Although used in academic research for several decades, 3D culture models have long been regarded expensive, cumbersome and unnecessary in drug development processes. Technical advances, coupled with recent observations showing that gene expression in 3D is much closer to clinical expression profiles than those seen in 2D, have renewed attention and generated hope in the feasibility of maturing organotypic 3D systems to therapy test platforms with greater power to predict clinical efficacies. Here we describe a standardized setup for reproducible, easy-handling culture, treatment and routine analysis of multicellular spheroids, the classical 3D culture system resembling many aspects of the pathophysiological situation in human tumor tissue. We discuss essential conceptual and practical considerations for an adequate establishment and use of spheroid-based drug screening platforms and also provide a list of human carcinoma cell lines, partly on the basis of the NCI-DTP 60-cell line screen, that produce treatable spheroids under identical culture conditions. In contrast to many other settings with which to achieve similar results, the protocol is particularly useful to be integrated into standardized large-scale drug test routines as it requires a minimum number of defined spheroids and a limited amount of drug. The estimated time to run the complete screening protocol described herein--including spheroid initiation, drug treatment and determination of the analytical end points (spheroid integrity, and cell survival through the acid phosphatase assay)--is about 170 h. Monitoring of spheroid growth kinetics to determine growth delay and regrowth, respectively, after drug treatment requires long-term culturing (> or =14 d).

TRAIL (also known as Apo-2L) is a member of the tumor necrosis factor (TNF) ligand family that rapidly induces apoptosis in a variety of transformed cell lines. The human receptor for TRAIL was found to be an undescribed member of the TNF-receptor family (designated death receptor-4, DR4) that contains a cytoplasmic "death domain" capable of engaging the cell suicide apparatus but not the nuclear factor kappa B pathway in the system studied. Unlike Fas, TNFR-1, and DR3, DR4 could not use FADD to transmit the death signal, suggesting the use of distinct proximal signaling machinery. Thus, the DR4-TRAIL axis defines another receptor-ligand pair involved in regulating cell suicide and tissue homeostasis.

Abstract. The secreted polypeptide transforming growth factor-/3 (TGF-fl) exerts its multiple activities through type I and II cell surface receptors. In epithelial cells, activation of the TGF-fl signal transduction pathways leads to inhibition of cell proliferation and an increase in extracellular matrix production. TGF-/3 is widely expressed during development and its biological activity has been implicated in epithelial-mesenchymal interactions, e.g., in branching morphogenesis of the lung, kidney, and mammary gland, and in inductive events between mammary epithelium and stroma.In the present study, we investigated the effects of TGF-/3 on mouse mammary epithelial cells in vitro. TGF-/3 reversibly induced an alteration in the differentiation of normal mammary epithelial NMuMG cells from epithelial to fibroblastic phenotype. The change in cell morphology correlated with (a) decreased expression of the epithelial markers E-cadherin, ZO-1, and desmoplakin I and II; (b) increased expression of mesenchymal markers, such as fibronectin; and (c) a fibroblast-like reorganization of actin fibers. This phenotypic differentiation displays the hallmarks of an epithelial to mesenchymal transdifferentiation event.Since NMuMG cells make high levels of the type I TGF-fl receptor Tsk7L, yet lack expression of the ALK-5/R4 type I receptor which has been reported to mediate TGF-/~ responsiveness, we evaluated the role of the Tsk7L receptor in TGF-~-mediated transdifferentiation. We generated NMuMG cells that stably overexpress a truncated Tsk7L type I receptor that lacks most of the cytoplasmic kinase domain, thus function as a dominant negative mutant. These transfected cells no longer underwent epithelial to mesenchymal morphological change upon exposure to TGF-fl, yet still displayed some TGF-fl-mediated responses.We conclude that TGF-fl has the ability to modulate E-cadherin expression and induce a reversible epithelial to mesenchymal transdifferentiation in epithelial cells. Unlike other transdifferentiating growth factors, such as bFGF and HGF, these changes are accompanied by growth inhibition. Our results also implicate the Tsk7L type I receptor as mediating the TGF-~-induced epithelial to mesenchymal transition.

Herpes simplex virus (HSV) 1 and 2 infect activated T lymphocytes by attachment of the HSV envelope glycoprotein D (gD) to the cellular herpesvirus entry mediator (HVEM), an orphan member of the tumor necrosis factor receptor superfamily. Here, we demonstrate that HVEM binds two cellular ligands, secreted lymphotoxin alpha (LTalpha) and LIGHT, a new member of the TNF superfamily. LIGHT is a 29 kDa type II transmembrane protein produced by activated T cells that also engages the receptor for the LTalphabeta heterotrimer but does not form complexes with either LTalpha or LTbeta. HSV1 gD inhibits the interaction of HVEM with LIGHT, and LIGHT and gD interfere with HVEM-dependent cell entry by HSV1. This characterizes herpesvirus gD as a membrane-bound viokine and establishes LIGHT-HVEM as integral components of the lymphotoxin cytokine-receptor system.

Over the past few years, establishment and adaptation of cell-based assays for drug development and testing has become an important topic in high-throughput screening (HTS). Most new assays are designed to rapidly detect specific cellular effects reflecting action at various targets. However, although more complex than cell-free biochemical test systems, HTS assays using monolayer or suspension cultures still reflect a highly artificial cellular environment and may thus have limited predictive value for the clinical efficacy of a compound. Today's strategies for drug discovery and development, be they hypothesis free or mechanism based, require facile, HTS-amenable test systems that mimic the human tissue environment with increasing accuracy in order to optimize preclinical and preanimal selection of the most active molecules from a large pool of potential effectors, for example, against solid tumors. Indeed, it is recognized that 3-dimensional cell culture systems better reflect the in vivo behavior of most cell types. However, these 3-D test systems have not yet been incorporated into mainstream drug development operations. This article addresses the relevance and potential of 3-D in vitro systems for drug development, with a focus on screening for novel antitumor drugs. Examples of 3-D cell models used in cancer research are given, and the advantages and limitations of these systems of intermediate complexity are discussed in comparison with both 2-D culture and in vivo models. The most commonly used 3-D cell culture systems, multicellular spheroids, are emphasized due to their advantages and potential for rapid development as HTS systems. Thus, multicellular tumor spheroids are an ideal basis for the next step in creating HTS assays, which are predictive of in vivo antitumor efficacy. (Journal of Biomolecular Screening 2004:273-285)