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Innovative in vitro models for breast cancer drug discovery
DDMOD-440; No of Pages 6
Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
Vol. xxx, No. xx 2017
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
DISEASE
MODELS
Innovative in vitro models for breast cancer drug discovery Elke Kaemmerer, Tayner E. Rodriguez Garzon, Aaron M. Lock, Carrie J. Lovitt, Vicky M. Averyz,* Discovery Biology, Griffith Institute for Drug Discovery, Griffith University, Building N27, Brisbane Innovation Park, Nathan, Queensland 4111, Australia
Abstract Breast cancer is a complex group of diseases and is one of the most common cancers diagnosed worldwide. Many studies have shown that tumour progression
Section editor: Dr. Robert M. Mader – Department of Medicine I, Comprehensive Cancer Center of the Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria
and drug responses vary due to tumour heterogeneity caused by genetic mutations and aberrant protein expression and are also directly affected by the local tumour microenvironment. To identify new targets, and/or evaluate potential new chemotherapeutics, with the ultimate goal of improving success rates and thus available treatment options, in vitro cell culture systems incorporating global tumour complexity are needed. This review provides an overview of the recent developments with respect to in vitro 2D, 3D and microfluidics cell culture techniques which mimic tumourigenesis, thus providing advanced model systems able to predict clinical drug responses.
mour progression and therapeutic responses [2]. Mimicking this complexity in an in vitro system is challenging, hence single cell monolayer cultures, based on immortalised cancer cells adhered to flat glass/polystyrene surfaces, have traditionally been used. Although these basic in vitro systems have merit and play a key role in early stage drug discovery, for identification of druggable molecular targets and extensive translational profiling chemotherapeutics, they all have limitations and are overly simplistic. Innovative in vitro models incorporating disease complexity are required to enable specific targeting of the key steps of tumour progression and to predict clinical outcomes. To meet this need, significant effort has been made to design more relevant in vitro models, encompassing 2D, 3D and microfluidics cell culture techniques.
Introduction
2D cell culture systems
Breast cancer is a heterogeneous collection of diseases with variations in genomic instability, gene expression and altered cell signalling [1]. Many of these parameters are affected by the tumour microenvironment, significantly impacting tu-
Monolayers of immortalised cell lines have been used since the 1950s for breast cancer drug discovery. Progress in microfabrication, as well as laboratory automation, resulted in miniaturised, downscaled assay formats, allowing for large high-throughput screening (HTS) campaigns using glass/ polystyrene multiwell tissue culture plates (e.g. 384 or
*Corresponding author: V.M. Vicky M.Avery (v.avery@griffith.edu.au) z URL: http://www.discoverybiology.org/.
1740-6757/$ © 2017 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ddmod.2017.02.002
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DDMOD-440; No of Pages 6 Drug Discovery Today: Disease Models |
Vol. xxx, No. xx 2017
(a)
2D co-culture
2D cell culture +/Surface topography or
2D microfluidics
Perfusion
+ / - co-culture
Cancer cells
Complexity
(b)
3D co-culture
Stromal cells
3D cell culture
+/Natural or bioengineered scaffold 3D microfluidics
Perfusion
+ / - co-culture
Complexity
Cell culture systems: Key advantages and disadvantages for drug discovery 2D culture systems + Integration of surface topography + Highly compatible with technologies for drug discovery + Cost effective - Lack physiological relevance
3D culture systems + Physiological architecture + Cell-to-matrix interactions + Moderately compatible with technologies for drug discovery - Labour/cost intensive
Microfluidic culture systems + Dynamic system + Small reagent volumes/cell numbers + Often not compatible with technologies for drug discovery - Labour/cost intensive, custom designs Drug Discovery Today: Disease Models
Figure 1. Advanced cell culture systems for cancer drug discovery. (a) More simplified 2D systems include nanostructured surfaces which provide cell culture substrates mimicking the natural tissue topography in vitro (left) or they can incorporate co-cultured cells and microfluidics technologies to add another level of complexity by representing tumour–stroma interactions and dynamic growth factor/chemokine and gas diffusion gradients (right). (b) 3D based model systems are able to duplicate the actual tumour architecture in an in vitro model (left) and in combination with co-culture and microfluidics techniques can very closely mimic the in vivo situation in vitro (right). Advantages and disadvantages of these advanced cell culture models are summarised in the text box.
1536 well plates) to be undertaken for drug discovery. Although cell cultures using cell lines on tissue-culture plates are still widely used throughout the early drug discovery stages, new concepts resulting in advanced cell culture systems with greater clinical relevance are now available and being increasingly utilised (Fig. 1).
Nanostructured topography Technological advances have furthered the development of existing cell culture systems containing flat, artificial surfaces into those with nanoscale substrates representing the natural e2
tissue topography, rather than a flat polystyrene/glass surface (Fig. 1A). These nanotopographic structures provide a biomimetic cell substrate, with precisely controlled topography, resulting in an innovative tool to explore changes in cell morphology, signalling, adhesion, migration, proliferation, and differentiation in response to variations in nanofeatures [3]. A study by Tavangar et al. assessed cellular response to nanostructures in breast cancer cells (MDA-MB-231) and mouse fibroblasts (NIH 3T3), cultured on a new hybrid nanomaterial platform of silicon nanostructures and immobilised gold/gold-palladium nanoparticles [4]. This study showed
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that cell adhesion and proliferation was selectively affected in breast cancer cells, but not in fibroblasts, suggesting a cell specific response to these nanostructures. Nanocolloids, which are sheets of graphene with a lateral dimension smaller than 100 nm are synthesised by chemical exfoliation of graphite nanofibers and are commercially available [5]. These systems are now being used to modulate or enhance the performance of induced pluripotent stem cells (iPSCs). Work by Chen et al., using nanocolloids made of graphene and graphene oxide to culture and differentiate iPSCs showed that the differentiation state, proliferation rate and level of cell adhesion of iPSCs can be modulated depending on the graphene-based nanomaterial used [6].
Induced pluripotent stem cells and co-cultures iPSCs technologies have the potential to recreate a more relevant cancer model than any model seen previously, primarily due to iPSCs being patient-derived, thus generating a more personalised therapy [7]. For example, mutations on BRCA1/2 responsible for hereditary breast cancer, cannot be modelled in mutated and artificially-induced human cell lines because of genomic instabilities, however generating iPSCs from patients carrying such mutations, enables the modelling and discovery of therapeutic targets in BRCA1 breast tumours [8]. Whilst bringing us closer to the patient situation a drawback can also be that the situation observed may be specific to the donor patient and not all patients. iPSCs can be used to identify critical molecular mechanisms accountable for disease initiation, progression [7] and even dissect mechanisms of drug toxicity. Burridge et al. used patient-derived cardiomyocytes iPSCs to identify molecular pathways that trigger doxorubicin-induced cardiotoxicity (DIC) in breast cancer patients [9], which may be developed into a diagnostic tool for DIC. Tumour–stroma interactions play a critical role in tumour formation, progression and drug responses. Hence co-culture systems have evolved substantially with 2D platforms more closely modelling the tumour stroma and thus bridging the gap with more complex tumour physiology. Co-culture systems have been used with a variety of immortalised cell lines, as well as primary cells, to study phenotypic selectivity, mechanisms of action, and target selectivity in drug discovery [10]. Breast cancer metastatic progression is one example of how co-culture systems can be used to discover therapeutic targets and evaluate inhibitors of chemotactic attraction that can block the chemotaxis between breast cancer cells subtypes [11]. It is evident that cross-talk between cell types in response to chemotactic gradients is crucial in cancer progression and that stromal cells are a key part of this. As a consequence, advanced co-culture systems now aim to address why tumour-associated stroma cells are essential for tumour cell invasion and metastasis. Innovative work using co-culture systems with breast cancer cells and stromal cells
Drug Discovery Today: Disease Models |
on nanostructured surfaces using polyelectrolyte multilayers (PEMs) and capillary force lithography (CFL), showed how this platform can be used to differentiate stages of breast tumour progression [12]. PEM are depositions of alternate polycation and polyanion layers for adsorption of polyelectrolytes which can be utilised to study permeability and adsorption, whereas CFL is a lithographic technique using the polymer melt concept to form nanosurface-patterns. This study used a co-culture of BT-474 cells and mesenchymal stem cells (MSCs) to model the invasive ductal carcinoma (IDC), and a co-culture of 21MT1 cells and MSCs cells were used to model invasive mammary carcinoma (IMC). Both models produced levels of miRNA levels comparable to the levels found in clinical samples and suggest this platform can be used to find potential targets for drug discovery [12].
3D cell culture systems 3D cell cultures have been increasingly utilised to further mimic the pathophysiological relevance of tumourigenesis and provide a more predictive in vitro model for breast and other cancers (Fig. 1B). They can be classified into two main categories: anchorage independent and anchorage dependent systems.
Anchorage independent systems Anchorage independent models are 3D cultures without external support thus formed through gravity, magnetic levitation or non-adherent, conical shaped or micro-patterned surfaces. These systems are able to represent the 3D cell architecture of the primary tumour and the oxygen and nutrient gradients present in vivo. Some of these systems are already commercially available and are compatible with HTS and suitable for co-culture approaches. A study by Jaganathan et al. introduced a scaffold-free co-culture system using magnetic levitation, to prepare heterogeneous micro-tumours consisting of different breast cancer cell lines and patientderived fibroblasts [13]. This system has the advantage of producing micro-tumours at an accelerated rate (<24 h) with different tumour densities and micro-tumour size to assess drug responses and penetration in a breast cancer model with tumour–stroma interactions. However, this system does not fully represent the non-cellular structure of the tumour microenvironment and cannot be easily manipulated.
Anchorage dependent systems Various studies have illustrated that in addition to cell–cell interactions, the biochemical and biophysical properties of the microenvironment have a major impact on cellular functions and the malignant phenotype [2,14], indicating the need for anchorage dependent 3D cell cultures. Anchorage dependent systems provide an external scaffold with material properties representing the structure and composition of the natural extracellular matrix (ECM). Therefor tumourigenic www.drugdiscoverytoday.com
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processes affected by cell–matrix adhesion, such as ligandinduced cell signalling or matrix degradation can be modelled in vitro, allowing the study of factors often associated with altered drug response, tumour progression and metastasis. Studies using breast cancer cells cultured in a lamin-rich ECM (lrECM) showed that these cells developed resistance to apoptosis inducing drugs [15] and showed a reduced response to molecular therapy (Herceptin) in HER2 positive breast cancer cells compared to cells grown as monolayers [16]. These and other studies identified integrin signalling, mainly through integrin b1 and b4, is altered in a 3D microenvironment compared to cells cultured as monolayers and is directly involved in mediating reduced drug response and promoting apoptosis resistance [15,16]. However, drug responses are even more complex, as illustrated by a study using polyethylene glycol (PEG) based micro-arrays with defined ECM protein coatings which revealed that matrix composition and cell–cell adhesion, through increased E-cadherin levels at high cell density, contribute independently in a 3D microenvironment to chemotherapeutic response [17].
Vol. xxx, No. xx 2017
and physiological relevance, whilst remaining compatible with HTS and automation. New input from the field of bioengineering using synthetic biomaterials [24], bio-printing, microfluidics techniques [25] and magnetic levitation [13] have resulted in a variety of new 3D model systems, with well defined, external controllable, tailorable properties, providing a complete new tool box for breast cancer research and drug discovery. An example of state of the art bioengineered model systems is shown with a 3D, multi cell type, tumourangiogenesis model system with external controllable ECM parameters based on synthetic starPEG and maleimide-functionalised heparin hydrogels. In this study, breast cancer cell lines (MCF-7 or MDA-MB-231) were cultured with endothelial cells (HUVECs) and MSCs in these synthetic hydrogel systems, and tumour growth, angiogenesis and responses to angiogenesis inhibitors and chemotherapeutics were assessed [26]. In addition, the application of iPSCs combined with 3D cell culture techniques, which are already used in drug testing for other diseases, has potential as a promising tool for breast cancer drug discovery leading to a more predictive, personalised therapy [27].
Recapturing tumour features and tumour progression Increased resistance to molecular therapy and radiotherapy in breast cancer is directly linked to alterations in cell metabolism which leads to the creation of a hypoxic microenvironment [18]. Using MDA-MB-231 cells in a 3D collagen hydrogel Szot et al. showed up-regulated expression of HIF1a and VEGF in response to oxygen and nutrient gradients in collagen hydrogels [19]. This study is an example demonstrating that the in vivo properties of breast cancer tumours can be recapitulated in a 3D in vitro model, providing a valuable tool to study the hypoxic microenvironment and tumour progression [19]. The presence of cancer stem cells, which are not affected by most anti-cancer therapies, represents an area of major concern for current treatment. Targeting the tumour stem cell population is thought to greatly improve the long term efficacy of treatment [20]. A study using MCF-7 cells in a collagen scaffold showed increased expression of pro-angiogenic factors, matrix metalloproteinases (MMPs) and cancer stem cell markers, as well as enhanced tumourigenic potential when implanted in mice [21]. This model provides a promising tool for identifying molecules targeting tumour progression and cancer stem cells.
Progress in 3D cell culture technologies Most anchorage dependent 3D model systems are utilised for basic research purposes as very few systems are commercially available and/or compatible with HTS and can be quite costintensive. Our laboratory established an in-house protocol for a miniaturised 3D assay for HTS high content imaging, based on cancer cells embedded in Matrigel1 [22,23]. These studies showed how advanced cell cultures can be used for compound testing in a model system with increased complexity e4
Microfluidics cell culture systems Despite the impressive advances made with both 2D and 3D cell culture systems, these are static models and are unable to replicate the fluid dynamics of the in vivo state. Conversely microfluidics devices can incorporate growth factor/chemokine and gas diffusion gradients into their design, are cost effective in that they use minimal reagent volumes and can be developed with HTS capability. These systems can be designed in 2D (Fig. 1A) to measure physical characteristics of tumour cells for the purposes of biomarker identification which can guide patient treatment programs or designed with greater physiological complexity in 3D with devices able to mimic the tumour microenvironment and vasculature in real time (Fig. 1B).
2D microfluidics An early 2D system consisted of branched microfluidic networks connected to seeding chambers under the control of a syringe pump that allowed the delivery of concentration gradients of chemical stimuli to cell monolayers specifically used to study the chemotherapeutic sensitivity of MCF-7 cells in response to endogenous glutathione levels [28]. Whilst this system could be adapted for profiling potential chemotherapeutics and exploring drug resistance mechanisms, it has limitations in analysing downstream tumourigenic events such as invasion and metastasis. Concurrently, Wang et al. and Mosadegh et al. developed similar devices designed to test growth factor (EGF and EGF/CXCR12)-induced chemotaxis of MDA-MB-231 cells embedded in a collagen-coated migration section of the device [29,30]. This study revealed a correlation between EGF concentration and cell migration
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and increased motility of MDA-MB-231 cells when treated with both EGF and CXCR12. A similar study investigated the chemokine CXCR12 and its role in the metastasis of MDAMB-231 cells [31]. Whereas the previous models consisted of tumour cell populations exposed to biochemical gradients of growth factors and chemokines, this later study better replicated the in vivo state by incorporating a layer of endothelial cells embedded on an artificial membrane. This accounted for the proximity of microvascular endothelium and how it impacts on the adhesion and metastasis of circulating tumour cells. Despite the increasing complexity of these systems and the incorporation of 3D tumour cultures, two-dimensional models are still being developed for biomarker identification and the detection of circulating tumour cells (CTCs) based on changes in metabolism, deformability and cell size. Two studies analysed the transit dynamics of luminal and basal breast cancer cells forcibly passed through a PDMS device containing microbarriers in order to determine cell stiffness and deformability [32,33]. They were able to distinguish invasive phenotypes and correlate tumour-initiating cells with the upregulation of cell surface markers and expression of metastasis-associated genes. An alternative study used a droplet-based fluorescence device that measured acid secretion from tumour cells to detect CTCs which has the potential to be used for monitoring disease progress in cancer patients [34].
3D microfluidics systems To enhance the biological relevance of microfluidics-based cancer models, groups have started using 3D tumour cultures in place of 2D monolayers. In a study investigating the effects of co-cultures on the effectiveness of drug treatment, Sabhachandani et al. developed a droplet-based microfluidics system comprised of sensitive and resistant MCF-7 spheroids cocultured with HS5 fibroblasts embedded in an alginate matrix [35]. This enabled the seeding of large numbers of spheroids of controlled size on a chip for the purposes of drug screening in higher throughput and has the capacity to include additional stromal components.
Advanced co-culture microfluidics The latest microfluidics systems are able to replicate the tumour microenvironment and its complex role in breast cancer progression. A study by Kim et al. embedded MDA-MB231 in a collagen matrix and exposed them to competing gradients of EGF and SDF-1a revealing a more sophisticated chemotactic movement reliant on the surrounding collagen fibres than that seen with earlier 2D models [36]. A parallel study focused on the interactions of heterogeneous tumour cell populations found within the tumour microenvironment
Drug Discovery Today: Disease Models |
and revealed the enhancement of metastatic potential of invasive tumour cells (MDA-MB-231) when co-cultured with epithelial-like tumour cells (MFC-7) in an artificial microfluidics scaffold [37]. Grolman et al. established a model for tumour-associated macrophages in the microenvironment and their role in causing breast cancer cells to intravasate into the bloodstream as a key initiating event in cancer progression [25]. Utilising MDA-MB-231 cells and RAW 264.7 mouse macrophages suspended in an alginate matrix of differing architectures a simple model, adaptable to HTS, has been developed aimed at profiling and optimising compounds able to disrupt the paracrine interaction between tumour cell and macrophage co-cultures, whilst assessing the influence of the stromal architecture [25]. To test an invasive mammary duct model of ductal carcinoma in situ (DCIS) Bishel et al. designed an artificial lumen consisting of collagen and Matrigel1 and lined with either cancerous (MCF10a.DCIS) or non-cancerous (MCF10a) mammary epithelial cells [38]. Co-culturing with HMFs induced an invasive phenotype consistent with the microenvironment. This model can be expanded to include other stromal cells, chemokine gradients and potential drug treatments. Bersini et al. and Jeon et al. developed triple negative breast cancer models for metastasis to bone tissue consisting of artificial HUVEC microvasculature in an ECM created by hBM-MSCs and osteo-differentiated hBM-MSCs to create an osteo-conditioned microenvironment in order to study extravasation processes, analyse molecular pathways and screen potential chemotherapeutics [39,40]. However, these tri-culture systems have yet to be made commercially available, are not currently HTS compatible and are cost-intensive compared to standard cultures platforms for drug testing.
Conclusion In the last decade considerable efforts to design more complex model systems for breast cancer research have been made, but only a few have been applied to drug discovery. Many model systems are not yet commercially available or are very expensive, labour intensive and not compatible with automated HTS platforms. Although progress will continue to be made and more innovative model systems will be integrated into routine drug testing, there will be no ‘one size fits all’ approach. Hence the most promising approach for improved cancer drug discovery will be the application of multiple models with increasing complexity throughout the drug discovery pipeline.
Conflict of interest The authors have no conflicts of interest to declare.
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Topics need to be addressed The likelihood of creating commercially available advanced, model systems with increased complexity and HTS capability for cancer drug discovery. The need for software to be developed specifically for analysing large data sets generated from 3D models and advanced microfluidics systems. Improvement is needed, especially for imagebased assays, current methods are limited and rely on 2D confocal microscopy algorithms. Increased application of iPSC cells and/or patient-derived primary cells, with defined source of origin and patient history, which has the potential to drive drug discovery towards personalised and targeted treatment approaches.
Acknowledgement E.K., T.R., A.L. and C.L. were funded by Cancer Therapeutics CRC Pty Ltd (CTx).
References [1] Vargo-Gogola T, Rosen JM. Modelling breast cancer: one size does not fit all. Nat Rev Cancer 2007;7:659–72. [2] Park CC, et al. The influence of the microenvironment on the malignant phenotype. Mol Med Today 2000;6:324–9. [3] Shi J, et al. Nanotechnology in drug delivery and tissue engineering: from discovery to applications. Nano Lett 2010;10:3223–30. [4] Tavangar A, et al. Noble hybrid nanostructures as efficient antiproliferative platforms for human breast cancer cell. ACS Appl Mater Interfaces 2016;8:10253–65. [5] Luo J, et al. Graphene oxide nanocolloids. J Am Chem Soc 2010;132:17667–69. [6] Chen GY, et al. A graphene-based platform for induced pluripotent stem cells culture and differentiation. Biomaterials 2012;33:418–27. [7] Kim JJ. Applications of iPSCs in cancer research. Biomark Insights 2015;10:125–31. [8] Soyombo AA, et al. Analysis of induced pluripotent stem cells from a BRCA1 mutant family. Stem Cell Rep 2013;1:336–49. [9] Burridge PW, et al. Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity. Nat Med 2016;22:547–56. [10] Berg EL, et al. Consideration of the cellular microenvironment: physiologically relevant co-culture systems in drug discovery. Adv Drug Deliv Rev 2014;69–70:190–204. [11] Arrigoni C, et al. In vitro co-culture models of breast cancer metastatic progression towards bone. Int J Mol Sci 2016;17. [12] Daverey A, et al. Breast cancer/stromal cells coculture on polyelectrolyte films emulates tumor stages and mirna profiles of clinical samples. Langmuir 2015;31:9991–10001. [13] Jaganathan H, et al. Three-dimensional in vitro co-culture model of breast tumor using magnetic levitation. Sci Rep 2014;4:6468. [14] Wirtz D, et al. The physics of cancer: the role of physical interactions and mechanical forces in metastasis. Nat Rev Cancer 2011;11:512–22.
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[15] Weaver VM, et al. beta4 integrin-dependent formation of polarized threedimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell 2002;2:205–16. [16] Weigelt B, et al. HER2 signaling pathway activation and response of breast cancer cells to HER2-targeting agents is dependent strongly on the 3D microenvironment. Breast Cancer Res Treat 2010;122:35–43. [17] Hakanson M, et al. Engineered 3D environments to elucidate the effect of environmental parameters on drug response in cancer. Integr Biol (Camb) 2011;3:31–8. [18] Ward C, et al. New strategies for targeting the hypoxic tumour microenvironment in breast cancer. Cancer Treat Rev 2013;39:171–9. [19] Szot CS, et al. 3D in vitro bioengineered tumors based on collagen I hydrogels. Biomaterials 2011;32:7905–12. [20] Liu S, Wicha MS. Targeting breast cancer stem cells. J Clin Oncol 2010;28:4006–12. [21] Chen L, et al. The enhancement of cancer stem cell properties of MCF-7 cells in 3D collagen scaffolds for modeling of cancer and anti-cancer drugs. Biomaterials 2012;33:1437–44. [22] Lovitt CJ, et al. Evaluation of chemotherapeutics in a three-dimensional breast cancer model. J Cancer Res Clin Oncol 2015;141:951–9. [23] Shelper TB, et al. Assessing drug efficacy in a miniaturized pancreatic cancer in vitro 3D cell culture model. Assay Drug Dev Technol 2016;14:367–80. [24] Lutolf MP, Hubbell JA. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat Biotechnol 2005;23:47–55. [25] Grolman JM, et al. Rapid 3D extrusion of synthetic tumor microenvironments. Adv Mater 2015;27:5512–7. [26] Bray LJ, et al. Multi-parametric hydrogels support 3D in vitro bioengineered microenvironment models of tumour angiogenesis. Biomaterials 2015;53:609–20. [27] Sirenko O, et al. Phenotypic characterization of toxic compound effects on liver spheroids derived from iPSC using confocal imaging and threedimensional image analysis. Assay Drug Dev Technol 2016;14:381–94. [28] Liu D, et al. Parallel microfluidic networks for studying cellular response to chemical modulation. J Biotechnol 2007;131:286–92. [29] Wang SJ, et al. Differential effects of EGF gradient profiles on MDA-MB231 breast cancer cell chemotaxis. Exp Cell Res 2004;300:180–9. [30] Mosadegh B, et al. Epidermal growth factor promotes breast cancer cell chemotaxis in CXCL12 gradients. Biotechnol Bioeng 2008;100:1205–13. [31] Song JW, et al. Microfluidic endothelium for studying the intravascular adhesion of metastatic breast cancer cells. PLoS One 2009;4:e5756. [32] Liu Z, et al. Microfluidic cytometric analysis of cancer cell transportability and invasiveness. Sci Rep 2015;5:14272. [33] Zhang W, et al. Microfluidics separation reveals the stem-cell-like deformability of tumor-initiating cells. Proc Natl Acad Sci U S A 2012;109:18707–12. [34] Del Ben F, et al. A method for detecting circulating tumor cells based on the measurement of single-cell metabolism in droplet-based microfluidics. Angew Chem Int Ed Engl 2016;55:8581–4. [35] Sabhachandani P, et al. Generation and functional assessment of 3D multicellular spheroids in droplet based microfluidics platform. Lab Chip 2016;16:497–505. [36] Kim BJ, et al. Cooperative roles of SDF-1alpha and EGF gradients on tumor cell migration revealed by a robust 3D microfluidic model. PLoS One 2013;8:e68422. [37] Shin Y, et al. Intratumoral phenotypic heterogeneity as an encourager of cancer invasion. Integr Biol (Camb) 2014;6:654–61. [38] Bischel LL, et al. Microfluidic model of ductal carcinoma in situ with 3D, organotypic structure. BMC Cancer 2015;15:12. [39] Bersini S, et al. A microfluidic 3D in vitro model for specificity of breast cancer metastasis to bone. Biomaterials 2014;35:2454–61. [40] Jeon JS, et al. Human 3D vascularized organotypic microfluidic assays to study breast cancer cell extravasation. Proc Natl Acad Sci U S A 2015;112:214–9.
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Drug Discovery Today: Disease Models
DRUG DISCOVERY
TODAY
Vol. xxx, No. xx 2017
Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Andrew McCulloch – University of California, SanDiego, USA
DISEASE
MODELS
Innovative in vitro models for breast cancer drug discovery Elke Kaemmerer, Tayner E. Rodriguez Garzon, Aaron M. Lock, Carrie J. Lovitt, Vicky M. Averyz,* Discovery Biology, Griffith Institute for Drug Discovery, Griffith University, Building N27, Brisbane Innovation Park, Nathan, Queensland 4111, Australia
Abstract Breast cancer is a complex group of diseases and is one of the most common cancers diagnosed worldwide. Many studies have shown that tumour progression
Section editor: Dr. Robert M. Mader – Department of Medicine I, Comprehensive Cancer Center of the Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria
and drug responses vary due to tumour heterogeneity caused by genetic mutations and aberrant protein expression and are also directly affected by the local tumour microenvironment. To identify new targets, and/or evaluate potential new chemotherapeutics, with the ultimate goal of improving success rates and thus available treatment options, in vitro cell culture systems incorporating global tumour complexity are needed. This review provides an overview of the recent developments with respect to in vitro 2D, 3D and microfluidics cell culture techniques which mimic tumourigenesis, thus providing advanced model systems able to predict clinical drug responses.
mour progression and therapeutic responses [2]. Mimicking this complexity in an in vitro system is challenging, hence single cell monolayer cultures, based on immortalised cancer cells adhered to flat glass/polystyrene surfaces, have traditionally been used. Although these basic in vitro systems have merit and play a key role in early stage drug discovery, for identification of druggable molecular targets and extensive translational profiling chemotherapeutics, they all have limitations and are overly simplistic. Innovative in vitro models incorporating disease complexity are required to enable specific targeting of the key steps of tumour progression and to predict clinical outcomes. To meet this need, significant effort has been made to design more relevant in vitro models, encompassing 2D, 3D and microfluidics cell culture techniques.
Introduction
2D cell culture systems
Breast cancer is a heterogeneous collection of diseases with variations in genomic instability, gene expression and altered cell signalling [1]. Many of these parameters are affected by the tumour microenvironment, significantly impacting tu-
Monolayers of immortalised cell lines have been used since the 1950s for breast cancer drug discovery. Progress in microfabrication, as well as laboratory automation, resulted in miniaturised, downscaled assay formats, allowing for large high-throughput screening (HTS) campaigns using glass/ polystyrene multiwell tissue culture plates (e.g. 384 or
*Corresponding author: V.M. Vicky M.Avery (v.avery@griffith.edu.au) z URL: http://www.discoverybiology.org/.
1740-6757/$ © 2017 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ddmod.2017.02.002
e1
Please cite this article in press as: Kaemmerer E, et al. Innovative in vitro models for breast cancer drug discovery, Drug Discov Today: Dis Model (2017), http://dx.doi.org/10.1016/j. ddmod.2017.02.002
DDMOD-440; No of Pages 6 Drug Discovery Today: Disease Models |
Vol. xxx, No. xx 2017
(a)
2D co-culture
2D cell culture +/Surface topography or
2D microfluidics
Perfusion
+ / - co-culture
Cancer cells
Complexity
(b)
3D co-culture
Stromal cells
3D cell culture
+/Natural or bioengineered scaffold 3D microfluidics
Perfusion
+ / - co-culture
Complexity
Cell culture systems: Key advantages and disadvantages for drug discovery 2D culture systems + Integration of surface topography + Highly compatible with technologies for drug discovery + Cost effective - Lack physiological relevance
3D culture systems + Physiological architecture + Cell-to-matrix interactions + Moderately compatible with technologies for drug discovery - Labour/cost intensive
Microfluidic culture systems + Dynamic system + Small reagent volumes/cell numbers + Often not compatible with technologies for drug discovery - Labour/cost intensive, custom designs Drug Discovery Today: Disease Models
Figure 1. Advanced cell culture systems for cancer drug discovery. (a) More simplified 2D systems include nanostructured surfaces which provide cell culture substrates mimicking the natural tissue topography in vitro (left) or they can incorporate co-cultured cells and microfluidics technologies to add another level of complexity by representing tumour–stroma interactions and dynamic growth factor/chemokine and gas diffusion gradients (right). (b) 3D based model systems are able to duplicate the actual tumour architecture in an in vitro model (left) and in combination with co-culture and microfluidics techniques can very closely mimic the in vivo situation in vitro (right). Advantages and disadvantages of these advanced cell culture models are summarised in the text box.
1536 well plates) to be undertaken for drug discovery. Although cell cultures using cell lines on tissue-culture plates are still widely used throughout the early drug discovery stages, new concepts resulting in advanced cell culture systems with greater clinical relevance are now available and being increasingly utilised (Fig. 1).
Nanostructured topography Technological advances have furthered the development of existing cell culture systems containing flat, artificial surfaces into those with nanoscale substrates representing the natural e2
tissue topography, rather than a flat polystyrene/glass surface (Fig. 1A). These nanotopographic structures provide a biomimetic cell substrate, with precisely controlled topography, resulting in an innovative tool to explore changes in cell morphology, signalling, adhesion, migration, proliferation, and differentiation in response to variations in nanofeatures [3]. A study by Tavangar et al. assessed cellular response to nanostructures in breast cancer cells (MDA-MB-231) and mouse fibroblasts (NIH 3T3), cultured on a new hybrid nanomaterial platform of silicon nanostructures and immobilised gold/gold-palladium nanoparticles [4]. This study showed
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that cell adhesion and proliferation was selectively affected in breast cancer cells, but not in fibroblasts, suggesting a cell specific response to these nanostructures. Nanocolloids, which are sheets of graphene with a lateral dimension smaller than 100 nm are synthesised by chemical exfoliation of graphite nanofibers and are commercially available [5]. These systems are now being used to modulate or enhance the performance of induced pluripotent stem cells (iPSCs). Work by Chen et al., using nanocolloids made of graphene and graphene oxide to culture and differentiate iPSCs showed that the differentiation state, proliferation rate and level of cell adhesion of iPSCs can be modulated depending on the graphene-based nanomaterial used [6].
Induced pluripotent stem cells and co-cultures iPSCs technologies have the potential to recreate a more relevant cancer model than any model seen previously, primarily due to iPSCs being patient-derived, thus generating a more personalised therapy [7]. For example, mutations on BRCA1/2 responsible for hereditary breast cancer, cannot be modelled in mutated and artificially-induced human cell lines because of genomic instabilities, however generating iPSCs from patients carrying such mutations, enables the modelling and discovery of therapeutic targets in BRCA1 breast tumours [8]. Whilst bringing us closer to the patient situation a drawback can also be that the situation observed may be specific to the donor patient and not all patients. iPSCs can be used to identify critical molecular mechanisms accountable for disease initiation, progression [7] and even dissect mechanisms of drug toxicity. Burridge et al. used patient-derived cardiomyocytes iPSCs to identify molecular pathways that trigger doxorubicin-induced cardiotoxicity (DIC) in breast cancer patients [9], which may be developed into a diagnostic tool for DIC. Tumour–stroma interactions play a critical role in tumour formation, progression and drug responses. Hence co-culture systems have evolved substantially with 2D platforms more closely modelling the tumour stroma and thus bridging the gap with more complex tumour physiology. Co-culture systems have been used with a variety of immortalised cell lines, as well as primary cells, to study phenotypic selectivity, mechanisms of action, and target selectivity in drug discovery [10]. Breast cancer metastatic progression is one example of how co-culture systems can be used to discover therapeutic targets and evaluate inhibitors of chemotactic attraction that can block the chemotaxis between breast cancer cells subtypes [11]. It is evident that cross-talk between cell types in response to chemotactic gradients is crucial in cancer progression and that stromal cells are a key part of this. As a consequence, advanced co-culture systems now aim to address why tumour-associated stroma cells are essential for tumour cell invasion and metastasis. Innovative work using co-culture systems with breast cancer cells and stromal cells
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on nanostructured surfaces using polyelectrolyte multilayers (PEMs) and capillary force lithography (CFL), showed how this platform can be used to differentiate stages of breast tumour progression [12]. PEM are depositions of alternate polycation and polyanion layers for adsorption of polyelectrolytes which can be utilised to study permeability and adsorption, whereas CFL is a lithographic technique using the polymer melt concept to form nanosurface-patterns. This study used a co-culture of BT-474 cells and mesenchymal stem cells (MSCs) to model the invasive ductal carcinoma (IDC), and a co-culture of 21MT1 cells and MSCs cells were used to model invasive mammary carcinoma (IMC). Both models produced levels of miRNA levels comparable to the levels found in clinical samples and suggest this platform can be used to find potential targets for drug discovery [12].
3D cell culture systems 3D cell cultures have been increasingly utilised to further mimic the pathophysiological relevance of tumourigenesis and provide a more predictive in vitro model for breast and other cancers (Fig. 1B). They can be classified into two main categories: anchorage independent and anchorage dependent systems.
Anchorage independent systems Anchorage independent models are 3D cultures without external support thus formed through gravity, magnetic levitation or non-adherent, conical shaped or micro-patterned surfaces. These systems are able to represent the 3D cell architecture of the primary tumour and the oxygen and nutrient gradients present in vivo. Some of these systems are already commercially available and are compatible with HTS and suitable for co-culture approaches. A study by Jaganathan et al. introduced a scaffold-free co-culture system using magnetic levitation, to prepare heterogeneous micro-tumours consisting of different breast cancer cell lines and patientderived fibroblasts [13]. This system has the advantage of producing micro-tumours at an accelerated rate (<24 h) with different tumour densities and micro-tumour size to assess drug responses and penetration in a breast cancer model with tumour–stroma interactions. However, this system does not fully represent the non-cellular structure of the tumour microenvironment and cannot be easily manipulated.
Anchorage dependent systems Various studies have illustrated that in addition to cell–cell interactions, the biochemical and biophysical properties of the microenvironment have a major impact on cellular functions and the malignant phenotype [2,14], indicating the need for anchorage dependent 3D cell cultures. Anchorage dependent systems provide an external scaffold with material properties representing the structure and composition of the natural extracellular matrix (ECM). Therefor tumourigenic www.drugdiscoverytoday.com
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processes affected by cell–matrix adhesion, such as ligandinduced cell signalling or matrix degradation can be modelled in vitro, allowing the study of factors often associated with altered drug response, tumour progression and metastasis. Studies using breast cancer cells cultured in a lamin-rich ECM (lrECM) showed that these cells developed resistance to apoptosis inducing drugs [15] and showed a reduced response to molecular therapy (Herceptin) in HER2 positive breast cancer cells compared to cells grown as monolayers [16]. These and other studies identified integrin signalling, mainly through integrin b1 and b4, is altered in a 3D microenvironment compared to cells cultured as monolayers and is directly involved in mediating reduced drug response and promoting apoptosis resistance [15,16]. However, drug responses are even more complex, as illustrated by a study using polyethylene glycol (PEG) based micro-arrays with defined ECM protein coatings which revealed that matrix composition and cell–cell adhesion, through increased E-cadherin levels at high cell density, contribute independently in a 3D microenvironment to chemotherapeutic response [17].
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and physiological relevance, whilst remaining compatible with HTS and automation. New input from the field of bioengineering using synthetic biomaterials [24], bio-printing, microfluidics techniques [25] and magnetic levitation [13] have resulted in a variety of new 3D model systems, with well defined, external controllable, tailorable properties, providing a complete new tool box for breast cancer research and drug discovery. An example of state of the art bioengineered model systems is shown with a 3D, multi cell type, tumourangiogenesis model system with external controllable ECM parameters based on synthetic starPEG and maleimide-functionalised heparin hydrogels. In this study, breast cancer cell lines (MCF-7 or MDA-MB-231) were cultured with endothelial cells (HUVECs) and MSCs in these synthetic hydrogel systems, and tumour growth, angiogenesis and responses to angiogenesis inhibitors and chemotherapeutics were assessed [26]. In addition, the application of iPSCs combined with 3D cell culture techniques, which are already used in drug testing for other diseases, has potential as a promising tool for breast cancer drug discovery leading to a more predictive, personalised therapy [27].
Recapturing tumour features and tumour progression Increased resistance to molecular therapy and radiotherapy in breast cancer is directly linked to alterations in cell metabolism which leads to the creation of a hypoxic microenvironment [18]. Using MDA-MB-231 cells in a 3D collagen hydrogel Szot et al. showed up-regulated expression of HIF1a and VEGF in response to oxygen and nutrient gradients in collagen hydrogels [19]. This study is an example demonstrating that the in vivo properties of breast cancer tumours can be recapitulated in a 3D in vitro model, providing a valuable tool to study the hypoxic microenvironment and tumour progression [19]. The presence of cancer stem cells, which are not affected by most anti-cancer therapies, represents an area of major concern for current treatment. Targeting the tumour stem cell population is thought to greatly improve the long term efficacy of treatment [20]. A study using MCF-7 cells in a collagen scaffold showed increased expression of pro-angiogenic factors, matrix metalloproteinases (MMPs) and cancer stem cell markers, as well as enhanced tumourigenic potential when implanted in mice [21]. This model provides a promising tool for identifying molecules targeting tumour progression and cancer stem cells.
Progress in 3D cell culture technologies Most anchorage dependent 3D model systems are utilised for basic research purposes as very few systems are commercially available and/or compatible with HTS and can be quite costintensive. Our laboratory established an in-house protocol for a miniaturised 3D assay for HTS high content imaging, based on cancer cells embedded in Matrigel1 [22,23]. These studies showed how advanced cell cultures can be used for compound testing in a model system with increased complexity e4
Microfluidics cell culture systems Despite the impressive advances made with both 2D and 3D cell culture systems, these are static models and are unable to replicate the fluid dynamics of the in vivo state. Conversely microfluidics devices can incorporate growth factor/chemokine and gas diffusion gradients into their design, are cost effective in that they use minimal reagent volumes and can be developed with HTS capability. These systems can be designed in 2D (Fig. 1A) to measure physical characteristics of tumour cells for the purposes of biomarker identification which can guide patient treatment programs or designed with greater physiological complexity in 3D with devices able to mimic the tumour microenvironment and vasculature in real time (Fig. 1B).
2D microfluidics An early 2D system consisted of branched microfluidic networks connected to seeding chambers under the control of a syringe pump that allowed the delivery of concentration gradients of chemical stimuli to cell monolayers specifically used to study the chemotherapeutic sensitivity of MCF-7 cells in response to endogenous glutathione levels [28]. Whilst this system could be adapted for profiling potential chemotherapeutics and exploring drug resistance mechanisms, it has limitations in analysing downstream tumourigenic events such as invasion and metastasis. Concurrently, Wang et al. and Mosadegh et al. developed similar devices designed to test growth factor (EGF and EGF/CXCR12)-induced chemotaxis of MDA-MB-231 cells embedded in a collagen-coated migration section of the device [29,30]. This study revealed a correlation between EGF concentration and cell migration
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and increased motility of MDA-MB-231 cells when treated with both EGF and CXCR12. A similar study investigated the chemokine CXCR12 and its role in the metastasis of MDAMB-231 cells [31]. Whereas the previous models consisted of tumour cell populations exposed to biochemical gradients of growth factors and chemokines, this later study better replicated the in vivo state by incorporating a layer of endothelial cells embedded on an artificial membrane. This accounted for the proximity of microvascular endothelium and how it impacts on the adhesion and metastasis of circulating tumour cells. Despite the increasing complexity of these systems and the incorporation of 3D tumour cultures, two-dimensional models are still being developed for biomarker identification and the detection of circulating tumour cells (CTCs) based on changes in metabolism, deformability and cell size. Two studies analysed the transit dynamics of luminal and basal breast cancer cells forcibly passed through a PDMS device containing microbarriers in order to determine cell stiffness and deformability [32,33]. They were able to distinguish invasive phenotypes and correlate tumour-initiating cells with the upregulation of cell surface markers and expression of metastasis-associated genes. An alternative study used a droplet-based fluorescence device that measured acid secretion from tumour cells to detect CTCs which has the potential to be used for monitoring disease progress in cancer patients [34].
3D microfluidics systems To enhance the biological relevance of microfluidics-based cancer models, groups have started using 3D tumour cultures in place of 2D monolayers. In a study investigating the effects of co-cultures on the effectiveness of drug treatment, Sabhachandani et al. developed a droplet-based microfluidics system comprised of sensitive and resistant MCF-7 spheroids cocultured with HS5 fibroblasts embedded in an alginate matrix [35]. This enabled the seeding of large numbers of spheroids of controlled size on a chip for the purposes of drug screening in higher throughput and has the capacity to include additional stromal components.
Advanced co-culture microfluidics The latest microfluidics systems are able to replicate the tumour microenvironment and its complex role in breast cancer progression. A study by Kim et al. embedded MDA-MB231 in a collagen matrix and exposed them to competing gradients of EGF and SDF-1a revealing a more sophisticated chemotactic movement reliant on the surrounding collagen fibres than that seen with earlier 2D models [36]. A parallel study focused on the interactions of heterogeneous tumour cell populations found within the tumour microenvironment
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and revealed the enhancement of metastatic potential of invasive tumour cells (MDA-MB-231) when co-cultured with epithelial-like tumour cells (MFC-7) in an artificial microfluidics scaffold [37]. Grolman et al. established a model for tumour-associated macrophages in the microenvironment and their role in causing breast cancer cells to intravasate into the bloodstream as a key initiating event in cancer progression [25]. Utilising MDA-MB-231 cells and RAW 264.7 mouse macrophages suspended in an alginate matrix of differing architectures a simple model, adaptable to HTS, has been developed aimed at profiling and optimising compounds able to disrupt the paracrine interaction between tumour cell and macrophage co-cultures, whilst assessing the influence of the stromal architecture [25]. To test an invasive mammary duct model of ductal carcinoma in situ (DCIS) Bishel et al. designed an artificial lumen consisting of collagen and Matrigel1 and lined with either cancerous (MCF10a.DCIS) or non-cancerous (MCF10a) mammary epithelial cells [38]. Co-culturing with HMFs induced an invasive phenotype consistent with the microenvironment. This model can be expanded to include other stromal cells, chemokine gradients and potential drug treatments. Bersini et al. and Jeon et al. developed triple negative breast cancer models for metastasis to bone tissue consisting of artificial HUVEC microvasculature in an ECM created by hBM-MSCs and osteo-differentiated hBM-MSCs to create an osteo-conditioned microenvironment in order to study extravasation processes, analyse molecular pathways and screen potential chemotherapeutics [39,40]. However, these tri-culture systems have yet to be made commercially available, are not currently HTS compatible and are cost-intensive compared to standard cultures platforms for drug testing.
Conclusion In the last decade considerable efforts to design more complex model systems for breast cancer research have been made, but only a few have been applied to drug discovery. Many model systems are not yet commercially available or are very expensive, labour intensive and not compatible with automated HTS platforms. Although progress will continue to be made and more innovative model systems will be integrated into routine drug testing, there will be no ‘one size fits all’ approach. Hence the most promising approach for improved cancer drug discovery will be the application of multiple models with increasing complexity throughout the drug discovery pipeline.
Conflict of interest The authors have no conflicts of interest to declare.
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Topics need to be addressed The likelihood of creating commercially available advanced, model systems with increased complexity and HTS capability for cancer drug discovery. The need for software to be developed specifically for analysing large data sets generated from 3D models and advanced microfluidics systems. Improvement is needed, especially for imagebased assays, current methods are limited and rely on 2D confocal microscopy algorithms. Increased application of iPSC cells and/or patient-derived primary cells, with defined source of origin and patient history, which has the potential to drive drug discovery towards personalised and targeted treatment approaches.
Acknowledgement E.K., T.R., A.L. and C.L. were funded by Cancer Therapeutics CRC Pty Ltd (CTx).
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