellular delivery device, which serves as a focal starting point for the sprouting of lumenized capillary-like structures that can be induced to form complex anastomosing networks. Formation of capillary anastomoses is associated with tensional remodeling of the collagen matrix and directional sprouting of outgrowing capillaries towards each other. To analyze whether directional sprouting is dependent on cytokine gradients or on endothelial cell-derived tractional forces transduced through the extracellular matrix, we designed a matrix tension generator that enables the application of defined tensional forces on the extracellular matrix. Using this matrix tension generator, causal evidence is presented that tensional forces on a fibrillar extracellular matrix such as type I collagen, but not fibrin, are sufficient to guide directional outgrowth of endothelial cells. RGD peptides but not control RAD peptides disrupted the integrity of sprouting capillary-like structures and induced detachment of outgrowing endothelial cells cultured on top of collagen gels, but did not inhibit primary outgrowth of endothelial cells. The data establish the endothelial cell spheroid-based three-dimensional angiogenesis technique as a standardized, highly reproducible quantitative assay for in vitro angiogenesis studies and demonstrate that integrin-dependent matrix tensional forces control directional capillary sprouting and network formation.

Paracrine interactions between endothelial cells (EC) and mural cells act as critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed at studying blood vessel maturation processes in a novel 3-dimensional spheroidal coculture system of EC and smooth muscle cells (SMC). Coculture spheroids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiological assembly of blood vessels with surface lining EC and underlying mural cells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexes of the EC surface layer, 2) a down-regulation of PDGF-B expression by cocultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furthermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-like sprouts in a VEGF-dependent manner in a 3-dimensional in gel angiogenesis assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-1 producing SMC, inducing lateral sheet migration as well as in gel sprouting angiogenesis. Taken together, the data establish the spheroidal EC/SMC system as a powerful cell culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell-mediated quiescence effects on endothelial cells.

PURPOSE: In glioma-in contrast to various other cancers-the impact of T-lymphocytes on clinical outcome is not clear. We investigated the clinical relevance and regulation of T-cell infiltration in glioma. EXPERIMENTAL DESIGN: T-cell subpopulations from entire sections of 93 WHO°II-IV gliomas were computationally identified using markers CD3, CD8, and Foxp3; survival analysis was then done on primary glioblastomas (pGBM). Endothelial cells expressing cellular adhesion molecules (CAM) were similarly computationally quantified from the same glioma tissues. Influence of prominent cytokines (as measured by ELISA from 53 WHO°II-IV glioma lysates) on CAM-expression in GBM-isolated endothelial cells was determined using flow cytometry. The functional relevance of the cytokine-mediated CAM regulation was tested in a transmigration assay using GBM-derived endothelial cells and autologous T-cells. RESULTS: Infiltration of all T-cell subsets increased in high-grade tumors. Most strikingly, within pGBM, elevated numbers of intratumoral effector T cells (T(eff), cytotoxic and helper) significantly correlated with a better survival; regulatory T cells were infrequently present and not associated with GBM patient outcome. Interestingly, increased infiltration of T(eff) cells was related to the expression of ICAM-1 on the vessel surface. Transmigration of autologous T cells in vitro was markedly reduced in the presence of CAM-blocking antibodies. We found that TGF-β molecules impeded transmigration and downregulated CAM-expression on GBM-isolated endothelial cells; blocking TGF-β receptor signaling increased transmigration. CONCLUSIONS: This study provides comprehensive and novel insights into occurrence and regulation of T-cell infiltration in glioma. Specifically, targeting TGF-β1 and TGF-β2 might improve intratumoral T-cell infiltration and thus enhance effectiveness of immunotherapeutic approaches.