Multicellular spheroids as an in vitro tumor model

doi:10.1016/S0304-3835(98)00198-0

Cancer Letters

Volume 131, Issue 1, 11 September 1998, Pages 29-34

https://doi.org/10.1016/S0304-3835(98)00198-0 Get rights and content

Abstract

Multicellular spheroids (MCS) have been used as an in vitro model system of micrometastases and avascular tumor regions for studying cell adhesion-dependent resistance to cytotoxic drugs and possible reversal by chemosensitizers and adhesion-reversing agents. Multicellular drug resistance has been linked to limited accessibility of cell subpopulations, active drug efflux, quiescence of cells in deeper layers due to cell contact inhibition and adverse microenvironmental conditions like acidic extracellular pH, hypoxia and nutritional depletion. The shortcomings of MCS as a tumor model include limited knowledge of the mechanisms leading to necrosis/apoptosis of core cells, the production of an extracellular matrix (ECM) by tumor cells instead of intratumoral normal cell populations and the complex relationship of MCS parameters like size, growth regulation, synthesis of ECM components and others on the origin and pretreatment of the tumor cells and specific culture conditions.

Introduction

Sutherland et al. 1, 2established multicellular spheroids (MCS) as an in vitro model for the systematic study of tumor response to therapy. MCS are spherically symmetric aggregates of cells analogous to tissues, with no artificial substrate for cell attachment. This system has been widely used as a model for microenvironmental effects on basic biological mechanisms, such as the regulation of proliferation, metabolism, differentiation, cell death, invasion, angiogenesis or immune response [3]. Other applications include the study of metastasis, embryogenesis and artificial tissue modeling [4]. One major advantage of three-dimensional cultures is their well defined geometry, which makes it possible to directly relate structure to function and gene expression. Compared to conventional monolayer cultures, cells in three-dimensional aggregates more closely resemble the in vivo situation with regard to cell shape and cell environment, which in turn can affect the gene expression and biological behavior of the cells and in addition MCS resemble an in vitro system of intermediate complexity between monolayer cultures in vitro and tumors in vivo. Increased resistance to ionizing radiation and later on to cytotoxic drugs by MCS was identified by Durand and Sutherland, who created the term contact effect for this phenomenon [1]. In the three following decades the cell biology of MCS and the knowledge of the mechanisms to be held responsible for the increased drug resistance of MCS advanced considerably 3, 4.

Section snippets

Cell biology of MCS

There exist different methods to initiate MCS from monolayer cultures of various cell lines derived from gliomas, fibroblasts and carcinoma cells (prostate, colon, breast, ovary, pancreas and others). MCS are formed in liquid overlay cultures (with the agarose coat preventing cell adhesion to culture flasks or dishes) upon mechanical stirring of cell suspensions in glass flasks or tissue culture vessels and in rotating wall microgravity vessels 5, 6. Stirring may select subpopulations of cells

ECM in MCS

The existence of an extracellular matrix was investigated in an early study in U-118 MG glioma cells and HTh-7 human thyroid cell spheroids [16]. The presence of fibronectin, laminin and collagen was detected by immunofluorescence staining and the presence of glycosaminoglycans was detected by radioactive labeling and extraction. The composition of an extracellular matrix (ECM) may be differently regulated in tumors, spheroids and monolayer cultures [4]. An ECM consists of a network of

Cell adhesion molecules in MCS

The cell surface contains specific molecules involved in cell–cell and cell–matrix binding and these receptor/ligands are additionally functional in the transduction of extracellular signals to intracellular molecules [23]. Four major families, namely CAMs (cell adhesion molecules), cadherins, selectins and integrins, and a number of other unrelated cell surface molecules, including the hyaluronic acid binding CD44 antigen, have been characterized. Integrin receptors are downregulated in

MCS and cytotoxic drugs

Cells isolated from MCS are generally more resistant to cytotoxic drugs than the same cells grown as monolayers 1, 2. A multicellular-mediated resistance to alkylating drugs was found in MCS from the EMT-6 mouse mammary tumors [26]. The same contact effect of MCS resistance previously found for ionizing radiation was held responsible for the resistance to cytotoxic drugs [27]. Intercellular communication via gap junctions, specific DNA packaging favoring increased repair in spheroids and other

Angiogenesis

MCS seem to be resistant to invasion by endothelial cells in co-culture. However, spheroids may be implanted into the peritoneal cavity of experimental animals where they eventually become vascularized and can be harvested for further studies [40]. VEGF (vascular endothelial growth factor) is upregulated in such glioma spheroids in hypoxic or hypoglycemic microenvironments [8]. Studies to develop an in vitro model of angiogenesis using MCS are in progress.

Conclusion

MCS have been studied in vitro as a representative model of avascular tumor regions in vivo and these investigations have provided a host of new results mainly concerning the role of the tumor microenvironment in response to therapeutic strategies. Most of this work has been done using several well established tumor lines derived from glioblastomas, prostate, mammary and other carcinomas. MCS are highly complex systems and their cellular properties are dependent on the origin of the tumor

Acknowledgements

This work was supported by a grant (#6960) from the Austrian National Bank.

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View full text

Multicellular spheroids as an in vitro tumor model

Abstract

Introduction

Section snippets

Cell biology of MCS

ECM in MCS

Cell adhesion molecules in MCS

MCS and cytotoxic drugs

Angiogenesis

Conclusion

Acknowledgements

Eur. J. Cancer Clin. Oncol.

Growth of nodular carcinomas in rodents compared with multi-cell spheroids in tissue culture

Growth

Growth of multicellular spheroids in tissue culture as a model of nodular carcinomas

J. Natl. Cancer Inst.

Cell and environment interactions in tumor microregions: the multicell spheroid model

Science

Three-dimensional cell cultures: from molecular mechanisms to clinical applications

Am. J. Physiol.

Cell culture for three-dimensional modeling in rotating-wall vessels: an application of simulated microgravity

J. Tissue Culture Methods

Physical mechanisms of cell damage in microcarrier cell culture bioreactors

Biotech. Bioeng.

Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implications for tumor angiogenesis

Proc. Natl. Acad. Sci. USA

Detection of hypoxic cells by monoclonal antibody recognizing 2-nitroimidazole adducts

Cancer Res.

Rhabdomyosarcoma spheroids with central proliferation and differentiation

Cancer Res.

Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation

J. Cell Biol.

Proliferation and oxygenation status of WiDr spheroids in different lactate and oxygen environments

Oncol. Rep.

Three-dimensional cell culture induces novel proliferative and metabolic alterations associated with oncogenic transformation

Int. J. Cancer

Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant C-H-ras oncogene expression

J. Cell Biol.

A retroviral wild-type p53 expression vector penetrates human lung cancer spheroids and inhibits growth by inducing apoptosis

Cancer Res.

Demonstration of a extracellular matrix in multicellular tumor spheroids

Cancer Res.

How and when do tumor cells metastasize?

Crit. Rev. Oncol.

Comparison of extracellular matrix in human osteosarcomas and melanomas growing as xenografts, multicellular spheroids, and monolayer cultures

Anticancer Res.

Extracellular matrices in multicellular spheroids of human glioma origin: increased incorporation of proteoglycans and fibronectin as compared to monolayer cultures

APMIS

Collagens, integrins and the mesenchymal drift in glioblastomas: a comparison of biopsy specimens, spheroid and early monolayer cultures

Int. J. Cancer