Issues to be considered when studying cancer in vitro

Critical Reviews in Oncology/Hematology 85 (2013) 95–111

Issues to be considered when studying cancer in vitro ∗ ˇ Beata Cunderlíková International Laser Centre, Ilkoviˇcova 3, 841 04 Bratislava, Slovakia Accepted 27 June 2012

Contents 1. 2.

3.

4.

5.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell modifications during in vitro cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Representativeness of tumour cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cell modifications due to culturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Cultured vs. uncultured cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lack of relevant interactions in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Extracellular matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Stromal cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Evidence from animal studies supporting importance of tumour cell environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Species specificity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lack of structural context in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Spatial organization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. ECM stiffness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Various cancer treatment approaches have shown promising results when tested preclinically. The results of clinical trials, however, are often disappointing. While searching for the reasons responsible for their failures, the relevance of experimental and preclinical models has to be taken into account. Possible factors that should be considered, including cell modifications during in vitro cultivation, lack of both the relevant interactions and the structural context in vitro have been summarized in the present review. © 2012 Elsevier Ireland Ltd. All rights reserved. Keywords: In vitro; Cell culturing; Extracellular matrix; Tumour microenvironment; Tissue stiffness

1. Introduction Intensive efforts within cancer research resulted in a number of novel anticancer agents. Positive results of treatments with some of them have been documented in patients. The most promising examples include anti-epidermal ∗

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growth factor receptor (EGFR) monoclonal antibodies in some patients with metastatic colorectal cancer [1] and metastatic breast cancer [2] or tyrosine kinase inhibitors in some patients with chronic myelogenous leukemia [3] or non-small cell lung cancer [4]. However, the efficacy of many of the newer biological and targeted therapies is confirmed in some individuals only. For example, only patients with colon tumours expressing wild-type KRAS or with HER2/neu-positive breast cancer are likely to benefit from

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the treatments with the anti-EGFR monoclonal antibodies [1]. Treatment with tyrosine kinase inhibitor (imatinib) is efficient in patients with BCR-ABL translocations [5]. Except of the encouraging achievements, however, negative results of clinical trials are also reported. To mention some of them, treatment with matrix metalloproteinase (MMP) inhibitors showed no survival advantage for the patients compared to conventional treatment modalities or even lead to poorer survival and had to be terminated [6–8]. Clinical trials with Fe-chelator Triapine® as anticancer drug, either alone in patients with metastatic renal cell carcinoma [9] or in combination with gemcitabine in patients with advanced pancreatic adenocarcinoma [10], were terminated as they did not give any benefit for the patients at the used doses and schedules. Clinical trials with antiEGFR agent-combining regimes in advanced non-small cell lung cancer patients showed no survival advantages over chemotherapy alone [11]. Based on in vitro observations of a dose–response effect, high-dose chemotherapy with stemcell transplantation has been evaluated for the treatment of various solid cancers and compared with conventional modalities. Although event-free survival was often found to be advantageous in favour of high-dose chemotherapy, no significant benefit in overall survival, apart from few exceptions, or even negative results were reported [12,13]. Current gene therapy has not proven very successful in clinical trials [14,15]. Understanding the reasons for the failures of clinical trials, in spite of promising preclinical results, is of high importance from scientific point of view. While searching for them, the relevance of experimental and preclinical models has to be considered. Predictions of treatment efficacies in clinical trials based on in vitro studies [16] and even preclinical mouse models [8] were reported to lead to disappointments in a number of cases. For example, Fe-chelator Dp44mT did not lead to Fe depletion within the tumour, despite its high activity at inhibiting Fe uptake from transferrin and inducing Fe mobilisation from cells in cultures [17]. Many of tumours that relapsed after treatment by radiotherapy appeared within irradiated regions, despite in vitro and preclinical data indicating that the doses used would kill >99% of both the tumour and endothelial cells [18]. Moreover, in a randomized trial of a regime combining application of a monoclonal antibody against EGFR, there was no correlation between EGFR expression and tumour response and/or symptom improvement in patients with non-small cell lung cancer [11]. In a phase III trial with a humanized monoclonal antibody against vascular endothelial growth factor (VEGF), VEGF expression, assessed by in situ hybridization in primary tumour samples, did not correlate with objective response in advanced breast cancer patients [11]. Apart from inconsistent findings between in vitro studies and clinical trials, there appear also reports that document discrepancies between results obtained under in vitro and in vivo (e.g. [19–26]) or ex vivo (e.g. [25,27]) conditions. In fact, many researchers realized limitations of usually used

in vitro and in vivo experimental systems already long time ago. It has been recognized that cell culture systems, and even animal carcinogenesis models, may not accurately represent the complexity and the true physiologic state of the diseased human tissue [28] and simplification inherent to vitro approaches might be achieved at the cost of physiological relevance [29]. In order to be able to correctly interpret findings of in vitro and preclinical experiments, it is crucial to understand the physiological and pathological relevance of the experimental conditions. Therefore, factors governing cell behaviour need to be precisely identified and the alterations of cells in cultures that are artifacts of culturing (e.g. cell modifications due to long-term culturing or cell processing) need to be distinguished. Unfortunately, inconsistency in experimental protocols and conditions used makes comparisons of the results among laboratories not easy. Issues that should be considered in experiments utilizing in vitro cell cultures and in interpreting their findings, especially when studying cancer, have been summarized in the present review.

2. Cell modifications during in vitro cultivation Use of cell cultures offers considerable simplification for biologically oriented research. Nevertheless, evidence suggests that cells undergo changes when placed in cultures [30–35] (Tables 1 and 2), which gives rise to doubts whether cell lines are representatives of their original tissues [36–38]. 2.1. Representativeness of tumour cell lines Many tumour cell lines have been derived from metastases, pleural or ascitic effusions (references in [36,39]). Such cells constitute a population of cells in a late stage of tumour evolution (references in [36]), which may differ from their corresponding primary tumours with respect to ploidy [36], antigenicity and immunogenicity and therefore the response to therapies [40,41]. Cell lines developed from primary tumours were claimed to be representatives of the tumour specimen from which they were derived [36,42,43]. Nevertheless, difficulties to establish even primary cultures from primary tumours [36,39,44–46] with majority of outgrowths arising from normal cells within the specimen [45,46] have often been reported. Success rates for cell line establishments from primary and metastatic breast cancers were 10% and 25%, respectively [47]. Low success rates in the range of 1–10% were reported for the establishment of leukaemia–lymphoma cell lines. It was suggested that the original primary cells might need to possess special features that make them prone to immortalization in vitro and only samples containing such cells may evolve into cell lines [48]. It was even mentioned that only rare specimens of breast cancer develop into cell lines [33,46]. Furthermore, the growth of primary carcinoma cells was found to be considerably slower than that of carcinoma cell lines (references in [45]). This is

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Table 1 Cell modifications observed during conventional cell culturing and processing. Influencing factor

Consequence

Reference

Substrate (culturing on plastic)

Cell dedifferentiation (loss of tissue specific gene expression, differentiated functions and structural organization) Modification of the expression of cell type markers Decrease in cell doubling time Chromosomal changes Development of features characteristic of transformation Reversion of properties of transformed phenotype to normal one Loss of chemoresistance-mediating factors Ploidy changes

[20,22,34,51,52,72,77–79]

Reduction of receptor binding sites Cell stability (possibly)

[98] [35]

Duration (extended time in culture/passaging)

Processing (extended storage at −70 ◦ C) (repeated treatment with EDTA-trypsin)

clearly opposite to the situation that we face within in vitro studies, where it is easy to culture tumour cell lines, while “normal” ones require more demanding conditions. 2.2. Cell modifications due to culturing Dedifferentiation on plastic. The changes in cell behaviour may be induced by environmental conditions during culturing, namely by extracellular matrix (ECM, [22,49–69]), cell culture substrate [70–75] as well as by composition of the cultivation medium [35,39,50,52,76]. Importantly, the most remarkable modifications compared to in vivo conditions were reported to occur in cells cultured on the most frequently used substrate-plastic. Dedifferentiation following cell culturing on plastic [20,22,51,52,72,77,78]

[39,76,84,85,99] [33,35] [33,35] [33] [75,104,105] [101] [36]

is accompanied by the loss of expression of tissue specific genes, differentiated functions as well as capability to form organized structures [34,51,77–79]. Re-appearance of various differentiated properties could be induced by providing the cells with microenvironment more closely mimicking in vivo conditions (references in [19,51,80], Fig. 1). Although these conclusions come mostly from the experiments with epithelial cells, the process of dedifferentiation has been rather extensively studied also in isolated adult rat cardiomyocytes [81–83]. The cells isolated from normal hearts acquired a number of properties of hypertrophic rather than normal myocytes in “long-term” cultures [82]. Vimentin appearance. Another property induced by cell culturing on plastic (Table 1), which is not consistent with the situation in vivo, is appearance of vimentin filaments

Table 2 Factors that should be considered when working with in vitro cell cultures. Representativeness of tissue Stability (permanent phenotypic changes due to processing, passaging, storage, medium composition, etc./genotypic drift) Context-dependent phenotype/dedifferentiation on plastic Cell-type specific behaviour

[36–46,248,249]a [33,35,36,76,98,99,102,103]

“Correct” ECM composition Matrigel (basement membrane) Type I collagen (tumour stroma) Fibroblast-derived matrices (normal, primed, activated stroma) Tissue-extracted ECM Artificial-material based matrices Co-cultures (fibroblasts, myoepithelial cells, astrocytes, macrophages, etc.) Source Type Abundance

[58,69,161,179] [150,151] [69,232,237,257] [152–154]

Structural context

3 Dimensionality ECM fibre architecture Tissue-relevant ECM stiffness

[69,100,108,161,202,227–237] [57,120,152–154,238,239] [157,237–243,247]

Others

Species dependency Hematopoietic/immune system Vascular/lymphatic system Interstitial fluid flow, local gradients, etc.

[219–223] [18,117,180,181] [161] [159]

Cell properties

Interactions cell–ECM (biochemical)

cell–cell

a

Examples of literature, including review articles with references to relevant seminal work, are included in the table.

[20,22,49–83] [58,59,63,139,161,250–256]

[155] [156–161] [66,67,130–137,144–147,199–207] [87,88,189–194] [152,182,185,186,188,194] [86,194]

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Components: conventional 2D culture

tumor epithelial cells non-tumor epithelial cells tumor-associated fibroblasts normal fibroblasts tumor-associated myoepithelial cells

D A

normal myoepithelial cells tumor-associated macrophages/other stromal cells normal macrophages/other stromal cells ECM fiber

3D culture mimicking in vivo microenvironment

basement membrane

Interactions: through direct contact through released substances

C normal ECM

activated ECM B

Fig. 1. Culturing of cells under conventional and in-vivo mimicking conditions. Components of an in vivo-mimicking culture as well as interactions important for cell behaviour and function well documented in the literature are shown. Additional factors that need to be considered are listed in Table 2. Depending on cell type and ECM used, the cells in 3D cultures may form different 3D structures, such as polarized acini with central lumens (A), polar reverted spheroids (B), disorganized colonies (C) or tubules (D).

in majority of cultured cells of epithelial and muscle origin [84,85]. The vimentin filaments could be sometimes detected even together with another type of intermediate filaments [84]. Vimentin is a major cytoskeletal component of mesenchymal cells and it is therefore frequently used as a marker of mesenchymal cells as well as cells undergoing epithelial– mesenchymal transition, whereas cytokeratins are used as markers of epithelial cells. Applications include identification and characterization of cell types [39,86–88] including cells with stem cell properties [89–92], evidence of epithelialmesenchymal transition [93] or detection of tumour micrometastases [94]. Expression of vimentin induced by cell culturing on plastic should be considered in these cases, but also with regard to the role of cytoskeleton in intracellular signalling and mechanotransduction [54,95–97]. Alterations due to cell processing. Alterations of the cultured cells could be caused by cell processing, as a consequence of extended storage at −70 ◦ C [98], repeated treatment with EDTA–trypsin [35], extended time in the culture and increased number of cultivation passages (Table 1). The latter two factors were reported to lead to decrease in doubling time and chromosomal changes [33,35], changes in adhesion complexes and increased potential to form tumours [33] or altered keratin expression [39,76,99] in epithelial cell lines, increase in proliferation of lipocytes [100] or to changes in phenotype of transformed fibroblasts [75]. Progressive loss of factors mediating chemoresistance on passaging of cells from metastases but not from primary tumours in monolayers was reported [101] and loss of tumourigenicity with cell passaging is also observed. Nevertheless, it was concluded that cell lines are relatively stable if they are kept under the same experimental conditions, however, when they are exposed to

different environments, different variants could be selected [102,103]. The appearance of cell variants with reversion of the properties of transformed cells to the parental or more normal phenotype [75,104,105] indicates rather poor control over the cell cultures. The reversion was found to be influenced by time of in vitro cell cultivation, cell density affecting cell spreading and cell shape [105], intercolony contact or by addition of substances such as cyclic AMP, DMSO and others to cell cultures [75]. Ploidy. A controversy has been noticed with regard to ploidy of the cultured cells. Selection in vitro for diploid cell populations mostly due to their better attachment to glass substrates has been reported [36]. On the other hand, majority of breast tumour cell lines was reported to be aneuploid [36]. Although these cell lines were usually established from metastasis and pleural effusions, and even after mutagenizing treatment procedures [36], suggesting that the cells may be originally aneuploid, the possibility of the evolution to aneuploidy could not be excluded [35]. Thus, it was mentioned that even cells from normal breast may become aneuploid in culture (references in [36]). In a study comparing cell lines with corresponding tumour tissues, poor correlation was noted in ploidy indices [42]. 2.3. Cultured vs. uncultured cells Distinct cell behaviour was documented when comparing cultured with uncultured cells [19,22,24,44,57] as well as primary cell cultures with cell lines [106–108]. Follicular lymphoma cells purified from lymph nodes of patients had little tendency to die in vivo, but they underwent apoptosis in vitro and expression of death suppressor bcl-xL declined

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rapidly within one day in culture [109]. Downregulation of bcl-xL and mcl-1 occurred also in germinal center B cells freshly purified from tonsils after several hours in culture [109]. Several attempts have been made to compare cultured tumour cells or tumour cell lines with cells from biopsies of various tumour tissues [30,37,38,110–112] by genomic or proteomic analyses. The comparisons have been hampered by cell heterogeneity of tissue biopsies containing non-tumour cells [113], still, modifications of cells occurring in vitro when cultures were established from primary tumours were suggested as an alternative explanations [30,37,38]. For example, comparison of protein profiles assayed by two-dimensional (2D) electrophoresis of non-invasive lowgrade superficial bladder transition cell carcinomas and their primary cultures has shown that short-term culturing of the lesions resulted in the altered expression of several proteins [30]. Differences in the expression of high-molecular-weight tropomyosins were found by the same method also when comparing tumour cells extracted from breast carcinoma tissues, non-cancerous human breast lesions and a number of breast carcinoma cell lines [37]. Different expression of several genes was found when serum-starved cells from marrow samples of patients with acute myeloid leukemia (AML) were compared with AML cell lines [112]. Further study compared tumour cells directly harvested from primary breast tumours by differential trypsinization, early culture passages of such cells and immortal cell lines frequently used in cancer research. Gene expression profiles of tumour cells harvested from primary breast tumour tissue were more consistent with those of the corresponding tumour cell cultures than with immortal cell lines [114]. In a study comparing broad panel of cell lines with primary tumour samples enriched for at least 50% malignant cells it was demonstrated that around 30% genes were differentially expressed in cell lines [115]. All these data indicate possibility of cell modification due to culturing that needs to be taken into account when interpreting experimental results. In case that differences in protein and gene expressions between cultured and uncultured cells are due to modifications of the cultured cells and not due to presence of contaminating cells or heterogeneity of cell population, then the results of experiments performed with cultured cells may not be applicable for in vivo situation if altered genes or proteins are involved in the studied pathway or process1 . 3. Lack of relevant interactions in vitro In the majority cases, cell culture experiments utilize monocultures, i.e. monolayers or suspensions of cells of one type (Fig. 1). In sharp contrast, solid tumours are not 1

For an example from author’s experience, when results obtained from experiments using cell line cultures were not consistent with those from studies utilizing non-cultured ex vivo samples and in vivo models, please see [25]. Modifications of the cells during cell culturing and/or lack of components of an in vivo system may be responsible.

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clones of cancer cells only. They are composed of multiple other cell types and ECM that constitute tumour stroma and together interact with the rest of the organism [116]. As a result, tumour behaviour is not dependent exclusively on the properties of the tumour comprising cancer cells, but it is affected by what happens outside them in the tumour microenvironment [117,118]. Several lines of evidence suggest that even though the initiating mutation in the cancers of epithelial origin usually occurs in the epithelium, it is the stroma that is involved in promotion, and in some cases even in initiation, of tumour progression [119]. The importance of the stroma of solid tumours has been recognized as summarized in several review papers [116,118,120]. However, behaviour of tumour cells is dependent on the stroma not only in solid tumours. The bone marrow microenvironment was suggested to contribute to drug resistance in variety of hematopoietic malignancies (for review see [121–123]). ECM, namely fibronectin [124–129], as well as stromal cells [130–135] are shown to be involved. 3.1. Extracellular matrix ECM produced in vitro. The ECM consists of multiple components, type and abundance of which depend on the stroma and epithelium within the specific tissue and interactions between them [108,136,137]. Therefore, although cells in vitro are able to produce their own ECM, this is likely to differ from the ECM that the cells sense in vivo, especially in the case of monocultures on plastic (e.g. [138]). Fibroblasts are believed to be the main source of ECM, however, other cell types are also capable of producing ECM components in vitro [67,71,136,139–143]. The ECM deposited by cell cultures may depend on cell type [67,139]. Importantly, while fibroblastic clone of spontaneously immortalized, nontumourigenic mouse mammary cell line in pure culture lacked deposited laminin on the cell surface, it was sporadically seen on the surface of the epithelial clone cultured alone. Co-culture of cells of the epithelial clone on a monolayer of cells of fibroblastic clone gave rise to a large increase in laminin deposition in contact areas between epithelial cell clusters and fibroblastic layer [144]. Likewise, deposition of ECM components in monocultures either of Sertoli or peritubular cells, was minimal in the absence of serum. It became appreciable in co-cultures of the two cell types [145]. In cultures of mammary epithelial cells alone, little or no extracellular fibrillar material could be detected, with some of it seen after two week incubation. However, it could be seen when mammary epithelial cells were grown on fibroblasts or adipocytes [146]. Moreover, co-culturing with breast adenocarcinoma cells was reported to enhance collagen synthesis by fibroblasts [147]. Thus, co-culturing of epithelial with mesenchymal cells considerably affects ECM production and deposition in cell culture. The presence of serum [145], duration of a culture [145], most probably due to increase in cell density and cellular contacts [63,136,148,149] and culture substrate [139] are other factors

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involved. For example, in vitro experiments showed that both the ultrastructure and ECM composition could be considerably altered when cells are plated on the matrix derived from the Engelbreth–Holm–Swarm mouse sarcoma as opposed to plastic, exemplified by production of collagen III and not of collagen I and remarkable changes in proteoglycan levels [100]. Deposition of collagen IV was found to be increased when mammary epithelial cells were cultured in collagen I matrices as compared to plastic [74,136]. In vivo ECM-mimicking cell cultures. With the intention to provide cultured cells with in vivo-like environment, several culture systems have been introduced (Fig. 1). They include matrices made of Matrigel, which consists of basement membrane components (procedures and modifications of this assay have been summarized in [150,151]), collagen I gels, ECM produced in vitro by fibroblasts [152–154] or extracted from tissues [155] as well as artificial materialbased matrices ([156–158], references in [159–161]). Use of these culture systems gave clear evidence for distinct cell behaviour and signalling, gene expression as well as response to therapies when compared to plastic (e.g. [162], reviewed in [161,163–167]). Due to distinct capabilities of individual integrins, mediating interactions between cells and the ECM, to interact with particular ECM ligands [168,169], the ECM composition ensures specific ECM-cell interaction and therefore cell behaviour [73,169–171]. These specific interactions are transmitted via the cytoskeleton to the nuclear matrix, where they affect gene expression ([49,95,97,172–174], reviewed in [175,176]), suggesting that the substrate used for cell culturing belongs to the factors responsible for gene expression in cultured cells. Consistent with this, dependence of cell behaviour on the type of ECM used for culture has been recognized (e.g. [59,61,65–67,155,177,178]) implying that if cell behaviour analyzed in vitro should aid in understanding of in vivo processes, correct type of ECM needs to be used [58,69,161,179], (Table 2). 3.2. Stromal cells Among stromal cells, fibroblasts, macrophages, neutrophils, mast cells, myeloid-derived suppressor cells, lymphocytes, endothelial cells, pericytes and adipocytes were reported to influence the fate of tumour cells [116–118]. Accordingly, they should be present in or available to culture model systems of tumour cells. There are obvious limitations to achieve this. Several cell types contributing to tumour stroma, such as bone marrow-derived cells, do not originally reside in the tumours. Instead, they are actively recruited to the tumour stroma by cancer cells [18,117,180,181]. Examples include tumour-associated macrophages, which promote tumour growth [18,116] or bone marrow-derived myeloid cells and endothelial progenitor cells, proposed in restoring tumour growth after treatment [18]. The other issue concerns choice of co-cultured cells with regard to their type, source and abundance. The properties of the

tumour-associated stromal cells differ from those of the same cell types within healthy body and even from their normal counterparts in adjacent normal tissue [116,182,183]. Normal and tumour-associated fibroblasts. Fibroblasts in particular seem to play a role in the initiation and progression of carcinomas [116,182,184], and accordingly most of the studies aimed to analyze their contribution to tumourigenesis. While fibroblasts from normal breast generally inhibit epithelial growth, fibroblasts from breast carcinomas can promote it [185,186]. Indeed, gene expression profiling identified many genes differently expressed in early passage cultures of normal breast fibroblasts as compared to fibroblasts from breast carcinomas [186]. Differences not only in mRNA but also in protein levels were reported [187]. Analysis of breast carcinoma-associated fibroblasts, their adjacent counterparts isolated from histologically noncancerous region of the same affected breast and fibroblasts isolated from normal breast tissue were shown to differ in various properties among each other, even fibroblasts isolated from noncancerous region of the affected breast were different from normal fibroblasts [182]. Not only tumour-derived fibroblasts seem to differ from normal ones. Differences in expression of fibroblast growth factor-2 and ability to induce increased epithelial cell proliferation and progesterone expression between carcinoma-associated fibroblasts derived from hormone independent as compared to hormonedependent mouse mammary tumours were reported [188]. In general, population of fibroblasts seems to be very heterogenous. Considerable variability in properties among fibroblasts derived from individual patients was noticed [86,185,186] and fibroblasts vary even among tissues and sites within the body [87,189–194]. Tissue source of fibroblasts was found to be critical in promoting the extent of tumour invasion, migration and the degree of tumour differentiation in the 3D culture model of esophageal squamous cell carcinoma [88]. While fetal skin fibroblasts supported well differentiated phenotype of EGFR, hTERT and p53transduced human esophageal epithelial cells, a tendency toward poorly differentiated phenotype and more aggressive invasion were observed in the presence of fetal esophageal fibroblasts. In contrast, human adult esophageal fibroblasts did not promote invasion of the epithelial cells [88]. It was also shown that different types of fibroblasts produced matrices of clearly distinct properties, with consequences for behaviour of cells cultured within them [57,59,152,195]. Presence of fetal esophageal fibroblasts in matrices composed of a collagen I: Matrigel mixtures resulted in greater increase in stiffness than in the case of fetal skin fibroblasts [88]. Experiments with 3D in vitro co-cultures documented importance of the ratio between epithelial cells to fibroblasts [86]. Even ratio of different types of fibroblasts influenced tumourigenesis. While pure TGF-␤-responsive and -unresponsive stromal fibroblasts supported growth of normal prostate and precancerous lesions, respectively, only their 1:1 mixtures resulted in development of prostatic adenocarcinomas [194].

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Other stromal cells modified in tumours. Myoepithelial cells [183] and tumour-associated macrophages (references in [116]) may be other examples of stromal cells with modified properties within tumours. Furthermore, tumour-specific autologous cytotoxic T cells were proposed to be modified by factors within tumour microenvironment, leading to suppression of their proliferative capability [196]. Substances released by stromal cells. The finding of T cell modifications by factors within tumour microenvironment emphasizes importance of production of substances by stromal cells within tumour microenvironment. Importantly, expression of some of these substances may be lost in cultured cells [57]. Analysis of MMP expression, mostly by in situ hybridization, revealed that stromal cell expression of MMPs, as a response to the tumour, is in general more common than their expression by tumour cells [6,7,197]. Tumour cells, tumour-associated fibroblasts, endothelial cells and various cells of the myeloid and lymphoid classes have been shown to supply proteolytic enzymes within a tumour microenvironment (references in [8]). Substances released by stromal cells within tumour microenvironment mediate communication among cells, serve as signals to modify properties and behaviour of the cells and ECM, activate cells within tumour or induce recruitment of distally located cells to the tumour site (reviewed in [198]). MMPs may trigger the cleavage of cytokines, their receptors (references in [196]) and ECM proteins and consequently affect cellular signalling and functions. The cleavage of ECM proteins may even result in generating fragments with new functions [6,108]. Cell–cell as well as cell–ECM interactions may be mediated by direct contact or by released substances acting in a dynamic and reciprocal manner [19,95,199,200]. Cell–cell interactions. Trials to mimic cell–cell interactions by co-culturing different cell types proved that interactions between different cell types may influence cell behaviour with regard to morphological and functional differentiation, protein secretion, ECM deposition and basement membrane formation, activation of factors, cell growth and development, gene expression, etc. ([201] and references in Table 3), often in cell-type specific manner [137,200,203]. Co-culturing of various hematopoietic tumour primary cells and cell lines with bone marrow stromal cells provided with evidence that bone marrow stromal cells through soluble factors and direct cell contact were responsible for drug resistance and protection from apoptosis of myeloma [130,131], lymphoma [132,133] and leukaemia cells [134]. Upregulation of survival genes in cancer cells could be induced by their co-culturing with astrocytes, leading to resistance to number of chemotherapeutic agents [205]. The above summarized data document complexity of the conditions in vivo, which, in the case of cancer, have been replaced by tumour cell monocultures in majority cases. There are many factors involved that operate in a specific, dynamic and reciprocal way and neglecting any of them may shift the whole process to direction different from the one which is relevant in vivo*.

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3.3. Evidence from animal studies supporting importance of tumour cell environment The importance of microenvironment for tumour cell behaviour has been proved by animal studies. When human mammary cancer cells premixed with either Matrigel, nulliparous or involution mammary matrices, were injected into nude mice, the ability of small tumours to metastasize out the mammary fat pad was found to be dependent on the source of the matrix [155]. Lung, liver and kidney metastases were increased when involution matrix was used. Furthermore, biological behaviour of tumours may be altered by the environment of the tumour cell growth or implantation site [210–213]. In general, the growth of tumour cells in the intraperitoneal cavity, the lymph node or the spleen was documented to lead to progression towards cell populations with a higher metastatic potential in different animal tumour systems [212]. Mammary cancer cell study. Injections of human mammary epithelial cell lines of increasing tumourigenic properties into mammary fat pad of nude mice resulted in increased tumour formation frequency as compared to subcutaneous (s.c.) injections [213]. Prostate cancer cell study. Prostate cancer cell line variants with different tumour growth and metastatic potential could be isolated based on cell ability to grow within the prostate or to produce lymph node metastases [211]. Leukemia-lymphoma study. Lymphomas, characterized by their localized involvement, could be transformed into leukaemia with systemic dissemination by the tissue microenvironment in which the neoplastic lymphocytes proliferated. Neoplastic lymphocytes capable of inducing leukaemia, proliferating as transplants in spleens and lymph nodes of allogeneic animals, retained the capacity to induce leukaemia with systemic manifestations when inoculated subcutaneously, but they lose this capacity when they proliferated at subcutaneous sites and regained it again when they proliferated intrasplenically [210,212]. Glioblastoma study. Data from animal study have revealed the potential of glioblastoma multiforme (GBM) to adapt to its changing microenvironment [214]. GBM tumours normally invade adjacent brain by infiltration of single cells into the brain parenchyma. In microenvironment lacking VEGF or MMP-9, this mode of invasion was found to be blocked and deep perivascular tumour invasion occurred instead [180,214]. Isograft vs. xenograft study. Furthermore, response to some treatments may depend on whether the tumour is induced in its natural anatomical site [215]. When isografts of rat mammary carcinoma in the Fischer rats were compared with xenografts of the same carcinoma and human mesothelioma in nude mice, the response to photodynamic therapy was different in each of them, for the same tumour in different hosts as well as for different tumours in the same host [23]. Some experimental evidence demonstrated that the organ microenvironment could regulate the level of gene

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Table 3 Cell–cell interactions studied in cell co-cultures. Studied cells

Co-cultured cells

Influenced cell behaviour/property

Reference

Primary luminal mammary epithelial cells, human Mammary epithelial cell clone, mouse Cultured mammary epithelial cells, mouse

Myoepithelial cells, human

Morphological differentiation, cell polarity

[66,67]

Fibroblastic cell clone, mouse

[144]

Cultured mammary epithelial cells, mouse Cultured endometrial epithelial cells, human

Adipocyte cell line, mouse

Morphological and functional differentiation, protein secretion, ECM protein deposition Morphological and functional differentiation, protein secretion, ECM deposition, basement membrane formation Functional differentiation, cell growth

[199]

Colorectal carcinoma cell lines, human Epithelial mammary tumour cell line, human Breast cancer cell line, human Lung cancer cell line, human Melanoma cell line, human Prostate cancer cell line, human Sertoli cells, rat

Mesenchymal cell strain, human

Morphological differentiation, ECM deposition, basement membrane formation, cell growth Morphological and functional differentiation

Normal skin fibroblasts, human

Tumour forming capacity

[184]

Astrocytes, mouse Astrocytes, mouse Astrocytes, mouse Macrophages, mouse Peritubular myoid cells, rat

[205] [205] [206] [207] [137] and references therein

Sertoli cells, rat Sertoli cells, rat Aortic endothelial cells, bovine

Testicular peritubular cells, rat Germ cells, rat Pericytes, smooth muscle cells, bovine Pericytes, smooth muscle cells, bovine Fibroblasts, human and mouse; retinal pigment epithelial cells, bovine; kidney epithelial cells, Madin–Darby canine Primary hepatocytes, rat (but not human) Mammary adenocarcinoma cell lines, human Stromal cell line, mouse

Protection from apoptosis, gene expression Protection from apoptosis, gene expression Protection from apoptosis IL-6 production increase Morphological and functional differentiation, protein secretion, basement membrane formation ECM deposition Functional differentiation TGF-␤ activation Inhibition of cell proliferation

[208]

Capillary endothelial cells, bovine

Lung microvessel endothelial cells, rat Fibroblasts, human B cell progenitors, LSK cells, mouse Myeloma cell lines, human Primary lymphoma cells, lymphoma cell lines, human Primary leukemia cells, leukemic cell lines, human

Adipocyte cell line, preadipocyte cell line, mouse

Cultured endometrial stromal cells, human

Bone marrow stromal cells, human Bone marrow stromal cell line, human Bone marrow stromal cell line, human

expression. For example, highly metastatic human prostate cancer cells implanted in nude mice at an ectopic site s.c. expressed lower levels of metastasis-related genes than those implanted in an orthotopic site into the prostate [211]. The activity of collagenolytic enzymes was shown to be influenced by tumour implantation site also in the rabbit carcinoma model [216]. Not only the nature of the implantation site, but a number of other factors need to be considered when comparing different tumour models, including hematopoietic and immune host response. Consistently, significant variation in utilization of bone-marrow derived cells and bioavailability of MMP-9 between different tumour models was reported [214].

[146]

[204]

[202]

[145] [203] References in [70]

Cell growth stimulation

Capillary morphogenesis

[209]

Stimulation of ECM protein synthesis

[147]

Cell development, gene expression

[200]

Drug resistance, protection from apoptosis

[130,131]

Drug resistance, protection from apoptosis

[132,133]

Drug resistance, protection from apoptosis

[134]

3.4. Species specificity Even animal models, which, contrary to in vitro models, represent intact systems, may not fully overcome limitations recognized particularly in the fields of immunotherapy and gene therapy [217]. Due to the complexity and the unique nature of the host–tumour immunorelationship and the fact that the immune systems in humans and animals are different [218], animal models do not seem to fully mimic the biology of human patients with cancer. That not only immune systems differ between animals and humans has been documented in several review papers [219–223]. This aspect should be also considered in in vitro models.

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4. Lack of structural context in vitro Although deficient composition of cell’s microenvironment is the first factor that one considers with regard to in vitro conditions, the lack of the correct structural context is another limitation of the most usually used in vitro conditions. 4.1. Spatial organization Physiologically crucial molecules are often immobilized on insoluble structural elements within the cell and nucleus [96,172]. Likewise, ECMs serve as cellular scaffolds that provide structure to tissues, bind and sequester signalling molecules, affecting their local concentration and biological activity [19,79,172]. Moreover, the macromolecules of the ECM act also as ligands for adhesion receptors of the cells that transduce signals to the cell interior. It has been detected long time ago that the biochemical signalling resulting from the specific interactions between integrins and ECM components is coupled with morphological changes of the cells [73,174]. The impact of cell spreading and cell shape has been documented in cell growth, differentiation, nuclear morphology and gene expression [97,224–226]. The importance of structural context for cell behaviour is documented by the fact that providing the cells with correct biochemical microenvironment in the form of ECM protein-forming coatings does not necessarily lead to in vivo-like cell behaviour. Cell behaviour under such conditions, and even on attached as opposed to relaxed ECM matrices, is usually similar to that seen on plastic [22,54,55,69,171]. Consistently, not only the ECM composition determines the functional behaviour of the cells in the tissue (references in [95]), but the organization and biophysical properties of the ECM also play an important role (Table 2). 2D vs. 3D cultures. Numerous studies with primary cultures of epithelial cells of different types, fibroblasts as well as various cell lines document distinct behaviour of the cells in 3D as compared to 2D environments [100,108,202,227–237]. Importantly, phenotype dependence on spatial organization appears in transformed and tumour cells as well [69,202,227,232–234]. The modifications concern cell morphology, gene expression, protein production, deposition of ECM components, proliferation and functional differentiation. Critical importance of tissue architecture has been shown also in resistance of mammary epithelial cells to apoptosis. Formation of polarized 3D structures, occurring in 3D basement membrane cultures, conferred protection to apoptosis in both non-malignant and malignant mammary epithelial cells, while both cell types exhibited apoptotic responsiveness in 2D monolayer cultures [65]. Improved clonogenic survival after ␥-irradiation in 3D collagen gel as compared to 2D plastic conditions was shown for human non-immortalized fibroblast strain, while opposite result was found for hamster transformed cell line (references in [225]). Architecture of ECM fibres. The synthesis, deposition, posttranslation modification, but also the architecture of

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the fibrillar collagen and ECM are altered in tumours [57,120,238,239] and various effects of ECM proteins were reported to be mediated by its architecture rather than solely by protein level [57,120]. The changed collagen organization may modify cell invasion by enabling cells to migrate along the collagen fibres or influence therapeutic efficacy by hindering diffusion of drugs through dense collagen networks (references in [116]). 4.2. ECM stiffness Evidence further suggests that even biophysical properties of the ECM, namely forces exerted through matrix stiffness provide information to the cells. Force can alter the structural properties of the cytoskeleton, which in turn can activate signalling events or induce binding of other structural proteins (references in [240]). Consistently, the changes in ECM stiffness affect cell morphology, integrin signalling, and the actin cytoskeleton to control the cell cycle [241] and physiological tissue stiffness acts as a cell-cycle inhibitor in mammary epithelial and vascular smooth muscle cells [157]. In general, ECM stiffness enhances cell growth and survival, influences differentiation and promotes migration [237,238,242] and could even direct lineage specification of stem cells (references in [243]). Increased ECM stiffness in tumours thus could influence treatment response (references in [237]) by eliciting diverse effects on cellular differentiation, gene expression, proliferation, survival and migration [120]. In conventional in vitro experiments, the cells are cultured on nondeformable substrates, which are far from to the elastic ECM that cells encounter in vivo [157], (Fig. 2). Milliprobe indentation and atomic force microscopy indicated elastic moduli of normal murine tissues such as isolated mammary glands, thoracic aortae, and femoral arteries ranging from 600 to 4300 Pa [157], skeletal muscles ∼12 kPa [244] or cartilage up to 1.3 MPa [245]. Stiffness scale for normal human solid tissues was reported to range from several hundreds of Pa for soft brain [246] to microstiffness in the order of MPa for cartilage and precalcified bone [242,245]. Unconfined compression analysis of normal and malignant breast tissues from MMTV-Her2/neu, Myc, and Ras transgenic mice revealed that normal mammary tissue was rather soft with elastic moduli as low as ∼167 Pa, whereas the stromal matrix adjacent to the transformed cells and the oncogene-induced tumour itself were stiffer [238], with average elastic moduli reaching values of ∼900 and ∼4000 Pa, respectively [237]. Different elastic moduli were reported for different tumour types. Murine carcinoma xenografts were shown to be the softest tumours with the lowest collagen contents compared to rigid murine glioblastomas and sarcomas [247]. Still, elastic moduli of plastic dishes and glass coverslips are several orders of magnitude higher, ∼109 to 1012 Pa [237,241]. Such a difference should be remembered as various cells, at least normal, show distinct responses over the substrate stiffness range that is relevant to that of in vivo tissues [243].

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Reviewers Andrea Sadlonova, Ph.D., Emory University, 1462 Clifton Road NE, DSB 405, Atlanta, GA 30322, United States. Elizabeth G.E., De Vries, M.D., Ph.D., University Medical Center Groningen, Department of Internal Medicine, Hanzeplein 1, NL-9713 RB Groningen, Netherlands. Galia Staneva, Ph.D., Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, BG-1113 Sofia, Bulgaria. Antoinette Wozniak, M.D., FACP, Wayne State University, Karmanos Cancer Institute, Department of HematologyOncology, 4100 John R, Detroit, MI 48201, United States.

Acknowledgements Due to space limitations, reviews have been cited throughout the article. I, therefore, apologize for the omission of many significant references. Helpful comments from reviewers are highly acknowledged. Many thanks to K.R.

References

Fig. 2. Stiffness of solid tissues and conventional culture supports.

5. Conclusion In order to interpret the results of in vitro experiments correctly, it is crucial to understand the physiological and pathological relevance of the experimental conditions. That monocultures in plastic dishes may not represent relevant in vitro models in cancer research has been acknowledged long time ago. Despite that, they still constitute a considerable part of current research. Complexity of cancer, however, necessitates work with more sophisticated culture systems that provide with in vivo-mimicking interactions and structural context (Table 2) as well as accompanying analyses of patient material and systems biology approaches. Apart from complex physiological aspects of tissues, such as vasculature, interstitial flows or local gradients, specificity of cell behaviour with regard to cell type and type of ECM needs to be considered when developing in vitro cancer cell models. This may require use of culture systems specifically generated for the studied condition/disease based on analyses of in vivo samples and choice of proper cellular and non-cellular components with in vivo corresponding properties in addition to standardized culture systems.

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Biography ˇ Beata Cunderlíková, Ph.D. graduated from the Comenius University in Bratislava, Slovakia. She took her post-graduate studies at the Comenius University in Bratislava and at the Institute for Cancer Research in Oslo, Norway and defended her doctoral theses at the Comenius University, Slovakia and at the University of Oslo, Norway. She was a fellow at the Department of Pathology, The Norwegian Radiumhospital/The Oslo University Hospital in Oslo, Norway. Since undergraduate studies she was mainly engaged in the field of photodynamic therapy of cancer. Currently, she is working as a researcher at the International Laser Centre in Bratislava, Slovakia.