The multilayered postconfluent cell culture as a model for drug screening

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Abstract

New drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumours and to decrease the use of experimental animals. In this paper, we review the characteristics and scope of a relatively simple cell-culture system with a three-dimensional organisation pattern — the multilayered postconfluent cell culture model. Solid tumour cell lines from diverse origins when grown in V-bottomed microtiter plates reach confluence in 3–5 days and then start to form multilayers. The initial exponential growth of the culture is followed by a plateau phase when cells reach confluence. This produces changes in the morphology of the cells. For some cell lines, it is possible to observe cell differentiation. A substantial advantage of the system is the use of the sulforodamine B (SRB) assay to determine relative cell growth or viability, which allows semiautomation of the experiments. Several experiments were performed to assess the differences and similarities between cells cultured as monolayers and multilayers, and eventually, compared with the results for solid tumours and some other models such as spheroids. Cell-cycle analysis for multilayers showed a lower S-phase arrest, which is accompanied by a decrease in the expression of cell-cycle-related proteins and a decrease in cellular nucleotide pools. Gene and protein expression of topoisomerase I, topoisomerase II and thymidylate synthase expression were lower for multilayers, but no substantial changes were observed for the expression of DT-diaphorase. P53 expression increased. Multilayer cultures present distinctive properties for drug transport across the membrane, drug accumulation and retention. In fact, the transport of antifolates across the membrane, accumulation of topotecan and gemcitabine-triphosphate are reduced in multilayers when compared with monolayers, which may be related to a decrease in drug penetration to the inner regions of the multilayers. Alteration of these pharmacodynamic parameters is directly related to a decrease in drug activity. The most powerful application of multilayers is in the assessment of cytotoxicity. Solid tumour cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The data show that multilayers are more resistant to the drugs than the corresponding monolayers, but there are substantial differences between the drugs depending on culture conditions, e.g. the difference was rather small for a drug such as cisplatin, miltefosine and EO9, a drug, which is activated under hypoxic conditions. Gemcitabine was active against ovarian cancer but not against colon cancer, resembling the in vivo situation. This observation was not evident with monolayer experiments. Another interesting application is the possibility to perform drug combination studies. The combination of gemcitabine and cisplatin proved to produce selective cell kill in H322 cells (non-small cell lung cancer cell line). Neither of the drugs was independently able to produce similar effects. In summary, multilayer cultures are relatively simple three-dimensional systems to study the effect of microenvironmental conditions on anticancer drug activity. The model might serve as a base for a more rigorous secondary in vitro screening.

Introduction

In the past 50 years, the mass screening of either synthetic derivatives or natural products has led to the discovery of the currently utilised anticancer drugs. Nowadays, because most cancers in advanced stages are not curable by chemotherapy, new drug development still continues to play a major role in the fight against cancer. Chemosensitivity testing in vitro is crucial in both random drug screening and the rational design of novel anticancer drugs. Until 1985, the National Cancer Institute (NCI; Bethesda, USA) primary screenings were performed preferentially on the in vivo L1210 and P388 murine leukaemia models [1], [2]. The major drawback of these systems is that selected drugs show remarkable clinical activity against human leukaemia and lymphoma, while presenting marginal or no activity against the commonest types of human solid tumours (e.g. lung, colon, mammary carcinomas). To overcome this disadvantage, the NCI in 1985 started to use an in vitro assay as the primary cancer screen with a new strategy, the disease-oriented approach. Hence, a panel of sixty human tumour cell lines, derived from diverse cancer types (lung, colon, breast, prostate, ovary, kidney, renal, ovarian, brain, melanoma and leukaemia) is currently employed for cancer drug discovery [1]. The ultimate goal of this disease-oriented screening is to facilitate the discovery of new compounds with potential cell line-specific antitumour activity. Among the many economical advantages, this system allows automation and avoids the use of laboratory animals. In this model, cells are grown in 96-well microtiter plates as subconfluent monolayer cultures.

It has been shown that drugs resulting from this screening system do not always present the same activity when they are further tested against solid tumours. In fact, this monolayer culture does not completely resemble the in vivo situation. Initial attempts to simulate in vivo microenvironment conditions led investigators to use postconfluent [3], [4], [5] or plateau phase tumour cell cultures [6], [7], [8]. Contrary to exponentially growing monolayer cultures, these systems have shown a degree of selectivity for anticancer drug testing. Although monolayers partially mimic cell–cell interactions and growth physiology, they have only a two-dimensional structure and not the three-dimensional structure of a solid tumour. Moreover, monolayer cultures are oversimplistic, since they do not mimic the complex and heterogeneous properties of tumours. This leads to incorrect predictions of efficacy when drugs are tested in vivo; in particular the poor blood supply to solid tumours introduces several factors that may influence the effectiveness of chemotherapy and radiotherapy. These factors include gradients of oxygen tension, extracellular pH, nutrients, catabolites, cell proliferation rates and drug penetration barriers, all of which vary as a function of distance from a supporting blood vessel (Fig. 1). In addition, the stressful conditions of this microenvironment can influence the expression and activity of specific molecular targets and hence, monolayer cultures used in clonogenic or non-clonogenic assays do not represent an accurate model for the screening of drugs against solid tumours. There is an increasing emphasis not only on the use of a greater variety of specific types of cancer, but also on new solid tumour models at the initial stages of screening.

Several experimental models that resemble the three-dimensional features of tumours have been developed, such as collagen-gel cultures, mesh-supported organoid cultures (also called histocultures) and multicellular spheroids. These models mimic the conditions of tumours and may form the basis of more rigorous secondary in vitro screening procedures designed to identify drugs with selectivity for solid tumours.

In the collagen-gel culture system, a gel prepared from rat tail collagen fibre provides the substrate for the embedding of a single cell suspension or a small tumour specimen (0.5 mm3) [9], [10]. The three-dimensional structure of the tumour can be preserved. Three-dimensional aggregates can be kept viable and proliferating for weeks showing very good degrees of resemblance compared with the in vivo tumours from which they were derived [11]. Histocultures are similarly based on embedding a tumour specimen in a cellulose or collagen containing sponge matrix [12], [13] that show a certain degree of complexity. This is due to their originating tumour cells, non-tumour cell subpopulations and extracellular matrix components. A large number of tumour cell types can be cultured with this system and, in some cases, can be kept viable for longer than a year [14]. Contrary to the models described above, the multicellular spheroid system has cells that are not embedded in a matrix. To obtain three-dimensional aggregates, single cell suspensions are usually kept in constant spinning movement [15], [16], [17], but spheroids can also be used in multiwell plates as used by Smith and colleagues [18], [19]. With this model, spheroids can contain different cell proliferation and metabolic gradients, such as an outer rim of viable and actively proliferating cells and an inner region containing necrotic regions [16].

The three-dimensional models described above usually show more resistance to treatment with anticancer drugs than cells growing as monolayers. They might be regarded as better models for the biological and biochemical characteristics of human solid tumours than monolayer subconfluent cell cultures. Collagen-gel matrix cultures, histocultures and spheroids offer unique opportunities for the study of human solid malignancies. Although the greater complexity of these models is appealing, they do possess the major drawback of more time and labour consuming culturing techniques. This is one of the main reasons why they have not been extensively used as screening systems [17], [20], [21], but their use has usually been limited to specific research questions, or as a secondary screening model. Especially, spheroids have been used most extensively for drug screening and testing the role of culture conditions.

In vitro chemosensitivity tests combining semiautomatic technology and more in vivo representative cell culture systems may be very useful in anticancer drug screening. This is the rationale for the development of the multilayered postconfluent cell culture model. In this system, tumour cells are grown in V-shaped wells showing an organisation pattern resembling that found in spheroids. This model combines semiautomatic techniques with a more complex cell culture system.

Section snippets

The multilayered postconfluent cell culture model

In this model, solid tumour cells are cultured in V-bottomed 96-well microtiter plates [22]. This particular shape allows cells to grow in a specific three-dimensional manner.

Biochemical and biological characterisation of the model

We have seen that cells cultured as monolayers or multilayers can display different characteristics in terms of growth and morphology. These differences may arise as a result of internal changes within the cells, and the use of more than one technique confirmed this conclusion.

Pharmacodynamics

The particular shape of multilayer cultures may affect the transport of drugs and provides an excellent model for pharmacodynamic studies. Factors such as the proliferative status of the cells influence the uptake, efflux or metabolism of drugs, and therefore alter their cytotoxic effects, as has also been described for other postconfluent cultures [8], [52]. Cell differentiation observed in multilayer cultures can affect carrier-mediated transport of drugs inside the cell.

Cytotoxicity determination and Rf factor

Cellular response is evaluated by measuring the absorbance of each well content (SRB assay) and using the parameters employed by the NCI [66]. The effect is defined as percentage of growth (PG), where 50% growth inhibition (IC50), total growth inhibition (TGI) and 50% cell killing (LC50) represent the concentration at which PG is +50, 0 and −50, respectively. These parameters are extrapolated from the dose–response curves. However, for multilayers the calculation of LC50 values is not always

Conclusion

Solid tumour cells cultured in V-bottomed microtiter plates display a pattern of organisation that mimics microenvironmental conditions occurring in the in vivo situation. We have observed marked differences in metabolism and sensitivity between the monolayers and multilayers, which are in favour of the multilayer system to predict selective chemosensitivity in solid tumours. A decrease in proliferative status or gene expression, changes in enzyme activity, modification of carrier-mediated

Reviewers

M.C. Bibby, Professor of Cancer Biology, Clinical Oncology Unit, University of Bradford, Bradford, Bradford, West Yorkshire BD7 1DP, UK.

Gary K. Smith, Department of Molecular Biochemistry, Glaxo Wellcome, 5 Moore Drive, Research Triangle Park, NC 27709, USA.

Acknowledgements

JMP acknowledges the Fundación Doctor Manuel Morales (La Palma, Islas Canarias, Spain) and the UICC for postdoctoral grants. This research was also supported by a grant from the ‘Platform Alternatives for Animals’ and the European Union by grant BMH4-CT96-0479.

José Manuel Padrón (1968) did his B.Sc. and Ph.D. in Pharmacy in 1991 and 1996, respectively, at the University of La Laguna (Canary Islands, Spain) on the asymmetric organic synthesis of biologically active compounds at the Instituto Universitario de Bio-Orgánica Antonio González under the supervision of Professor Vı́ctor S. Martı́n. Two Scholarships from the Fundación Dr Manuel Morales and from the UICC, enabled him to perform part of the present work as a Post Doctoral Fellow at the Vrije

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    José Manuel Padrón (1968) did his B.Sc. and Ph.D. in Pharmacy in 1991 and 1996, respectively, at the University of La Laguna (Canary Islands, Spain) on the asymmetric organic synthesis of biologically active compounds at the Instituto Universitario de Bio-Orgánica Antonio González under the supervision of Professor Vı́ctor S. Martı́n. Two Scholarships from the Fundación Dr Manuel Morales and from the UICC, enabled him to perform part of the present work as a Post Doctoral Fellow at the Vrije Universiteit in the group of Dr Peters. Since 1998 he is a TMR Fellow at the Life Sciences Competence Centre of DSM Research.

    Dr Godefridus J. Peters (1952) is head of the division of Pharmacology of the Department of Medical Oncology of the University Hospital Vrije Universiteit in Amsterdam, The Netherlands. In 1992 he was appointed as associate professor. His research interests include the pharmacology of various anticancer agents, including antimetabolites, platinum analogues, and drug combinations. For (pre) clinical drug development, both animal models and the present alternative methodology have been developed with the aim to optimise treatment by determining parameters of response.

    1

    Present address: DSM Research, FC-OCB, P.O. Box 18, 6160 MD Geleen, The Netherlands.

    2

    Present address: CPOBA-UNICAMP, Campinas, Brazil.

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