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Multicellular Spheroids: a Three-Dimensional in Vitro Culture System to Study Tumour Biology

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Multicellular Spheroids: a Three-Dimensional in Vitro Culture System to Study Tumour Biology Int. J. Exp. Path. (1998), 79, 1–23 Current Status Review Multicellular spheroids: a three-dimensional in vitro culture system to study tumour biology LEONI A. KUNZ-SCHUGHART*, MARINA KREUTZ† AND RUTH KNUECHEL* *Institute of Pathology and †Department of Haematology/Oncology, University of Regensburg, Regensburg, Germany Received for publication 15 October 1997 Accepted for publication 3 December 1997 Summary. The growth of tumour cells as three-dimensional multicellular spheroids in vitro has led to important insights in tumour biology, since properties of the in vivo-tumour such as proliferation or nutrient gradients, can be studied under controlled conditions. While this review starts with an update of recent data on spheroid monocultures, especially concerning tumour microenvironment and therapeutic modalities, the main emphasis is put on the spectrum of heterologous cultures which have evolved in previous years. This type of culture includes tumour cell interaction with endothelial, fibroblast or immunocompetent cells. The relation of the spheroid culture model to other types of three-dimensional culture and our critical evaluation and presentation of the technical aspects of growing and analysing spheroids are included in the text. These topics are chosed to help the experimental pathologist design experiments with tumour spheroids and to stimulate discussion. Keywords: Multicellular spheroids, in vitro, three-dimensional culture system, tumour Introduction major advantage of establishing three-dimensional, spherical aggregates from permanent cell lines is that Over the past three decades three-dimensional (3-D) cell basic mechanisms of cell growth, proliferation, and dif- cultures of both normal and malignant origin have been ferentiation can be studied in a reproducible format with progressively used as in vitro systems in organogenesis an internal environment dictated by the metabolism and and biomedical research. In contrast to conventional, adaptive responses of the cells. Thus, aggregates pos- homogeneous monolayer or suspension cultures, sess a three-dimensionally organized complex network three-dimensional cultures may preserve specific bio- displaying cell-to-cell and cell-to-matrix interactions in chemical and morphological features similar to the conjunction with a well-defined morphological and phy- corresponding tissues in vivo and can stay in a siological geometry affecting internal criteria such as cell differentiated, active functional state for many weeks. A shape, size, distribution of organelles, or enzymatic activity. This ‘secular heterogeneity’ (Heppner & Miller Correspondence: Prof. Dr R. Knuechel, Institute of Pathology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D- 1989) should be distinguished from clonal cell hetero- 93053 Regensburg, Germany. Fax: þ49 941 944 6602, E- geneity and is reflected in 3-D rather than 2-D culture mail: [email protected] systems. q 1998 Blackwell Science Ltd 1 2 L. A. Kunz-Schughart et al. Since its first introduction in the early seventies by represent an important cell population to be considered Sutherland and coworkers (Sutherland et al. 1970a,b; as negative or positive affectors of tumour cell growth Inch et al. 1970), multicellular tumour spheroids (MCTS) and propagation (Mantovani 1992). have gained fundamental importance in experimental Besides recent data on classical topics of research on cancer research with the vast majority of previous stu- MCTS monocultures, this review will highlight work with dies focusing on tumour cell response to various treat- heterologous cocultures. An overview on the technical ment modalities (for reviews see: Mueller-Klieser 1987; aspects of working with homologous and heterologous Sutherland 1988; Knuechel & Sutherland 1990; Carlsson cultures is presented to provide insight into the practi- 1992; Carlsson & Nederman 1992; Freyer 1992). In cality of the tissue culture method. We intend to show addition to therapeutically orientated investigations, the that a three-dimensional controlled model system, use of MCTS has expanded to other areas of basic cell including both normal and tumour cells and their products biology and pathophysiology such as intercellular com- will offer understanding of tumour micromilieu appro- munication, cell invasion, angiogenesis, and neovascu- priate to test new anticancer strategies in vitro, allowing larization (reviewed by: Bracke et al. 1992; Engebraaten for two therapeutic targets, the tumour cell and its 1992; Neeman et al. 1997). Similarly, investigations on environment. microenvironmental and epigenetic mechanisms involved in the regulation of cell proliferation, differentia- Spheroid monocultures tion, cell death, and gene expression have been intensified within the last 10 years (Acker & Carlsson Technical aspects I 1992; Knuechel & Sutherland 1992; Mueller-Klieser 1997). Diverse techniques for cultivation of tumour spheroids Although 3-D culture systems such as multicellular have been described in the literature (for review: Mueller- tumour spheroid monocultures are considered to mimic Klieser 1987; Lund-Johansen et al. 1992). Spinner flask the in vivo micromilieu of small avascular tumours, much cultures are most widely used since large numbers of of the biological complexity of the in vivo situation is lost. spheroids may be generated at the same time reaching In organogenesis for example, numerous reports have diameters up to 1–2 mm. Other approaches to avoid been published dealing with the role of mesenchyme and effects caused by stagnant medium are roller bottles or its extracellular matrix. It is well known that fibroblastic roller tubes. With all methods spherical aggregates of mesenchyme is not only critical to the development of the tumour cells form spontaneously in the absence of integumental, urinary, gastrointestinal, skeletal and uro- attachment to another substrate either directly in the genital systems pre- and postnatally but also induces flasks or during an initiation interval in agar-coated or specific patterns of epithelial morphogenesis and cyto- microbacteriological culture dishes. For rapid harvesting differentiation such as the expression of tissue specific of spheroids of approximately the same size, cultures secretory proteins (for review: Cunha et al. 1991). In may be pipetted through a sieve of nylon meshes prior to addition, the intricate macromolecular network of the the transfer into spinner flasks or prior to the final ECM which is predominantly produced and secreted by experiment. Thus, one can easily culture large numbers stromal cells has been shown to play a crucial role in cell/ of spheroids to obtain precise growth data while each epithelial differentiation and tissue-specific gene expres- individual spheroid is essentially identical to others in the sion acting as positive or negative regulator. For par- complex, yet ordered, internal physiology and morphol- enchymal organs the microenvironment can thus be ogy. However, for monitoring and manipulating single defined as a unit of function consisting of the epithelium, spheroids at particular growth stages, cultivation of its underlying basement membrane, and the subjacent aggregates on a stationary, nonadherent surface such stroma. Nevertheless, fibroblasts have been investigated as agar or agarose utilizing 96-well plates is recom- as targets of tumour cell invasion rather than in tumour mended. These so-called agar or liquid overlay cultures cell differentiation. The possibility that emerging or may be kept on a gyratory shaker following a defined established carcinomas may be regulated by their con- initiation time in order to guarantee optimum nutrient nective tissue environment has come into focus only supply. In 1990, Koide et al. (1990) described a novel, recently. semiadhesive substratum method for spheroid cultiva- In addition to endothelial cells which are mainly tion taking advantage of the spontaneous detachment of involved in tumour angiogenesis and neovascularization small aggregates formed on semiadhesive substrates (for review: Folkman 1990; Mareel et al. 1990; Bracke et such as proteoglycans or positively charged polystyrene. al. 1992), tumour-associated immunocompetent cells Spheroid diameters to estimate spheroid volume q 1998 Blackwell Science Ltd, International Journal of Experimental Pathology, 79, 1–23 Heterologous 3-D cocultures 3 growth curves are conventionally measured with a reti- resonance microimaging, i.e. to provide information on cule manipulated by a micrometer screw in the eyepiece intracellular water-diffusion coefficients in individual of an inverted microscope. More automatic methods for MCTS (Sillerud et al. 1990; Neeman et al. 1991). Other measurements of growth curves or for size-dependent methods such as the Lowry technique or biolumines- sorting of spheroids are based on microscope-image- cence used to investigate steady state levels of ATP, analyser, flow cytometry or laser-diffraction particle sizer. lactate, and glucose have to be carried out on shock- The Gompertz equation first introduced by Winsor (1932) frozen/fixed spheroid sections (Walenta et al. 1990; has been shown to mathematically describe not only Teutsch et al. 1995). Since the sensitivity of the latter early tumour growth as summarized in the early 60s by methods is clearly beyond cellular level, selective spher- Laird (1964, 1965) but has also been applied to a variety oid dissociation prior to single cell analysis by flow of spheroid types in the literature (e.g. Marusic
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