ADR-12600; No of Pages 8 Advanced Drug Delivery Reviews xxx (2014) xxx–xxx

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In vitro modeling of the prostate cancer microenvironment☆,☆☆ Stuart J. Ellem a,⁎, Elena M. De-Juan-Pardo b, Gail P. Risbridger a a b

Prostate Cancer Research Group, Department of Anatomy and Developmental Biology, Monash University, Victoria, Australia Regenerative Medicine Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia

a r t i c l e

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Article history: Accepted 29 April 2014 Available online xxxx Keywords: Prostate Prostate cancer Microenvironment Stroma Scaffolds Matrix 2D 3D

a b s t r a c t Prostate cancer is the most commonly diagnosed malignancy in men and advanced disease is incurable. Model systems are a fundamental tool for research and many in vitro models of prostate cancer use cancer cell lines in monoculture. Although these have yielded significant insight they are inherently limited by virtue of their two-dimensional (2D) growth and inability to include the influence of tumour microenvironment. These major limitations can be overcome with the development of newer systems that more faithfully recreate and mimic the complex in vivo multi-cellular, three-dimensional (3D) microenvironment. This article presents the current state of in vitro models for prostate cancer, with particular emphasis on 3D systems and the challenges that remain before their potential to advance our understanding of prostate disease and aid in the development and testing of new therapeutic agents can be realised. © 2014 Published by Elsevier B.V.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . The Prostate Tumour Microenvironment . . . . . . . . Current Models . . . . . . . . . . . . . . . . . . . 3.1. Two dimensional models (2D) . . . . . . . . . 3.1.1. Co-cultures & advanced 2D models (2.5D) 3.2. Three dimensional models (3D) . . . . . . . . . 3.2.1. Spheroids (aka prostaspheres) . . . . . 4. Advanced bioengineered models . . . . . . . . . . . 5. Ex vivo explant culture . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Prostate cancer (PCa) is the most commonly diagnosed cancer and second leading cause of cancer death in men throughout the Western ☆ Financial support: SJE: APP1003247 (Australian National Health and Medical Research Council); GPR: APP1002648 (Australian National Health and Medical Research Council); SJE & GPR: NCG4712 (Movember / Prostate Cancer Foundation of Australia). ☆☆ This review is part of the Advanced Drug Delivery Reviews theme issue on "Engineering of Tumor Microenvironments". ⁎ Corresponding author at: Prostate Cancer Research Group, Department of Anatomy and Developmental Biology, Monash University, Wellington Road, Clayton, Victoria, Australia, 3800. Tel.:+61 3 9902 9514; fax:+61 3 9902 9223. E-mail address: [email protected] (S.J. Ellem).

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world. Although this represents a significant and growing problem with an aging population, the mechanisms of PCa disease initiation, progression and metastasis remain poorly understood. Research towards this has been particularly hampered by the lack of robust and biologically relevant model systems. In particular, the lack of reliable in vitro models that accurately recapitulate the complex three-dimensional (3D) microenvironment of the prostate has been a major impediment to furthering our understanding of prostate disease, as well as the development and testing of new therapeutic agents. Animal models have been the foundation of PCa research, however, these typically bear limited relevance to human disease and are hampered by additional significant limitations. The only nonhuman mammals known to develop prostate cancer naturally are nonhuman

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Please cite this article as: S.J. Ellem, et al., In vitro modeling of the prostate cancer microenvironment, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.04.008

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primates and dogs [1,2]. Both models, however, are highly limited due to significant expense and long tumour latencies. Experimental rodent models have also been developed and used extensively to elucidate discrete mechanisms of prostate carcinogenesis. These include a variety of transgenic mouse (eg, TRAMP, LADY, myc, Pten, and more) [3–7] and rat models (spontaneous, hormone or chemically induced) [8–10]. Ultimately, and without exception, each of these animal models is universally limited by its nonhuman origin, significantly restricting its relevance and application to human disease. In vivo prostate cancer models of human origin typically consist of primary cell or tissue slice grafts [11], multiple cancer cell lines as grafts [12–15], as well as cancer tissue xenografts [16–19]. While these models address the nonhuman nature and fundamental limitation of animal models, they suffer from limitations relating to expense as well as experimental and tumour latency. The combination of these three key factors – expense, long tumour latency and nonhuman origin – has represented the major hurdle for models of prostate cancer that, to date, can only be collectively overcome through the use of in vitro models. In vitro models, however, come with the caveat that some inherent advantages of in vivo systems, such as being able to follow the natural progression of PCa and/or metastasis, are lost. Traditionally, the vast majority of in vitro models of prostate cancer have almost exclusively consisted of immortalised cancer cell lines in monoculture [13,20]. While these cell lines and model systems have significantly advanced our understanding of the mechanisms of PCa, they remain poorly representative of human disease in vivo due to the highly abnormal culture environment and inherent inability to incorporate parameters of disease development and progression, such as invasion, and multicellular interaction. Appropriate in vitro experimental models suitable for the analysis of cell growth and interaction, homeostasis, EMT, invasion and metastasis, are becoming increasingly important for basic research and the development of new therapeutics. However, the need for rapid, highthroughput screening in drug development and testing has resulted in most contemporary platforms remaining based on two-dimensional (2D) monoculture cell assays. Using these systems, and animal models, anticancer drugs screened for PCa can show significant promise in the laboratory, but ultimately have little or no impact or benefit on the survival of patients [21]. A key factor underlying this discrepancy is that 2D

in vitro models do not faithfully recreate the complex multi-cellular, 3D tumour microenvironment seen in vivo in humans [22,23]. It is the combined lack of these attributes/features that is the underlying reason for the limited predictive power of 2D systems in terms of clinical efficacy when used for drug testing and discovery [22]. While progress has being made in this area with 3D models such as spheroids providing a more accurate biological readout, the current model systems still only represent a highly limited reconstruction of the native prostatic heterogeneity and complex in vivo architecture (Fig. 1). Ultimately, the development of more robust and effective in vitro PCa models that accurately mimic the in vivo tumour niche microenvironment is of vital importance for drug discovery, drug testing, and to advance our knowledge of PCa biology. 2. The Prostate Tumour Microenvironment The prostate and PCa are both highly heterogeneous tissues. In addition to the luminal epithelial and tumour cells that have been the typical and traditional basis of in vitro models, the prostate is also comprised of basal cells and a small number of neuroendocrine cells in the epithelium, which itself is surrounded by stroma tissue that also plays a major role in cancer cell growth, survival, invasion and metastatic progression [24]. The prostatic stroma is primarily composed of smooth muscle and extracellular matrix, but also consists of nerves, lymphatics and the blood vessels of the organ. Other cell types present include stromal cells (fibroblasts and myofibroblasts), endothelial cells, pericytes and inflammatory cells (including resident mast cells); collectively these form the prostate microenvironment (Fig. 1). It is the combined effect and interaction of these components that define prostate tumourigenesis, progression, invasion and the potential to respond to various therapeutics. Prostate fibroblasts form particularly important stromal components that have a well-established role in driving tumourigenesis. Very early studies showed morphological changes identified by pathology in prostate carcinoma associated fibroblasts (CAFs) compared to normal prostatic fibroblasts (NPFs), while recent work has unequivocally shown that CAFs can induce transformation and tumourigenesis in benign epithelia, whereas NPFs do not [25–28]. Inflammation also has a well-documented role in the development and progression of many

Fig. 1. Prostate architecture and relevance of in vitro model systems. The prostate and prostate cancer are highly heterogeneous tissues, consisting of multiple compartments and cell types within which cell–cell and cell–matrix interactions define cell behaviour and response to therapy. Current in vitro models, ranging from simple 2D monoculture to complex bioengineered 3D systems, harbour intrinsic advantages and limitations and vary significantly in their recapitulation of the in vivo tissue architecture, biological relevance, and drug response.

Please cite this article as: S.J. Ellem, et al., In vitro modeling of the prostate cancer microenvironment, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.04.008

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cancers; inflammation also has an emerging role in PCa [29,30]. With significant roles in disease initiation and progression postulated for tissue resident immune cells such as mast cells [31], as well as recruited populations like macrophages [32], the potential importance of the immune contribution to PCa cannot be understated. Angiogenesis and the important role of the vasculature are well-established hallmarks of cancer [33,34]. These are potentially no less important in PCa, although the specific role and importance of angiogenesis in prostate tumour development and progression remain controversial [35]. These three key factors – the tumour stroma, inflammation and vasculature – each play key and well-documented roles in prostate tumour development and progression. Despite this, they are universally absent in the vast majority of in vitro models. The same is true for the other components of the prostate microenvironment, such as the nerves, ECM and smooth muscle, which are also important. For example, nerves also form a small but essential part of the microenvironment that is often overlooked, yet the specific interaction of nerves and PCa cells in the microenvironment promotes perineural invasion [36], while autonomic nerve fibres may also regulate cancer development and dissemination [37]. In short, each of the many components of the stroma plays a vital role in normal growth and homeostasis, but also has the potential to play key roles in the development and progression of PCa. Thus, each component of the tissue architecture is important and has the potential to play a contributory role in carcinogenesis, either by itself or via interaction with another component (eg, macrophages and CAFs [38]). An effective and relevant in vitro model ideally, therefore, must consider and incorporate these aspects [39]. This has been sorely lacking in contemporary PCa models, of which the most routinely used rely on 2D monoculture comprised exclusively of epithelial/tumour cells. 3. Current Models Currently within the prostate cancer field there are a myriad of models that span the spectrum of complexity. These range from the traditional and simple 2D monocultures that have been the backbone of prostate and PCa research for decades, to the more complex and recently developed 3D models that attempt to recreate the in vivo tissue architecture (Fig. 1). Each system possesses its own inherent benefits and limitations that contribute to its overall suitability for research into prostate biology and/or drug development and testing. 3.1. Two dimensional models (2D) The study of PCa, particularly regarding preclinical drug discovery and testing, is heavily dependent on the use of 2D monoculture models, almost exclusively based on immortalised cancer cell lines. The characteristics of the many PCa cell lines (and sub-lines) in laboratory use have been extensively described, with their properties and suitability for particular research purposes thoroughly detailed [13,20]. A wealth of information has been gained from utilising these PCa cell lines, particularly the metastasis-derived, most commonly used and traditional mainstays, DU-145, PC-3 and LNCaP. In these systems, cells are typically grown in monoculture (2D) in hard, flat polystyrene culture dishes at body temperature (37C), with medium that is often supplemented with bovine serum, L-glutamine, various other growth factors and antibiotics, to aid cell growth. Whilst this ultimately represents a highly abnormal microenvironment, it is the simplicity of this culture system that contributes to the major strength of these models. The PCa cell lines themselves are also readily available, simple to use, and exhibit consistency in their behaviour and response. This combination of simplicity and accessibility permits the ready reproduction of research findings in different laboratories. PCa cell lines also provide a particularly useful model for identifying prospective gene targets in a fast and efficient manner, as well as for

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defining discrete intracellular molecular mechanisms without confounding paracrine influences. 2D monolayer cultures also possess other very significant inherent limitations, not least of which is that they represent the most reductionist model of tissue and cancer available. Specifically, these systems are mono-cellular, lack a relevant basement membrane, have defective or no ECM deposition, no tertiary structure, and no stromal or inflammatory components. Coupled with the inherently abnormal culture environment (including a hard, flat plastic surface and high serum concentrations), these models provide a very poor representation of in vivo tumour biology. Consequently, cell behaviour is altered, with differences in cell morphology, polarity, receptor expression, proliferation, migration, apoptosis, differentiation status, oncogene expression, interaction with the ECM (including the basement membrane) and overall cellular architecture, occurring between cells grown as 2D monolayer cultures and what is observed in vivo (ie, 3D) [40]. Indeed, the complete absence of microenvironmental cues significantly limits the interpretation of data from 2D systems, primarily as reciprocal paracrine signalling mechanisms and direct cell interactions significantly modulate tumour cell behaviour. This is particularly highlighted when considering cell proliferation, one of the most common readouts from these systems. When cultured in 2D, cells show a high proliferation rate that is abnormal whilst cells cultured in 3D proliferate at a slower rate that is more reminiscent of proliferation in vivo [41–43]. This is a direct consequence of multiple parameters, including the physical environment and high serum levels, but particularly a lack of any paracrine influence from stromal constituents [44,45]. The utilisation of prostate cancer cell lines in research is also hampered by a number of other contributory factors. A large proportion of research is still undertaken using the three ‘classic’ cell lines, LNCaP, PC3, and DU145. The limited number of these cell lines itself is an issue, is relatively few compared to other cancers, and prevents any accurate reflection of the diversity that is seen in human prostate cancer. Additionally, these cell lines all show loss [46], or mutation [47], of the androgen receptor (AR), thus meaning they are not representative of primary disease, where androgen ablation and targeting AR signalling is front-line therapy. Additionally, these, and many of the other prostate cell lines, were derived from advanced cancer and/or metastatic lesions, and, therefore, are not suitable for elucidating the multistep process of carcinogenesis and tumour progression. Furthermore, the prolonged culture and repeated passage of these cell lines renders them increasingly different from their tumour of origin, with cells of increasing passage showing progressive genotypic and phenotypic change, including alterations in morphology, response to stimuli, growth rates, protein expression and migration [48–50]. Despite these many and significant limitations, cell-line-based PCa models remain the primary, and, in many cases, the only screening methodology used for the preclinical assessment of drug efficacy, largely due to their simplicity, ease of use and reproducibility. Cell-line based 2D models are also ideal for defining discrete intracellular signalling mechanisms, free from confounding paracrine influences. This, however, is very much a double-edged sword due to the caveat that 2D culture does not faithfully recapitulate the physiological behaviour of cells in vivo. Indeed, the correlation of results from 2D cultures to in vivo situations can be particularly poor, with anti-cancer drugs having been demonstrated to produce markedly different effects on cells when cultured in 2D versus 3D [21,51]. Thus, models that retain the benefits of 2D culture whilst incorporating components of the in vivo 3D environment are of immediate interest. 3.1.1. Co-cultures & advanced 2D models (2.5D) The many fundamental limitations of traditional 2D monocultures and a growing appreciation of the important interplay between epithelial/tumour cells and their microenvironment, particularly epithelial– stromal cell interaction and the role of the ECM, have led to the refinement of 2D models to specifically include one, or more, of these

Please cite this article as: S.J. Ellem, et al., In vitro modeling of the prostate cancer microenvironment, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.04.008

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parameters. The development of these pseudo-3D (or 2.5D) models represents a significant advancement, particularly as specific discrete mechanisms relating to multi-cell and ECM interaction can now be examined and quantified in vitro. There are now increasing recognition and appreciation of the important role that the prostatic stroma plays in tissue homeostasis, but also disease initiation and progression. Previously, this role of the stroma could only be investigated using complex tissue recombination and/or xenograft models [25–27]. Recently, however, we described a novel co-culture model that enables the role of the stroma to be investigated and quantitatively measured in vitro. This system, incorporating stromal fibroblasts, benign epithelium and stromal ECM deposition, now allows us to examine discrete mechanisms and the influence of the stroma on epithelial transformation and tumourigenesis, with CAFs, but not NPFs, inducing discrete malignant changes in benign epithelial cells [28]. This and other similar models that incorporate both stromal and epithelial components [52] have the potential to provide significant new insight regarding the role of the stroma in PCa development and progression; they are ideal for defining specific cellular interactions (eg, fibroblasts + epithelium) and paracrine signalling mechanisms. These models are also likely to have significant impact on drug development and testing, something that has largely remained limited to tumour cell lines in 2D culture. In particular, the contribution of the stroma may influence drug sensitivity and development of resistance [53], which may be tested, and accounted for, using systems that incorporate interactions with the stroma. While representing a significant step forward from traditional 2D monocultures, these models still remain limited by virtue of their lack of tertiary structure and limited composition. More specifically, they still do not replicate the original structural milieu in terms of glandular organisation and do not reflect the intrinsic heterogeneity that is characteristic of prostate tumours. Further refinements to these models, as well as the development of more complex 3D systems, are essential to better mimic in vivo situations and obtain an accurate representation of cell and tumour behaviour, in vitro. 3.2. Three dimensional models (3D) The influence and role of the surrounding environment are critical components of the prostate cancer ecosystem, playing vital roles in tumour development and progression. In particular, the composition and 3D architecture of the tissue, ECM and chemical milieu can all exert strong effects modulating cell proliferation, invasion, morphology, and signalling [40], but also drug response and efficacy [54]. Many of these important cues are lacking in, and are unable to be factored into, traditional 2D monolayer cultures, producing results and responses that do not reflect in vivo behaviour. To better examine this and overcome some of the inherent limitations of 2D systems, several methods have been devised to generate 3D cell cultures that better mimic physiological tissues and cell interaction. Of these, the growth of multicellular spheroids in purified ECM gels or hanging drops has demonstrated the most utility. 3.2.1. Spheroids (aka prostaspheres) Spheroids are spherical multicellular aggregates that are grown within a 3D culture system. These systems have proven to be a particularly effective model for 3D culture [55,56], particularly for solid tumours such as brain [57], breast (mammospheres; [58–60]) and prostate (prostaspheres; [61–65]). Spheroids, in particular, serve as excellent physiologic tumour models by virtue of their 3D growth and ability to develop of discrete intracellular/intramatrix interactions. Tissue spheroids closely mimic avascular tumours and micrometastases [66] and demonstrate properties of cell survival, growth and behaviour that are reminiscent of the in vivo situation. They also exhibit some of the in vivo microenvironmental characteristics of solid tumours that are fundamental to tumour progression, including

anchorage-independent growth and the presence of intra-tumoural hypoxia and nutrient gradients. These properties of spheroids are particularly relevant to PCa studies, with PCa possessing a microenvironment that is characterised by hypoxia, acidosis, and nutrient deprivation. Since spheroids reproduce the tumour microenvironment more accurately than conventional monolayer systems, they provide more accurate and meaningful biological readouts compared to 2D models [55]. Thus, spheroids have significant potential to advance our understanding of prostate cancer biology, a point highlighted over 40 years ago when the growth of multi-cell spheroids was first described [23]. Of the many techniques for spheroid formation that have been developed, prostaspheres produced from cells grown suspended in a purified ECM gel/matrix, or within a ‘hanging drop’, represent the most commonly used systems in the field. 3.2.1.1. Purified ECM gels (Matrigel, collagen, etc.). When cells are cultured within an anchorage-independent, 3D matrix, spheroids – prostaspheres – are formed. This technique, established over two decades ago for prostate [67], relies on the culture of epithelial populations in a purified ECM matrix, such as collagen or Matrigel. These matrices support specific processes such as cell polarity, cell–cell and cell–matrix interaction, and re-expression of differentiation markers [67]. This model system is reliant upon the presence of cells with stem-like properties, which have the capacity to survive and proliferate to form spheroids via clonal expansion in 3D culture [63], whereas differentiated cells fail to survive. In this system normal/benign prostate epithelial cells differentiate into well-polarized hollow spheroids, a hallmark of functional, glandular epithelial cells. In contrast, tumour cells usually show a defective differentiation program, and form atypical spheroids with disorganized architecture (demonstrated particularly prominently in breast cancer [68]). 3.2.1.2. Hanging drop. Tissue spheroids may also be grown in hanging drop cultures. Rather than being encased in an ECM gel, the cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids [69,70]. The natural reaggregation of monodispersed cells is achieved simply with the aid of gravity enforced self-assembly, via a literal ‘hanging drop’ or by preventing attachment to the culture surface. This is a technique whose primary strengths derive from its simplicity; the model typically does not require specialised equipment and can be easily adapted to include the addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell–cell or cell–ECM interaction. 3.2.1.3. Implantation of Spheroid Cultures in 3D Gels. Some groups have used hanging drop cultures to form initial cancer spheroids, and have further implanted them into a 3D gel during the gelation process. This allows forming gels of desired properties around the spheroids for further studies of tumour invasiveness or drug testing under controlled microenvironmental conditions. This model presents both advantages: the simplicity to form spheroids of controlled cell numbers thanks to the hanging drop method and the controlled microenvironment created with either natural ECM gels or synthetic ones. However, the potential of these hybrid systems is yet to be explored for studies of prostate tumour progression. 3.2.1.4. Advantages & Limitations. Each of these spheroid based 3D in vitro techniques is fundamentally different, possessing its own strengths and limitations. However, both remain particularly well suited to assessing specific parameters of tumour biology, such as cell survival, growth and behaviour, within a more in-vivo-like, 3D, construct. ECM matrix based prostasphere assays have a unique strength for the study of prostate stem cells. This system allows for the potential functional isolation of prostate stem cells, expansion of stem cell numbers in vitro, and the ability to manipulate stem cells in vitro which

Please cite this article as: S.J. Ellem, et al., In vitro modeling of the prostate cancer microenvironment, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.04.008

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provides research opportunities to identify regulation of stem cell and progenitor proliferation and differentiation. In contrast, the simplicity of hanging drop models means that these techniques can be readily adapted for high-throughput systems, with hanging drop kits and protocols now readily available from Biotechnology companies (including up to 384 well plates). Recent advances in micro-fluidic technology also offer significant potential to improve these systems even further, allowing incorporation and integration of physiologically relevant elements without extra complexity. Platforms based upon these technologies are very likely to become important parts of high-throughput systems in the future, particularly for parallel experiments, multiplexing, screening, and drug testing purposes. Indeed, various groups have already developed and described spheroid based systems utilising technologies such as microwell arrays and microfluidic devices [71–74]. These spheroid systems are also sufficiently flexible to allow coculture of two (or more) different cell populations so as to elucidate the role of cell–cell or cell–ECM interactions; that is, they may also serve as co-culture models and thus better approximate the in vivo tissue architecture. Multiple cell types, such as stromal fibroblasts, nerve ganglia or endothelial cells, can be seeded within a matrix gel to influence spheroid growth and define specific roles or interactions in PCa [36]. Additionally, multiple cells can be seeded into hanging drops to form heterogeneous aggregates, a procedure with proven utility for examining angiogenesis and blood vessel maturation [75–77]. While the advantages of 3D spheroid cultures are appreciated and widely known, typical spheroid culture methods, such as those described, also suffer from a number of significant limitations. Technically, the methods are often tedious, difficult to handle, and/or produce nonuniform samples. Experimentally, the models can also suffer from problems such as efficiency of forming spheroids, long-term culture, control of spheroid size, necrotic cores in large spheroids, and poor control of cell distribution. While these systems do provide a significant advance over 2D models in representing in vivo tissue, they also remain poorly representative of the natural heterogeneity of the prostate; there is no control of assembly, structure, and only very limited spatial or glandular organisation. Overall, spheroid-based 3D culture models have yielded some important information regarding prostate and PCa biology, highlighting the role of cell–cell interactions, and the importance of the stroma and ECM, in the microenvironment. However, the combined limitations and complications of these models, such as those described, have hindered the adoption of 3D spheroid cultures into routine research use. Consequently, alternate avenues for 3D culture have been explored to better approximate and examine human in vivo prostate and PCa tissue, in vitro. 4. Advanced bioengineered models The growing appreciation of the interplay between PCa cells and their microenvironment, along with the importance of the 3D arrangement of the tissue, has led to the development of a number of different 3D models. Invariably, however, these models do not provide a faithful recreation of the in vivo structure of the prostate. The development of novel biomaterials and tissue engineering strategies, widely used for the purpose of wound healing or bone regeneration, offers novel and unique avenues to overcome this limitation and design a better in vitro recapitulation of the in vivo prostate architecture [78,79]. The underlying rationale for the development of these advanced bioengineered models is the utilisation of already existing technology and platforms developed for tissue engineering strategies, which can be further adapted and optimised to recapitulate the complexity of the prostate tumour microenvironment. A great variety of both natural and synthetic biomaterials have been extensively used as scaffolds for tissue engineering applications; they are starting to be also indispensable for advanced 3D in vitro cancer models. Polymeric

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scaffolds can be made from natural (collagen, hyaluronic acid, elastin, glyocsaminoglycans, etc.) or synthetic (polycaprolactone [PCL], polylactide [PLA], polyglycolide [PGA] and co-polymers [PLGA, PLG]) polymers to form various 3D structures, like meshes, fibres and sponges [80,81]. Cell behaviour, such as migration, proliferation and aggregation, can all be influenced by the various properties of these biomaterials, including chemical composition, biodegradability, 3D structure, hydrophobicity/hydrophilicity, mechanical properties and porosity; each of these parameters having the ability to be specified and varied in the case of synthetic polymers [81]. Thus, they offer significant potential to mimic aspects of the natural microenvironment as they can be functionalised and designed according to requirements. Their properties can be tuned up to provide an optimised framework for cell adhesion and growth in 3D and the recreation of the prostatic structure [78]. Due to the control and flexibility scaffolds afford, these systems offer the potential to faithfully recapitulate different stages of PCa progression, from benign tissue to initial tumour development, and, ultimately, to metastasis. While this area of research and the development of scaffold-based PCa models is still in its infancy, it is in this latter arena, metastasis, where significant advances are already being made using these techniques. In particular, tissue engineering and scaffold-based systems to model the PCa-bone metastatic niche are already providing significant new insight [82–84]. With further development these systems offer significant promise to produce fundamental new insight into prostate biology. Engineered 3D models will be of particular utility to study multiple cell–cell interactions and their effects within a heterogeneous 3D microenvironment, including complex processes such as angiogenesis. However, synthetic scaffolds by their very nature are not made of natural components and to achieve an in vivo-like structure with the complexity of the prostate, containing all the essential microenvironmental cues, is a vastly challenging task. Consequently, alternate in vitro systems that maintain the original 3D morphology and structure of the prostate – ex vivo explants – have garnered renewed interest. 5. Ex vivo explant culture Instead of relying on the in vitro reconstruction of in vivo tissue architecture, an alternative 3D model system that possesses significant potential, particularly for PCa research and drug discovery, is the ex vivo culture of human patient tissues. Unlike spheroid and other reconstructed models, ex vivo culture of primary patient tissues retains the original tissue architecture, microenvironment and heterogeneity that are characteristic of PCa, thus potentially providing a more clinically relevant system [85]. A variety of differing methods have been developed over a number of years, although all have the same basic premise of in vitro culture of small (~1–2 mm) samples or slices (~200–300 μm thick) of primary patient tissue [85]. The methodology can vary, with different techniques based on culture either in solution [86–88], supported on a metallic grid [89–91], or atop a sponge/gel [92–94]. In particular, the latter adaption of culturing explants atop a gelatin sponge has proven to be particularly effective, with improved viability and culture duration (N 1 week), as well as preservation of tissue structure and steroid receptor expression [94]. Regardless of the specific methodology used, these ex vivo techniques harbour very significant benefits and limitations. Largest of the limiting factors is the high dependency on ready access to patient tissues, not only in sufficient quantities, but also of reliable viability (ie, small latency post-surgery to laboratory). Additional complications also arise from the heterogeneous nature of the tissue source and the subsequent need for histopathological controls and verification of the tissue content of explants. Despite these factors, the reliance and utilisation of primary patient tissues are also key strengths of these methods. In particular, the maintenance of the 3D in vivo tissue structure and cell interactions provides a far more accurate biological

Please cite this article as: S.J. Ellem, et al., In vitro modeling of the prostate cancer microenvironment, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.04.008

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response, rendering this system particularly suited to pre-clinical drug testing and the effect on specific cell parameters (such as proliferation and apoptosis), while utilising patient tissues also provides the ability to examine specific disease states, including CRPC and metastatic tumours. Overall, ex vivo explant based techniques have also been of significant benefit to the field. They have proven of particular utility examining hormone action [90,91,95], irradiation and chemotherapeutics [96, 97], as well as novel drugs/therapeutics [98,99]. Furthermore, the key feature and reliance on primary patient tissues present the opportunity to examine a particular patients’ response to therapy, providing the possibility to adapt these methods in the future for personalised medicine.

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[8] [9]

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6. Conclusions There is a great need to advance in the development of more physiologically relevant in vitro model systems of cancer. Currently, PCa research, drug development and testing remain almost exclusively based around traditional 2D monoculture models that utilise highly abnormal, immortalised cancer cell lines. These systems cannot consider or incorporate the tissue microenvironment that plays a prominent role in the mechanisms and processes of tumour development and growth, particularly in PCa. Of particular importance in prostate biology are reciprocal mechanisms of paracrine signalling between the various cell types and compartments of the microenvironment. Paracrine signalling between the stroma and epithelium is of fundamental importance in PCa and the aberrant paracrine signalling of many factors, including TGF-β, Wnt/β-catenin, AR, FGF, and more, has been demonstrated to drive prostate tumour development and progression [100–103]. These mechanisms are lacking in the 2D cancer cell line models that underpin the vast majority of PCa research, highlighting their limited biological relevance. To date, only the advanced 2D and bioengineered 3D systems discussed in this review offer the potential to examine, modulate and define these mechanisms and their contribution to PCa in vitro. It is imperative for any model to accurately mimic the key players and characteristics of the in vivo tumour niche in vitro to better understand disease aetiology and progression and to improve preclinical drug development and screening. 3D bioengineered multicellular model systems represent the ideal tools to achieve this, with the ability to mimic the complex cellular structure of PCa to varying degrees and assess mechanisms of tumour growth, EMT, invasion and metastasis. However, as for any other models, due to the very fact that they are models, they also present intrinsic drawbacks. The improved biological relevance of these systems frequently comes at the expense of increased complexity and lower throughput, thus the utilisation of the different 3D models for research and/or drug testing must be tempered by this. Ultimately, it will only be with the development of new platforms that provide a high degree of biological relevance, while remaining cost effective and providing sufficient throughput for high content screening, that any significant advancement in PCa therapeutics will be achieved.

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Please cite this article as: S.J. Ellem, et al., In vitro modeling of the prostate cancer microenvironment, Adv. Drug Deliv. Rev. (2014), http:// dx.doi.org/10.1016/j.addr.2014.04.008