Engineering Mammary Gland in Vitro Models for Cancer Diagnostics

Review pubs.acs.org/molcularpharmaceutics

Engineering Mammary Gland in Vitro Models for Cancer Diagnostics and Therapy Jonathan J. Campbell,*,† Robert D. Hume,‡ and Christine J. Watson*,‡ †

Department of Materials Science and Metallurgy, University of Cambridge, 27 Charles Babbage Road, Cambridge CB3 0FS, U.K. Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP. U.K.

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ABSTRACT: Breast cancer is a complex disease with many distinct subtypes being recognized on the basis of histological features and molecular signatures. It is difficult to predict how cancers will respond to therapy, which results in many women receiving unnecessary or inappropriate treatment. Advances in materials science and tissue engineering are leading the development of complex in vitro 3D breast tissue models that will increase our understanding of normal development and tumorigenic mechanisms. Ultimately, platforms that support primary tissue culture could readily be adapted to form high-throughput drug screening tools for personalized medicine. This review will summarize the control of mammary gland phenotype within in vitro 3D environments, in the context of a detailed analysis of mammary gland development and stem and progenitor cell controlled tumorigenesis. KEYWORDS: breast cancer, mammary gland, in vitro tissue models, 3D, stem cell niche, high-throughput screening, biomaterials

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INTRODUCTION Breast cancer is the most common cancer in the UK, with over 50,000 new cases diagnosed each year.1 A recent wide-ranging study2 identified 10 key areas to focus future breast cancer research, including understanding how cancers grow and spread and assessing patients’ response to therapy (Table 1). Advances in tissue engineering and materials science have enabled researchers to develop robust organotypic in vitro tissue models, with controlled biochemical, mechanical, and spatial parameters that can be tuned to explore simultaneous features of development and tumorigenesis. These could be deployed to enhance patient-specific diagnosis and therapeutic management, in tandem with the rapid advances in our understanding of genetic and epigenetic control of cancer pathogenesis. Breast cancer may have multiple cells-of-origin, giving rise to different categories of tumors that require specific therapeutic strategies. Experimentation to uncover cell signaling and molecular deregulation in cancer, together with pharmacological drug testing, is at present highly dependent on the generation of large numbers of specific in vivo animal models while certain subtypes of breast cancer, such as triple negative breast cancer (BC) remain lethal in a high proportion of cases.3 It is likely that elucidating the complex drug combinations required to effectively manage such a disease will benefit from high-throughput platforms to assess potency on a patientspecific basis. In vitro 3D systems that aim to recapitulate aspects of the in vivo microenvironment will potentially increase the quantity of research material available, enabling populationwide screening with faster output, lower cost, and experimental work load that can rapidly assimilate patient-specific data. Considerable challenges remain before such approaches are in © XXXX American Chemical Society

Table 1. Tops 10 Gaps Identified for Translating Current Knowledge of Breast Cancer Research and Treatment into Clinical Improvementa 1. understanding the specific functions and contextual interactions of genetic and epigenetic changes in the normal breast and the development of cancer*b 2. effective and sustainable lifestyle changes (diet, exercise, and weight) alongside chemopreventive strategies 3. tailored screening approaches including clinically actionable tests* 4. molecular drivers behind breast cancer subtypes, treatment resistance, and metastasis* 5. mechanisms of tumor heterogeneity, tumor dormancy, de novo or acquired resistance; how to target the key nodes in these dynamic processes* 6. validated markers of chemosensitivity and radiosensitivity* 7. interactions, duration, sequencing, and optimal combinations of therapy for improved individualization of treatment* 8. Optimized multimodality imaging for diagnosis and therapeutic monitoring should enable better evaluation of primary and metastatic disease.* 9. interventions and support to improve the survivorship experience including physical symptoms such as hot flushes and lymphoedema 10. clinically annotated tissues for translational research including tumor, nontumor, and blood based materials from primary cancers, relapsed and metastatic disease* a Adapted with permisson from ref 2. Copyright 2013 XXX. b*, could be enhanced through the development of 3D high-throughput in vitro models.

Special Issue: Engineered Biomimetic Tissue Platforms for in Vitro Drug Evaluation Received: February 10, 2014 Revised: April 9, 2014 Accepted: April 14, 2014

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dx.doi.org/10.1021/mp500121c | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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Figure 1. Ultrastructure of the murine mammary gland. (A) Whole-mount and (B) higher power micrograph (10 day gestation) and (C) schematic showing a branched epithelium (e) composed of ducts (d) and acini (a) within an adipocyte-rich fatpad ( f p) with a prominent lymph node (l). Bars = 1 mm and 50 μm.

widespread clinical use however. Notably, the refinement and standardization of in vitro methodology to explore critical mechanisms of malignancy will enable high-throughput systems that replicate microenvironments precisely over many times, increasing the power of experimental or diagnostic studies. Development of the Mammary Gland Is Mediated through Stem Cells. In vitro models of the mammary gland must accommodate aspects of physiological development if they are to be used as tools for studying disease mechanisms. It is worthy of note at this stage that many of the vital mechanisms of development, such as epithelial−mesenchymal transition (EMT), are required for cancer pathogenesis and spread. The mammary gland is a highly active organ with marked regenerative potential. The mature gland comprises a branching bilayered epithelium of inner luminal cells enveloping ducts and lobuloalveolar structures surrounded by a thin myoepithelium, enveloped within an adipose rich stroma (Figure 1). Unique among tissues, it has distinct stages of development within the embryo as well as in the female adult during puberty and pregnancy. Although a complete description of these processes are beyond the focus of this review, it is likely that each stage is reliant upon the coordination of stem cell (SC) pools by surrounding biochemical signaling gradients initiated by hormones or upon autocrine and paracrine signals from adjacent differentiated progeny or stromal tissues. In the embryo, specialized regions of ectoderm organize into adjacent, ventrally located ridges, known as milk lines that display activated Wnt signaling,4,5 an established regulator of SC pluripotency.6 These form as a result of Tbx-3 expression, a member of the T-box family of transcription factors, and an important promoter of embryonic stem cell (ESC) proliferation,7 the expression of which is focused beneath the milk line in a dorso-ventral manner by fibrobastic growth factor (FGF)10 and laterally by bone morphogenic protein (BMP)-4. Later, ectodermal cells coalesce along each milk line to form thickened regions known as placodes under the control of canonical Wnt signaling and members of the TNF superfamily. These begin to penetrate into the mesenchyme to form a rudimentary branched gland known as a mammary sprout (for reviews, see refs 8 and 9). This initial anlagen is controlled by parathyroid hormone related protein (PTHrP)10 originating within the invading mammary bud, which specifies BMP receptor 1A upregulation in the subjacent mesenchyme, rendering it sensitive to BMP4 stimulation and differentiation programs initiated through SMAD phosphorylation. In addition, hair follicle development is suppressed through Msx-2 expression within the developing nipple.11

Lumenogenesis proceeds within the mammary sprout through a process of cavitation.12 In this model, apoptosis proceeds via loss of integrin mediated extracellular matrix (ECM) contact.13 The mammary epithelium is highly dependent upon the composition of basement membrane (BM) proteins, specifically laminin species, for its apico-basal polarity and function.14 Indeed, laminin has been identified as a tumor suppressor in breast tissue.15 BM deposition within the human embryonic colon and pancreas is positively regulated by SMAD4,16,17 and it is likely that BM is synthesized at this stage of development through cooperative signaling between epithelial and stromal cells. Apoptosis plays a major role within lumenogenesis of the mammary sprout and later within the terminal end bud of the invading duct, mediated through a member of the BCL-2 protein family BIM; however, other mechanisms are thought to contribute to the process including autophagy.18 Embryonic mammary gland development ceases soon after the appearance of white adipose tissue at E18 in mice. At puberty, systemic rises is estrogen give rise to club-like thickenings up to 10 cell layers deep, known as terminal end buds (TEB). At this stage, the TEB is a combination of two progenitor cell compartments. Central rapidly dividing body cells will give rise to a single layer of trailing luminal cells that characteristically express cytokeratin (K)18, while peripheral cap cells contribute an enveloping myoepithelial compartment that stains positively for K14 and smooth muscle actin. It has been suggested that much of the proliferative potential of the gland is supplied by transit amplifying cells.19 The TEB invades the fatty stroma and branches by bifurcation until complete occupation of the fatpad by epithelium, in cooperation with side-branching mechanisms from the primary ducts. The regulation of branching pattern is broadly stochastic20 but is dependent on specific signaling pathways and spatial gradients utilizing numerous growth factors such as hepatocyte growth factor (HGF), fibroblastic growth factors (FGF), and transforming growth factor-β (TGF-β),21,22 together with enzymatic digestion of the ECM, under endocrine control via growth hormone (GH) and estrogen. The importance of the stroma to branching morphogenesis was highlighted in early work grafting the primary mammary epithelium within salivary gland stroma, noting an adoption in the epithelial branching pattern of the target organ.23 Indeed elements of mammary stromal−epithelial signaling are both tissue- and species-specific. Monitoring epithelial outgrowth in cleared murine fatpads − a conservative assay for stem cell and tumorigenic potential − is not possible with human mammary epithelial cells.24 The successful de novo expansion of B



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under physiological conditions. Their model describes postnatal lineage restriction of basal and luminal stem cells, each supplying their respective compartments in contrast to embryonic basal K14+ cells that can contribute to both lineages.41 However, live-tracing techniques using the confetti multicolored cell fate reporting system have demonstrated bipotent stem cell activity confined to the basal keratin5+ compartment.42 Furthermore, this activity is maintained into pregnancy, noted by the clonal nature of large lobuloalveolar units in the macroscopic gland. Stem Cells and Breast Cancer. Stem cells, characterized by their proliferative and self-renewal capacity, have been implicated as key players in cancer development. Broadly, tumor cells that display metastatic outgrowth can be considered cancer stem cells (reviewed in ref 43); however, evidence that directly implicates stem cells in tumorigenesis is as yet limited to a number of tissues including intestinal and prostate cancers.44 The “cell of origin” theory requires genetic tumorinducing mutation often in the SC compartment that leads to malignancy. However, it is possible that more mature cells within the lineage sustain tumor-inducing mutations and are programmed to adopt features of SCs, namely, high selfrenewal capacity and proliferation potential. Subsequent rounds of cell division within this cell leads to the accumulation of multiple descendant clones.45 Further mutation within the hierarchy of daughter cells leads to heterogeneous accumulation of genetically distinct clones. This common feature of tumorigenesis typical in breast cancer46 has important clinical consequences resulting in wide data variation at diagnosis and variable response to treatment. Cell signal deregulation in the SC niche is highly complex and can include interactions with stromal and immune cells and disruption of important signaling pathways critical to development, including Wnt,47 hedgehog,48 and Notch regulation. For example, inhibition of notch4 and notch1 signaling has been shown to promote mammary luminal cell fate, concomitantly reducing the stem cell pool and tumor formation rate in vivo.49 Notch signaling is highly spatial and operates between neighboring cells, where upon activation the transmembrane receptor is cleaved and transits to the nucleus to regulate cell differentiation programs and oncogenic targets. Notch2 receptor tracing has been shown to label spatially controlled mammary progenitor populations that may control lineage hierarchy and branching.50 Breast cancers are broadly classified as luminal or basal subtypes depending on their phenotypic profile, typically displaying the distinct expression of K18 or K14, respectively. This classification has subsequently been expanded on a genetic basis.51 In normal tissue preparations, isolated basal cells can readily repopulate cleared mammary fatpads, demonstrating measurable SC frequency within this cell fraction;52,53 however, in tumors, increasing numbers of studies suggest plasticity between luminal and basal SC and progenitor compartments.54,55 For example, BRCA-1 mutation results in a highgrade basal-type carcinoma suggesting origin in the K14+ basal compartment,56 although more recently, it has been traced to a luminal ER-progenitor subset.57 Basal cell regenerative capacity of the mammary gland has been shown to be dependent on the expression of the Myc transcription factor, an oncogene signaling through Wnt-β-catenin pathways.58 Interestingly, Myc is implicated in ESC pluripotency59 and is also highly expressed in luminal progenitor cells.60 While overexpression of Myc leads to proliferation in a range of breast cancer cell lines,

coengrafted human epithelial cells with irradiated human mammary stromal fibroblasts25 is a finding that has important consequences for future human high-throughput mammary gland models. Specific examples of stromal−epithelial cross-talk critical for mammary development are highlighted by the actions of pituitary GH acting upon its receptor GH-r in the mammary stroma, in turn initiating the stromal expression of insulin-like growth factor (IGF) acting upon an essential IGFreceptor in the mammary epithelium (reviewed in ref 26). In addition, FGF-10 being distributed throughout the juvenile adipose fatpad signals through FGF-R2b complexes in the invading TEB. Attenuation of FGF-10/FGF-2b signaling has been shown to lead to a 40% reduction in ductal outgrowth compared to that of a wild-type control.27 Extracellular matrix composition and its matrix metalloproteinase (MMP) mediated enzymatic clearance by the invading TEB in conjunction with a locally cooperative stroma has been shown to regulate branching.28 Stromal epidermal growth factor receptor (EGF-r) is activated by amphiregulin (Areg) originating in the TEB as an inactive transmembrane precursor, itself activated through cleavage by the matrix metalloproteinase Adam-17. Adam-17 expression is spatially controlled at the invasive front by the localized downregulation of its inhibitor, tissue inhibitors of matrixmetalloproteinase (TIMP)-3.29 Additional metalloproteinase action at the TEB, comes from collagenolytic MMP14 in conjunction with MMP2.30,31 MMPs act on the ECM, degrading and remodeling the microenvironment and releasing morphogens, although the isolated action of MMP-3 as a side-branching regulator has been demonstrated in the absence of growth factors.32 Among the constituents of the ECM that regulate patterning in numerous organs is heparin sulfate (HS) proteoglycan, which regulates the distribution of many signaling biomolecules by binding in an electrostatic manner33 and is known to regulate mammary branching.34,35 HS is known to regulate FGF7 and FGF10 spatial distribution and potency as a morphogen in lacrimal gland and salivary gland branching.36 At pregnancy, increased progesterone (P) and prolactin (Prl) trigger tertiary branching and the development of milkproducing lobuloalveolar structures. The progesterone receptor is activated in a proportion of mammary epithelial cells by the circulating hormone where it induces the expression of Wnt4, leading to increased branching and alveolar-type proliferation of the mammary epithelium.37 In a similar manner, its actions are also mediated through the activated NF-κB ligand (RANKL),38 which acts in a paracrine manner triggering the hyperproliferation of adjacent mammary epithelium, now shown to be Elf5+ mammary luminal cells.39 Interestingly, the mutually exclusive expression of PR and Elf-5 in mammary epithelial cells and the reciprocal balance of a mammary stem cell compartment expressing CD61+/CD29+ by these two factors demonstrate that side-branching is a possible consequence of positional stem cell niche regulation. It has been argued recently that progesterone treatment leads to a dramatic expansion in the CD49fhi stem cell-enriched population. In this study, diestrous mammary epithelial cells (MECs) demonstrated a 7.6-fold enrichment in mammary gland repopulating unit frequency compared to those obtained at estrous.40 The nature of SC and progenitor activity within the luminal and basal compartments throughout embryogenesis and adulthood has remained controversial. Van Keymeulen et al. argue that the demonstration of multipotential FACs sorted MECs by transplantation assays is artificial and not what occurs C



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Figure 2. Schematic illustrating some of the regulating factors controlling mammary gland phenotype with progression from normal tissue to tumorigenesis. The epithelium (green) moves through an EMT becoming more disorganized and liable to metastasis, in association with the changing phenotype of the mammary stroma, represented by the influx of tumor-associated macrophages and cancer-associated fibroblasts (CAFs) and increasing tissue stiffness. In vitro models can be adapted to represent such situations by modulating scaffold chemistry, coculture cell types, matrix stiffness, or organization. Migration, invasion and proliferation, morphology, and stiffness can be readily modeled as outputs.

control of trophic factors secreted by inflammatory macrophages.72 Tumour-associated macrophages play important roles in changing the biochemical milieu of the cancer microenvironment, promoting angiogenesis and cancer cell invasion (for a review see73). Lastly, breast tissue stroma composed largely of adipocytes can contribute to the inflammatory profile of cancer as a disease. It can be seen that accommodation of such complexity into a single model will not be possible; however, multifunctional assays that simultaneously measure response factors have been demonstrated with success, and it is possible to visualize highthroughput assays accommodating aspects of such environments. It is possible to design in vitro experiments that facilitate tumorigenesis or metastasis, modeling the movement of cells away from regions of primary disease. In this regard, the link between stem cells and cancer development could be exploited when tissue engineering in vitro platforms for drug discovery. The control of stem cell phenotype ex vivo has been studied for decades for cell therapy treatments and remains a core technique in tissue engineering.74,75 Biochemical Control of Mammary Tissue Phenotype in Vitro. Robust high-throughput assays must by definition place stringent emphasis on model repeatability. Strategies modeling drug response in vitro commonly focus on basic cytological methods such as cell proliferation, cell morphology, or movement described by migration or invasion. These methods are highly applicable to high-throughput methods in 2D multiwell plates or transwell chambers with great standardization in substrate chemistry and compatibility with absorbance, luminescence, or fluorescence systems as well as visual observation. Such systems are easily employed for simple

it inhibited the metastatic potential by modulating cell−ECM interaction through integrin receptors.61 Metastatic potential is essential for the spread of cancer and is highly dependent upon EMT processes, allowing epithelial cells, phenotypically dependent upon cell−cell interactions, to adopt more invasive migratory profiles62 driven in part by Wnt signaling elements.63,64 The finding that overexpressing proEMT transcription factors twist and snail in normal mammary epithelial cells leads to the adoption of mammary stem cell characteristics65 is critical evidence linking SC and cancer regulation. On closer inspection of a basal mammary SC population isolated on the basis of CD49high/CD61+, this group identified the transcription factor Slug as capable of stem cell enrichment in both basal and luminal cell compartments and increasing metastatic potential when overexpressed in conjunction with Sox9.66 The regulation of the SC niche by stromal cells is highly applicable to in vitro models because their contribution can be monitored in isolation, reflected by the increasing number of culture strategies combining stromal and epithelial tissues.67−69 The residual tumor-supporting capacity of normal stroma has been reported, where fibroblasts expressing a high level of PGRE2 isolated from normal reduction mammoplasty material were capable of supporting tumor formation in an equivalent manner to those isolated from regions of tumor.70 Likewise, human mesenchymal cells isolated from bone marrow have been implicated in supporting cancer stem cell self-renewal as measured by aldehyde dehydrogenase activity (ALDEFLOUR, Stemcell technologies SARL) through a C-X-C signaling motif −7 (CXCL7)/IL-6 signaling feedback loop.71 Mesenchymal stem cells (MSCs) migrate to sites of tissue injury under the D



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measures of cell response; however, comparisons to in vivo tissue complexity are limited. We now know that the control of tissue phenotype is highly complex and dependent upon the intricate spatial 3D arrangement of differing cell types in combination with varied ECM biochemistry and physical properties (for a summary, see Figure 2). Delivering biocompatible 3D environments to cells in culture can either be achieved through the “top-down” use of natural biomaterial preparations, often with multiple bioactive constituents, or using “bottom-up” functionalized synthetic polymer biomaterials with more defined chemistry, but a more limited presentation of signaling motifs, challenging the maintenance of matrix-dependent cell types. Cells integrate with their biochemical environment via cell− cell contacts through tight junctions, cadherins, and desmosomes and through the extracellular matrix with integrin receptors and other specialized transmembrane adhesion receptors. In the mammary gland, luminal and basal cells display different ratios of integrin receptors that can be used as a basis for sorting stem and progenitor cells.53 Integrin receptors tether cells to the ECM through focal adhesion kinase complexes connecting in turn to elements of the cell cytoskeleton. There is thus mechanical continuity between intercommunicating cells and their ECM.76 Integrins are composed of alpha and beta subunits, the variation of which controls ligand recognition; however, some proteins such as collagen type I and fibronectin have multiple recognition motifs for a range of integrins. The most promiscuous integrin subunit β1 pairs with a large number of α integrin subunits,77 and β1 knockout studies demonstrate its critical involvement in appropriate mammary development and function, including controlling epithelial polarity through integrin-linked kinase signaling78,79 and in correctly orienting basal cell division.80 It also has important roles in controlling metastatic spread81 and drug response.82 To date, recapitulating the complex ECM environment of glandular tissue has relied on the use of the reconstituted basement membrane (rBM). These preparations derived from Engelbreth Host-Swarm (EHS) tumors in mice are complex mixtures of proteins, notably laminin and collagen IV, and proteoglycans in combination with growth factors and cytokines. Because of their tumor derivation, rBM could be considered a false environment for normal tissue growth, and care should be taken if interpreting data in cancer studies.83 However, they do impart the correct cell-matrix signals, promoting epithelial polarity and differentiation through βcasein expression in mammary epithelia.84 Normal primary mammary epithelial cells and cell lines typically form growtharrested acini within these gels with morphology comparable to that of mammary alveoli, termed mammospheres, whereas cancer cell lines maintain proliferative rates to form disorganized cell masses.85 They are also deployed in physiological invasion studies, where they can coat substrates to enhance assay function, modeling the transit of malignant cells through the epithelial basement membrane or the endothelial lining of capillaries. Although tumor cells are less dependent on cell-matrix integration for survival, the in vitro colony formation assay performed in biochemically inert matrices such as soft agar has long been used as an assay for stem cell potential and tumorigenicity. There is increasing evidence that breast cancer cells respond differentially to 3D environments over 2D conformations. A panel of 25 breast cancer cell lines

demonstrated significant regulation of genes associated with signal transducer activity and enzyme regulator activity when grown in 3D rBM gels versus 2D tissue culture plastic.86 More recently, the expression of human epidermal growth factor receptor 2 (HER2), an important distinguishing marker in breast cancer classification was shown to be regulated depending on cell culture in 2D and 3D rBM preparations.87 The utilization of rBM has been adapted for high-throughput in vitro approaches as pure preparations88 and in hybrid systems.89 It is noteworthy that pure rBM gels exhibit a low, temperature-dependent elastic modulus relative to that of other natural and synthetic hydrogels90 and high degradation rates91,92 limiting their usefulness for long-term experiments. Although rBM gels remain ill-defined, attempts to clarify cellECM signals using pure protein preparations for the maintenance of mammary epithelial tissues in vitro can significantly modulate phenotype. For example, Collagen IV, a critical protein-organizing basement membrane assembly93 induces an EMT in the normal MCF-10A cell line, upregulating vimentin and down-regulating E-cadherin.94 In another study, silencing RNA techniques directed toward α5 and α6 integrin mediated EMT induced by fibronectin and laminin, respectively.95 Pure collagen preparations support both ductal and acinar formation depending on the cultured cell type96 and coculture strategies,97 and they support the expression of MMPs,98 which has been shown to be critical for cancer cell invasion, an activity not recapitulated by culture in rBM gels.99 However, collagen hydrogels are susceptible to significant cell-mediated contraction profoundly altering the microenvironment over short periods in culture.100 Other natural biomaterials have been utilized for studies of mammary epithelial and stem cell maintenance. Hyaluronic acid (HA) is a highly charged nonsulfated glycosaminoglycan of variable chain length with important biophysical and biochemical properties that have been exploited in in vitro systems,68,101 including increased tissue hydration and scaffold stability and cell-signaling pathways through specific CD44 and RHAMM receptors. It is processed by transmembrane hyaluronic acid synthases 1−3 (HAS) where it contributes to pericellular matrix formation. HAS-2 has been shown to be an intermediate in TGF-β-induced EMT in NMuMG cells102 and extracellular HA triggers CD44 translocation to the nucleus and lysyl oxidase transcription, up-regulating Twist transcription.103 CD44, expressed by myoepithelial cells in the breast, signals to luminal cells and is responsible for maintaining a bilayered organization of tissue.104 Properties of HA are dependent upon chain length, with high-molecular weight variants regulating EMT105 and short chain HA fragments supporting and triggering apoptosis.106 Its pericellular localization has been found to promote tumor cell survival in a breast cancer model,107 whereas its stromal concentration is found to increase in a range of cancers where it promotes invasion and angiogenesis.108 Polymeric biomaterials offer exquisite control over physicochemical substrate properties and are amenable to scale-up, making them ideal candidates for high-throughput technologies. Although many polymeric substrate chemistries have been explored for the culture of adult mesenchymal cell types109 and embryonic stem cells,110,111 their application to mammary epithelial cell culture has so far been limited. The adhesive environment is critical to establish cell function within these substrates, which require chemical modification with functional ligands. Indeed, the response of JIMT-1 breast cancer cells E



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produced by cell protrusions.128 Tunable substrate stiffness has now been incorporated into high-throughput assays in conjunction with other biochemical regulators of cell fate. A soft-lithographic technique has been used to produce microwells in hydrogels of varying stiffness, spotted with proteins in an array format.109 Such techniques can be used to explore the relationship of multiple niche regulators and could readily be adapted to studies of cancer pathogenesis. Cell and tissue geometry control stem cell phenotype and tissue organization.129,130 Lithographic approaches have been used to cast microchambers within collagen gel preparations to simulate 3D ductal structures in vitro. This approach, which is amenable to high-throughput integration, has been used to model epithelial branching, with branch point initiation predicted to be dependent upon the concentration profile of diffusible inhibitors. Thus, with exposure to a branching trigger stimulus, cell frequency was shown to increase at the tips of elongated structures relative to their midpoint or at the convex edge of a curved elongated structure.22 This versatile model has been expanded to demonstrate the self-arranging property of MMP14 expressing mammary epithelial cells dependent upon ROCK signaling. MMP14hi cells, which represent the most motile group of cells in the population, localize to tubule ends, whereas MMP3-expressing cells remained diffuse throughout the specimen.131 Similarly, noninvasive tumor cell lines proliferated to a greater extent at the tips of elongated structures where they adopted invasive morphologies,132 modeled as regions of high mechanical stress. The orientation of the ECM dictates how cells behave and is particularly important in the control TEB invasion. A recent in vivo experiment133 has shown that mammary epithelia display randomly arranged morphologies at an early stage of development but become progressively more oriented along the long axis of the invading gland as development proceeds. This group postulated that ECM fiber orientation determined branching pattern and utilized mechanically oriented collagen type I fibers in an in vitro model to demonstrate that fiber direction correlated with the orientation of branched cellular protrusions. It is noteworthy that the striking orientation of K5+ mammary myoepithelium along the major axis of the gland has been visualized in a recent study42 As well as the mechanical environment, 3D models of mammary gland and disease are highly dependent upon the maintenance of nutrient delivery, gas tension, and pH. The utilization of bioreactors for improved nutrient delivery to 3D cell cultures or the spatial control of morphogens remains a promising approach to improve tissue function; however, although miniaturization is ongoing,134 their widespread adoption would require significant material investment and technical expertise. Among the promising approaches recently described, the combination of soft lithography techniques to cast microscale channels to accommodate cell cultures, deliver nutrients, or cast solute gradients135 are highly promising and could be scaled to high-throughput approaches with minimal adaptation. Recently, such an approach was used to culture MCF-10A cells within small matrigel castings, in conjunction with a gravity fed flow chamber, eliminating the need for a complex pumping apparatus and improving handling of the culture chamber.136 Although optimization of nutrient delivery is critical to successful in vitro culture, cancers are often a solid mass with a hypoxic core. Recapitulating this scenario in vitro may improve physiological tumor microenvironments by seeding the breast

exposed to a panel of compounds when seeded in poly(2hydroxyethyl methacrylate) coated plates was similar in profile to that measured for cells grown on 2D tissue culture plastic.112 In addition, cells cultured in 3D rBM gels displayed a higher sensitivity to drug exposure with gene array expression profiles clustering with cell xenograft material. The functionalization of synthetic polymers can be achieved by incorporating bioactive ligands onto inert matrices. The modification of poly(ethylene) glycol (PEG) hydrogels with YIGSR or RGD ligands was reported to support a similar frequency of polar mammary acinar structures to PEG gels modified with laminin 1 chains.113 PEG matrices can be readily mixed with natural biomaterials to control resultant matrix stiffness or supply spatially controlled functional ligands. In a recent study, an RGD sequence containing cyclodextrins within PEG/matrigel cross-linked matrices supported the 3D dissemination of single cells from seeded tumor explants.114 Such models provide tunable systems that incorporate the stromal, pro-invasive quality of pure collagen preparations with the functional tissue support of rBM gels. Synthetic systems for normal mammary tissue models will need to incorporate degradation pathways to accommodate growing glandular tissues. Interestingly, MCF-10A cells exhibited improved viability in degraded PEG based hydrogels to nondegraded preparations.113 Recognizing the importance of MMP degradation to mammary gland development, the incorporation of MMP cleavable substrates into synthetic polymeric hydrogels115 will become a critical factor in the success of such materials for mammary gland models. Physical Control of Mammary Tissue Phenotype in Vitro. Matrix stiffness is a well-characterized bioactive regulator and has been shown to influence mammary epithelial phenotype and 3D tissue morphology.116−119 Increased mammographic density is a prognostic factor in breast cancer120 where stiffening of the ECM occurs as a consequence of increased matrix deposition or increased cross-linking.117,121 Weaver and co-workers showed that collagen matrix stiffness correlates with tumor cell invasion in a breast model mediated through integrin−ECM interactions.122 A separate group demonstrated that increased invasive phenotype, driven through a FAK-Rho kinase−MAPK feedback mechanism, occurred as a consequence of exposure to stiffer collagen matrices.123 This study was repeated on PEG-functionalized hydrogels to demonstrate that such cell responses were not the consequence of an increased concentration of signaling motifs on higher density gels. More recent work demonstrated that STAT5 phosphorylation by Prl exposure may be modulated by matrix stiffness.117 The application of Ingber’s tensegrity model,76,124 integrating the ECM and cell mechanically through the cytoskeleton, has been demonstrated using particle tracking rheometry, measuring MCF-10A cellular stiffness on substrates of varied compliance.125 Interestingly, this study correlated cell stiffening in response to matrix stiffening as a consequence of HER2/neu overexpression. The regulation of stem cell differentiation by the mechanical environment is now a major topic in mechanobiology,126 and its relationship to cancer stem cell niche regulation is an area of increasing research activity. In this regard, the measurement of cell stiffness on a per-cell basis may increase our understanding of heterogeneous cell systems and help identify invasive cell types. This can be achieved by seeding cells on ultrathin substrates that can be deformed by individual cells127 or micropillars that measurably deform depending on local force F



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cancer cell lines in bulk hydrogels.137 Hypoxia has been demonstrated in such models by the up-regulation of hypoxiainducible factor (HIF)-1α and increased angiogenic potential by up-regulation of vascular endothelial growth factor (VEGF).138 In a novel approach, layered paper-supported 3D hydrogel models controlled the spatial delivery of O2 and nutrients to a hypoxic core, mimicking the necrotic cores of tumors in vivo and tumor spheroids in vitro. Such a model can be readily assembled to produce a full-depth cellularized tissue mass and deconstructed to aid analysis.139 A more complex hypoxic model has been synthesized and has been used to test a cancer therapeutic. An example of one such study used a cationic photosensitizer EtNBS to treat hypoxic regions deep inside in vitro 3D models of metastatic ovarian cancer.140

E.; Bundred, N. J.; Burchell, J. M.; Campbell, A. M.; Carroll, J. S.; Clarke, R. B.; Coles, C. E.; Cook, G. J. R.; Cox, A.; Curtin, N. J.; Dekker, L. V.; Silva, I. D. S.; Duffy, S. W.; Easton, D. F.; Eccles, D. M.; Edwards, D. R.; Edwards, J.; Evans, D.; Fenlon, D. F.; Flanagan, J. M.; Foster, C.; Gallagher, W. M.; Garcia-Closas, M.; Gee, J. M. W.; Gescher, A. J.; Goh, V.; Groves, A. M.; Harvey, A. J.; Harvie, M.; Hennessy, B. T.; Hiscox, S.; Holen, I.; Howell, S. J.; Howell, A.; Hubbard, G.; Hulbert-Williams, N.; Hunter, M. S.; Jasani, B.; Jones, L. J.; Key, T. J.; Kirwan, C. C.; Kong, A.; Kunkler, I. H.; Langdon, S. P.; Leach, M. O.; Mann, D. J.; Marshall, J. F.; Martin, L.; Martin, S. G.; Macdougall, J. E.; Miles, D. W.; Miller, W. R.; Morris, J. R.; Moss, S. M.; Mullan, P.; Natrajan, R.; O’Connor, J. P. B.; O’Connor, R.; Palmieri, C.; Pharoah, P. D. P.; Rakha, E. A.; Reed, E.; Robinson, S. P.; Sahai, E.; Saxton, J. M.; Schmid, P.; Smalley, M. J.; Speirs, V.; Stein, R.; Stingl, J.; Streuli, C. H.; Tutt, A. N. J.; Velikova, G.; Walker, R. A.; Watson, C. J.; Williams, K. J.; Young, L. S.; Thompson, A. M. Critical Research Gaps and Translational Priorities for the Successful Prevention and Treatment of Breast Cancer. Breast. Cancer Res. 2013, 15, R92. (3) Dent, R.; Trudeau, M.; Pritchard, K. I.; Hanna, W. M.; Kahn, H. K.; Sawka, C. A.; Lickley, L. A.; Rawlinson, E.; Sun, P.; Narod, S. A. Triple-Negative Breast Cancer: Clinical Features and Patterns of Recurrence. Clin. Cancer Res. 2007, 13, 4429−4434. (4) Veltmaat, J. M.; Van Veelen, W.; Thiery, J. P.; Bellusci, S. Identification of the Mammary Line in Mouse by Wnt10b Expression. Dev. Dyn. 2004, 229, 349−356. (5) Chu, E. Y.; Hens, J.; Andl, T.; Kairo, A.; Yamaguchi, T. P.; Brisken, C.; Glick, A.; Wysolmerski, J. J.; Millar, S. E. Canonical WNT Signaling Promotes Mammary Placode Development and Is Essential for Initiation of Mammary Gland Morphogenesis. Development 2004, 131, 4819−4829. (6) Sato, N.; Meijer, L.; Skaltsounis, L.; Greengard, P.; Brivanlou, A. H. Maintenance of Pluripotency in Human and Mouse Embryonic Stem Cells Through Activation of Wnt Signaling by a Pharmacological GSK-3-Specific Inhibitor. Nat. Med. 2004, 10, 55−63. (7) Esmailpour, T.; Huang, T. TBX3 Promotes Human Embryonic Stem Cell Proliferation and Neuroepithelial Differentiation in a Differentiation Stage-Dependent Manner. Stem Cells 2012, 30, 2152− 2163. (8) Watson, C. J.; Khaled, W. T. Mammary Development in the Embryo and Adult: A Journey of Morphogenesis and Commitment. Development 2008, 135, 995−1003. (9) Cowin, P.; Wysolmerski, J. Molecular Mechanisms Guiding Embryonic Mammary Gland Development. Cold Spring Harbor Perspect. Biol. 2010, 2, a003251−a003251. (10) Brennan, K. R.; Brown, A. M. C. Wnt Proteins in Mammary Development and Cancer. J. Mammary Gland Biol. 2004, 9, 119−131. (11) Hens, J. R.; Dann, P.; Zhang, J.-P.; Harris, S.; Robinson, G. W.; Wysolmerski, J. BMP4 and PTHrP Interact to Stimulate Ductal Outgrowth During Embryonic Mammary Development and to Inhibit Hair Follicle Induction. Development 2007, 134, 1221−1230. (12) Coucouvanis, E.; Martin, G. R. Signals for Death and Survival: A Two-Step Mechanism for Cavitation in the Vertebrate Embryo. Cell 1995, 83, 279−287. (13) Frisch, S. M.; Francis, H. Disruption of Epithelial Cell-Matrix Interactions Induces Apoptosis. J. Cel. Biol. 1994, 124, 619−626. (14) Gudjonsson, T.; Rønnov-Jessen, L.; Villadsen, R.; Rank, F.; Bissell, M. J.; Petersen, O. W. Normal and Tumor-Derived Myoepithelial Cells Differ in Their Ability to Interact with Luminal Breast Epithelial Cells for Polarity and Basement Membrane Deposition. J. Cel. Sci. 2002, 115, 39−50. (15) Martin, K. J.; Kwan, C. P.; Nagasaki, K.; Zhang, X.; O’Hare, M. J.; Kaelin, C. M.; Burgeson, R. E.; Pardee, A. B.; Sager, R. DownRegulation of Laminin-5 in Breast Carcinoma Cells. Mol. Med. 1998, 4, 602−613. (16) Zapatka, M.; Zboralski, D.; Radacz, Y.; Böckmann, M.; Arnold, C.; Schöneck, A.; Hoppe, S.; Tannapfel, A.; Schmiegel, W.; SimonAssmann, P.; Schwarte-Waldhoff, I. Basement Membrane Component

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CONCLUSIONS AND FUTURE DIRECTIONS In recent times, advances in materials science and engineering have accelerated the development of 3D in vitro models of the mammary gland significantly over the fundamental advance offered by the utilization of rBM gels to control epithelial polarity and function. We are now starting to witness the introduction of highly tractable models that will be capable of scale-up for the high-throughput approaches necessary to address some of the more complex issues of cancer as a disease, such as cell heterogeneity and drug resistance. However, there are still fundamental issues to consider, such as the selection of biomaterials, adequate perfusion of a 3D tissue sample in vitro, fabrication strategies, and usability by researchers and biomedical scientists. Natural biomaterials are subject to species and batch variation, and with time, the use of recombinant technology to engineer human specific proteins will become cheaper. For example, plant-derived human collagen type I prepared from transgenic tobacco plants was recently used to culture skin fibroblasts and activated THP-1 macrophages where it resulted in lower IL-1β production.141 High-throughput approaches will require miniaturization of tissue analogues and culture vessels, to allow massive replication of sample size with minimal material cost. However, tissue-response parameters may be dependent on scale,142 and studies of a cancer tissue mass must balance tissue scale with heterogeneity. Problems of miniaturization have been highlighted by tissue microarray (TMA) technologies, where small cores of tissue from multiple sites are assembled on a slide for histological or in situ hybridization analyses. It is possible to introduce diagnostic error simply by missing the tumor site, even if multiple random sites of a biopsy specimen are selected.143 In addition, future models for diagnostic and drug discovery applications must be able to support human primary cell lines as well as being capable of accommodating the multiple cell types present within the gland. Such approaches are in development.

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AUTHOR INFORMATION

Corresponding Authors

*(J.J.C.) E-mail: [email protected]. *(C.J.W.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Breast cancer campaign website. breastcancercampaign.org. (2) Eccles, S. A.; Aboagye, E. O.; Ali, S.; Anderson, A. S.; Armes, J.; Berditchevski, F.; Blaydes, J. P.; Brennan, K.; Brown, N. J.; Bryant, H. G



Review

Laminin-5 Is a Target of the Tumor Suppressor Smad4. Oncogene 2006, 26, 1417−1427. (17) Costello, I.; Biondi, C. A.; Taylor, J. M.; Bikoff, E. K.; Robertson, E. J. Smad4-Dependent Pathways Control Basement Membrane Deposition and Endodermal Cell Migration at Early Stages of Mouse Development. BMC Dev. Biol. 2009, 9, 54. (18) Mailleux, A.; Overholtzer, M.; Brugge, J. Lumen Formation During Mammary Epithelial Morphogenesis: Insights From in Vitro and in Vivo Models. Cell Cycle 2008, 7, 57. (19) Smalley, M.; Ashworth, A. Stem Cells and Breast Cancer: A Field in Transit. Nat. Rev. Cancer 2003, 3, 832−844. (20) Affolter, M.; Zeller, R.; Caussinus, E. Tissue Remodelling Through Branching Morphogenesis. Nat. Rev. Mol. Cell Biol. 2009, 10, 831−842. (21) Roarty, K.; Serra, R. Wnt5a Is Required for Proper Mammary Gland Development and TGF-Beta-Mediated Inhibition of Ductal Growth. Development 2007, 134, 3929−3939. (22) Nelson, C. M.; Vanduijn, M. M.; Inman, J. L.; Fletcher, D. A.; Bissell, M. J. Tissue Geometry Determines Sites of Mammary Branching Morphogenesis in Organotypic Cultures. Science 2006, 314, 298−300. (23) Sakakura, T.; Nishizuka, Y.; Dawe, C. J. MesenchymeDependent Morphogenesis and Epithelium-Specific Cytodifferentiation in Mouse Mammary Gland. Science 1976, 194, 1439−1441. (24) Sheffield, L. G.; Welsch, C. W. Transplantation of Human Breast Epithelia to Mammary-Gland-Free Fat-Pads of Athymic Nude Mice: Influence of Mammotrophic Hormones on Growth of Breast Epithelia. Int. J. Cancer 1988, 41, 713−719. (25) Kuperwasser, C.; Chavarria, T.; Wu, M.; Magrane, G.; Gray, J. W.; Carey, L.; Richardson, A.; Weinberg, R. A. Reconstruction of Functionally Normal and Malignant Human Breast Tissues in Mice. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4966−4971. (26) Wiseman, B. S.; Werb, Z. Stromal Effects on Mammary Gland Development and Breast Cancer. Science 2002, 296, 1046−1049. (27) Parsa, S.; Ramasamy, S.; De Langhe, S.; Gupte, V.; Haigh, J.; Medina, D.; Bellusci, S. Terminal End Bud Maintenance in Mammary Gland Is Dependent Upon FGFR2b Signaling. Dev. Biol. 2008, 317, 121−131. (28) Sternlicht, M. D.; Kouros-Mehr, H.; Lu, P.; Werb, Z. Hormonal and Local Control of Mammary Branching Morphogenesis. Differentiation 2006, 74, 365−381. (29) Sternlicht, M. D.; Sunnarborg, S. W. The ADAM17− Amphiregulin−EGFR Axis in Mammary Development and Cancer. J. Mammary Gland Biol. 2008, 13, 181−194. (30) Wiseman, B. S.; Sternlicht, M. D.; Lund, L. R.; Alexander, C. M.; Mott, J.; Bissell, M. J.; Soloway, P.; Itohara, S.; Werb, Z. Site-Specific Inductive and Inhibitory Activities of MMP-2 and MMP-3 Orchestrate Mammary Gland Branching Morphogenesis. J. Cel. Biol. 2003, 162, 1123−1133. (31) Kouros-Mehr, H.; Werb, Z. Candidate Regulators of Mammary Branching Morphogenesis Identified by Genome-Wide Transcript Analysis. Dev. Dyn. 2006, 235, 3404−3412. (32) Simian, M.; Hirai, Y.; Navre, M.; Werb, Z.; Lochter, A.; Bissell, M. J. The Interplay of Matrix Metalloproteinases, Morphogens and Growth Factors Is Necessary for Branching of Mammary Epithelial Cells. Development 2001, 128, 3117−3131. (33) Bishop, J. R.; Schuksz, M.; Esko, J. D. Heparan Sulphate Proteoglycans Fine-Tune Mammalian Physiology. Nature 2007, 446, 1030−1037. (34) Crawford, B. E.; Garner, O. B.; Bishop, J. R.; Zhang, D. Y.; Bush, K. T.; Nigam, S. K.; Esko, J. D. Loss of the Heparan Sulfate Sulfotransferase, Ndst1, in Mammary Epithelial Cells Selectively Blocks Lobuloalveolar Development in Mice. PLoS One 2010, 5, e10691. (35) Garner, O. B.; Bush, K. T.; Nigam, K. B.; Yamaguchi, Y.; Xu, D.; Esko, J. D.; Nigam, S. K. Developmental Biology. Dev. Biol. 2011, 355, 394−403.

(36) Makarenkova, H.; Mohammadi, M. Differential Interactions of FGFs with Heparan Sulfate Control Gradient Formation and Branching Morphogenesis. Sci. Signal. 2009, 2, ra55. (37) Boras-Granic, K.; Wysolmerski, J. J. Wnt Signaling in Breast Organogenesis. Organogenesis 2008, 4, 116−122. (38) Fernandez-Valdivia, R.; Mukherjee, A.; Ying, Y.; Li, J.; Paquet, M.; DeMayo, F. J.; Lydon, J. P. The RANKL Signaling Axis Is Sufficient to Elicit Ductal Side-Branching and Alveologenesis in the Mammary Gland of the Virgin Mouse. Dev. Biol. 2009, 328, 127−139. (39) Lee, H. J.; Gallego-Ortega, D.; Ledger, A.; Schramek, D.; Joshi, P.; Szwarc, M. M.; Cho, C.; Lydon, J. P.; Khokha, R.; Penninger, J. M.; Ormandy, C. J. Progesterone Drives Mammary Secretory Differentiation via RankL-Mediated Induction of Elf5 in Luminal Progenitor Cells. Development 2013, 140, 1397−1401. (40) Joshi, P. A.; Jackson, H. W.; Beristain, A. G.; Di Grappa, M. A.; Mote, P. A.; Clarke, C. L.; Stingl, J.; Waterhouse, P. D.; Khokha, R. Progesterone Induces Adult Mammary Stem Cell Expansion. Nature 2010, 465, 803−807. (41) Keymeulen, A. V.; Rocha, A. S.; Ousset, M.; Beck, B.; Bouvencourt, G.; Rock, J.; Sharma, N.; Dekoninck, S.; Blanpain, C. Distinct Stem Cells Contribute to Mammary Gland Development and Maintenance. Nature 2011, 1−7. (42) Rios, A. C.; Fu, N. Y.; Lindeman, G. J.; Visvader, J. E. In Situ Identification of Bipotent Stem Cells in the Mammary Gland. Nature 2014, 1−19. (43) Giancotti, F. G. Mechanisms Governing Metastatic Dormancy and Reactivation. Cell 2013, 155, 750−764. (44) Visvader, J. E. Cells of Origin in Cancer. Nature 2011, 469, 314−322. (45) Marjanovic, N. D.; Weinberg, R. A.; Chaffer, C. L. Cell Plasticity and Heterogeneity in Cancer. Clin.Chem. 2013, 59, 168−179. (46) Asselin-Labat, M.-L.; Vaillant, F.; Sheridan, J. M.; Pal, B.; Wu, D.; Simpson, E. R.; Yasuda, H.; Smyth, G. K.; Martin, T. J.; Lindeman, G. J.; Visvader, J. E. Control of Mammary Stem Cell Function by Steroid Hormone Signalling. Nature 2010, 465, 798−802. (47) Smalley, M. J.; Dale, T. C. Wnt Signalling in Mammalian Development and Cancer. Cancer Metastasis Rev. 1999, 18, 215−230. (48) Gupta, S.; Takebe, N.; Lorusso, P. Targeting the Hedgehog Pathway in Cancer. Ther. Adv. Med. Oncol. 2010, 2, 237−250. (49) Harrison, H.; Farnie, G.; Howell, S. J.; Rock, R. E.; Stylianou, S.; Brennan, K. R.; Bundred, N. J.; Clarke, R. B. Regulation of Breast Cancer Stem Cell Activity by Signaling Through the Notch4 Receptor. Cancer Res. 2010, 70, 709−718. (50) Šale, S.; Lafkas, D.; Artavanis-Tsakonas, S. Notch2 Genetic Fate Mapping Reveals Two Previously Unrecognized Mammary Epithelial Lineages. Nat. Cell Biol. 2013, 15, 1−11. (51) Perou, C. M.; Sørlie, T.; Eisen, M. B.; van de Rijn, M.; Jeffrey, S. S.; Rees, C. A.; Pollack, J. R.; Ross, D. T.; Johnsen, H.; Akslen, L. A.; Fluge, O.; Pergamenschikov, A.; Williams, C.; Zhu, S. X.; Lønning, P. E.; Børresen-Dale, A. L.; Brown, P. O.; Botstein, D. Molecular Portraits of Human Breast Tumours. Nature 2000, 406, 747−752. (52) Shackleton, M.; Vaillant, F.; Simpson, K. J.; Stingl, J.; Smyth, G. K.; Asselin-Labat, M.-L.; Wu, L.; Lindeman, G. J.; Visvader, J. E. Generation of a Functional Mammary Gland From a Single Stem Cell. Nature 2006, 439, 84−88. (53) Stingl, J.; Eirew, P.; Ricketson, I.; Shackleton, M.; Vaillant, F.; Choi, D.; Li, H. I.; Eaves, C. J. Purification and Unique Properties of Mammary Epithelial Stem Cells. Nature 2006, 439, 993−997. (54) Chaffer, C. L.; Weinberg, R. A. Cancer Cell of Origin: Spotlight on Luminal Progenitors. Cell Stem Cell 2010, 7, 271−272. (55) Medema, J. P. Cancer Stem Cells: the Challenges Ahead. Nat. Cell Biol. 2013, 15, 338−344. (56) Liu, X.; Holstege, H.; van der Gulden, H.; Treur-Mulder, M.; Zevenhoven, J.; Velds, A.; Kerkhoven, R. M.; van Vliet, M. H.; Wessels, L. F. A.; Peterse, J. L.; Berns, A.; Jonkers, J. Somatic Loss of BRCA1 and P53 in Mice Induces Mammary Tumors with Features of Human BRCA1-Mutated Basal-Like Breast Cancer. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12111−12116. H



Review

(57) Molyneux, G.; Geyer, F. C.; Magnay, F.-A.; McCarthy, A.; Kendrick, H.; Natrajan, R.; MacKay, A.; Grigoriadis, A.; Tutt, A.; Ashworth, A.; Reis-Filho, J. S.; Smalley, M. J. BRCA1 Basal-Like Breast Cancers Originate From Luminal Epithelial Progenitors and Not From Basal Stem Cells. Cell Stem Cell 2010, 7, 403−417. (58) Moumen, M.; Chiche, A. L.; Decraene, C.; Petit, V. R.; Gandarillas, A.; Deugnier, M.-A.; Glukhova, M. A.; Faraldo, M. M. Myc Is Required for B-Catenin-Mediated Mammary Stem Cell Amplification and Tumorigenesis. Mol. Cancer 2013, 12, 1−1. (59) Kim, J.; Woo, A. J.; Chu, J.; Snow, J. W.; Fujiwara, Y.; Kim, C. G.; Cantor, A. B.; Orkin, S. H. A Myc Network Accounts for Similarities between Embryonic Stem and Cancer Cell Transcription Programs. Cell 2010, 143, 313−324. (60) Moumen, M.; Chiche, A.; Deugnier, M.-A.; Petit, V.; Gandarillas, A.; Glukhova, M. A.; Faraldo, M. M. The Proto-Oncogene Myc Is Essential for Mammary Stem Cell Function. Stem Cells 2012, 30, 1246−1254. (61) Liu, H.; Radisky, D. C.; Yang, D.; Xu, R.; Radisky, E. S.; Bissell, M. J.; Bishop, J. M. MYC Suppresses Cancer Metastasis by Direct Transcriptional Silencing of Av and B3 Integrin Subunits. Nat. Cell Biol. 2012, 14, 567−574. (62) Kalluri, R.; Weinberg, R. A. The Basics of EpithelialMesenchymal Transition. J. Clin. Invest. 2009, 119, 1420−1428. (63) Wu, Y.; Ginther, C.; Kim, J.; Mosher, N.; Chung, S.; Slamon, D.; Vadgama, J. V. Expression of Wnt3 Activates Wnt/-Catenin Pathway and Promotes EMT-Like Phenotype in Trastuzumab-Resistant HER2Overexpressing Breast Cancer Cells. Mol. Cancer Res. 2012, 10, 1597− 1606. (64) Wu, Z.-Q.; Li, X.-Y.; Hu, C. Y.; Ford, M.; Kleer, C. G.; Weiss, S. J. Canonical Wnt Signaling Regulates Slug Activity and Links Epithelial-Mesenchymal Transition with Epigenetic Breast Cancer 1, Early Onset (BRCA1) Repression. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 16654−16659. (65) Mani, S. A.; Guo, W.; Liao, M.-J.; Eaton, E. N.; Ayyanan, A.; Zhou, A. Y.; Brooks, M.; Reinhard, F.; Zhang, C. C.; Shipitsin, M.; Campbell, L. L.; Polyak, K.; Brisken, C.; Yang, J.; Weinberg, R. A. The Epithelial-Mesenchymal Transition Generates Cells with Properties of Stem Cells. Cell 2008, 133, 704−715. (66) Guo, W.; Keckesova, Z.; Donaher, J. L.; Shibue, T.; Tischler, V.; Reinhardt, F.; Itzkovitz, S.; Noske, A.; Zürrer-Härdi, U.; Bell, G.; Tam, W. L.; Mani, S. A.; van Oudenaarden, A.; Weinberg, R. A. Slug and Sox9 Cooperatively Determine the Mammary Stem Cell State. Cell 2012, 148, 1015−1028. (67) Nyga, A.; Cheema, U.; Loizidou, M. 3D Tumour Models: Novel in Vitro Approaches to Cancer Studies. J. Cell Commun. Signaling 2011, 5, 239−248. (68) Campbell, J. J.; Davidenko, N.; Caffarel, M. M.; Cameron, R. E.; Watson, C. J. A Multifunctional 3D Co-Culture System for Studies of Mammary Tissue Morphogenesis and Stem Cell Biology. PLoS One 2011, 6, e25661. (69) Wang, X.; Kaplan, D. L. Hormone-Responsive 3D Multicellular Culture Model of Human Breast Tissue. Biomaterials 2012, 33, 3411− 3420. (70) Rudnick, J. A.; Arendt, L. M.; Klebba, I.; Hinds, J. W.; Iyer, V.; Gupta, P. B.; Naber, S. P.; Kuperwasser, C. Functional Heterogeneity of Breast Fibroblasts Is Defined by a Prostaglandin Secretory Phenotype That Promotes Expansion of Cancer-Stem Like Cells. PLoS One 2011, 6, e24605. (71) Liu, S.; Ginestier, C.; Ou, S. J.; Clouthier, S. G.; Patel, S. H.; Monville, F.; Korkaya, H.; Heath, A.; Dutcher, J.; Kleer, C. G.; Jung, Y.; Dontu, G.; Taichman, R.; Wicha, M. S. Breast Cancer Stem Cells Are Regulated by Mesenchymal Stem Cells Through Cytokine Networks. Cancer Res. 2011, 71, 614−624. (72) Anton, K.; Banerjee, D.; Glod, J. Macrophage-Associated Mesenchymal Stem Cells Assume an Activated, Migratory, ProInflammatory Phenotype with Increased IL-6 and CXCL10 Secretion. PLoS One 2012, 7, e35036. (73) Pollard, J. W. Tumour-Educated Macrophages Promote Tumour Progression and Metastasis. Nat. Rev. Cancer 2004, 4, 71−78.

(74) Dimarino, A. M.; Caplan, A. I.; Bonfield, T. L. Mesenchymal Stem Cells in Tissue Repair. Front. Immunol. 2013, 4, 201. (75) Caplan, A. I. Adult Mesenchymal Stem Cells for Tissue Engineering Versus Regenerative Medicine. J. Cell. Physiol. 2007, 213, 341−347. (76) Ingber, D. E. Cellular Mechanotransduction: Putting All the Pieces Together Again. FASEB J. 2006, 20, 811−827. (77) Humphries, J. D. Integrin Ligands at a Glance. J. Cel. Sci. 2006, 119, 3901−3903. (78) Akhtar, N.; Streuli, C. H. An Integrin-ILK-Microtubule Network Orients Cell Polarity and Lumen Formation in Glandular Epithelium. Nat. Cell Biol. 2012, 15, 17−27. (79) Yu, W.; Datta, A.; Leroy, P.; O’Brien, L. E.; Mak, G. B1-Integrin Orients Epithelial Polarity via Rac1 and Laminin. Mol. Biol. Cell 2005, 16, 433−445. (80) Taddei, I.; Deugnier, M.-A.; Faraldo, M. M.; Petit, V.; Bouvard, D.; Medina, D.; Fässler, R.; Thiery, J. P.; Glukhova, M. A. B1 Integrin Deletion From the Basal Compartment of the Mammary Epithelium Affects Stem Cells. Nat. Cell Biol. 2008, 10, 716−722. (81) Huck, L.; Pontier, S. M.; Zuo, D. M.; Muller, W. J. Beta1Integrin Is Dispensable for the Induction of ErbB2Mammary Tumors but Plays a Critical Role in the Metastatic Phase of Tumor Progression. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 15559−15564. (82) Weigelt, B.; Lo, A.; Park, C.; Gray, J. 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. (83) Vukicevic, S.; Kleinman, H. K.; Luyten, F. P.; Roberts, A. B.; Roche, N. S.; Reddi, A. H. Identification of Multiple Active Growth Factors in Basement Membrane Matrigel Suggests Caution in Interpretation of Cellular Activity Related to Extracellular Matrix Components. Exp. Cell Res. 1992, 202, 1−8. (84) Li, M. L.; Aggeler, J.; Farson, D. A.; Hatier, C.; Hassell, J.; Bissell, M. J. Influence of a Reconstituted Basement Membrane and Its Components on Casein Gene Expression and Secretion in Mouse Mammary Epithelial Cells. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 136− 140. (85) Petersen, O. W.; Rønnov-Jessen, L.; Howlett, A. R.; Bissell, M. J. Interaction with Basement Membrane Serves to Rapidly Distinguish Growth and Differentiation Pattern of Normal and Malignant Human Breast Epithelial Cells. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9064− 9068. (86) Kenny, P. A.; Lee, G. Y.; Myers, C. A.; Neve, R. M.; Semeiks, J. R.; Spellman, P. T.; Lorenz, K.; Lee, E. H.; Barcellos-Hoff, M. H.; Petersen, O. W.; Gray, J. W.; Bissell, M. J. The Morphologies of Breast Cancer Cell Lines in Three-Dimensional Assays Correlate with Their Profiles of Gene Expression. Mol. Oncol. 2007, 1, 84−96. (87) Weigelt, B.; Lo, A. T.; Park, C. C.; Gray, J. W.; Bissell, M. J. 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. 2009, 1−9. (88) Desbordes, S. C.; Studer, L. Adapting Human Pluripotent Stem Cells to High-Throughput and High-Content Screening. Nat. Protoc. 2013, 8, 111−130. (89) Krause, S.; Maffini, M. V.; Soto, A. M.; Sonnenschein, C. A Novel 3D in VitroCulture Model to Study Stromal−Epithelial Interactions in the Mammary Gland. Tissue Eng., Part C 2008, 14, 261−271. (90) Soofi, S. S.; Last, J. A.; Liliensiek, S. J.; Nealey, P. F.; Murphy, C. J. The Elastic Modulus of Matrigel as Determined by Atomic Force Microscopy. J. Struct. Biol. 2009, 167, 216−219. (91) Zhang, W.; Ahluwalia, I. P.; Yelick, P. C. Three Dimensional Dental Epithelial-Mesenchymal Constructs of Predetermined Size and Shape for Tooth Regeneration. Biomaterials 2010, 31, 7995−8003. (92) Claffey, K. P.; Abrams, K.; Shih, S. C.; Brown, L. F.; Mullen, A.; Keough, M. Fibroblast Growth Factor 2 Activation of Stromal Cell Vascular Endothelial Growth Factor Expression and Angiogenesis. Lab. Invest. 2001, 81, 61−75. I



Review

(93) Pöschl, E.; Schlötzer-Schrehardt, U.; Brachvogel, B.; Saito, K.; Ninomiya, Y.; Mayer, U. Collagen IV Is Essential for Basement Membrane Stability but Dispensable for Initiation of Its Assembly During Early Development. Development 2004, 131, 1619−1628. (94) Neira, R. E.; Salazar, E. P. Native Type IV Collagen Induces an Epithelial to Mesenchymal Transition-Like Process in Mammary Epithelial Cells MCF10A. Int. J. Biochem. Cell Biol. 2012, 44, 2194− 2203. (95) Chen, Q. K.; Lee, K.; Radisky, D. C.; Nelson, C. M. Extracellular Matrix Proteins Regulate Epithelial-Mesenchymal Transition in Mammary Epithelial Cells. Differentiation 2013, 86, 126−132. (96) Debnath, J.; Brugge, J. S. Modelling Glandular Epithelial Cancers in Three-Dimensional Cultures. Nat. Rev. Cancer 2005, 5, 675−688. (97) Berdichevsky, F.; Alford, D.; D’Souza, B. Branching Morphogenesis of Human Mammary Epithelial Cells in Collagen Gels. J. Cel. Sci. 1994, 107, 3557−3568. (98) Guo, C. Type I Collagen-Induced MMP-2 Activation Coincides with Up-Regulation of Membrane Type 1-Matrix Metalloproteinase and TIMP-2 in Cardiac Fibroblasts. J. Biol. Chem. 2003, 278, 46699− 46708. (99) Sodek, K. L.; Brown, T. J.; Ringuette, M. J. Collagen I but Not Matrigel Matrices Provide an MMP-Dependent Barrier to Ovarian Cancer Cell Penetration. BMC Cancer 2008, DOI: 10.1186/14712407-8-223. (100) Gentleman, E.; Nauman, E. A.; Livesay, G. A.; Dee, K. C. Collagen Composite Biomaterials Resist Contraction While Allowing Development of Adipocytic Soft Tissue in Vitro. Tissue Eng. 2006, 12, 1639−1649. (101) Gerecht, S.; Burdick, J.; Ferreira, L.; Townsend, S.; Langer, R. Hyaluronic Acid Hydrogel for Controlled Self-Renewal and Differentiation of Human Embryonic Stem Cells. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 11298. (102) Porsch, H.; Bernert, B.; cacute, M. M.; Theocharis, A. D.; Heldin, C.-H.; Heldin, P. Efficient TGFb-Induced Epithelial− Mesenchymal Transition Depends on Hyaluronan Synthase HAS2. Oncogene 2012, 32, 4355−4365. (103) El-Haibi, C. P.; Bell, G. W.; Zhang, J.; Collmann, A. Y.; Wood, D.; Scherber, C. M.; Csizmadia, E.; Mariani, O.; Zhu, C.; Campagne, A.; Toner, M.; Bhatia, S. N.; Irimia, D.; Vincent-Salomon, A.; Karnoub, A. E. Critical Role for Lysyl Oxidase in Mesenchymal Stem CellDriven Breast Cancer Malignancy. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 17460−17465. (104) Louderbough, J. M. V.; Brown, J. A.; Nagle, R. B.; Schroeder, J. A. CD44 Promotes Epithelial Mammary Gland Development and Exhibits Altered Localization During Cancer Progression. Genes Cancer 2012, 2, 771−781. (105) Craig, E. A.; Parker, P.; Camenisch, T. D. Size-Dependent Regulation of Snail2 by Hyaluronan: Its Role in Cellular Invasion. Glycobiology 2009, 19, 890−898. (106) Alaniz, L.; Garcia, M.; Gallo-Rodriguez, C.; Agusti, R.; SterinSpeziale, N.; Hajos, S.; Alvarez, E. Hyaluronan Oligosaccharides Induce Cell Death Through PI3-K/Akt Pathway Independently of NFκB Transcription Factor. Glycobiology 2006, 16, 359−367. (107) Koyama, H.; Hibi, T.; Isogai, Z.; Yoneda, M.; Fujimori, M.; Amano, J.; Kawakubo, M.; Kannagi, R.; Kimata, K.; Taniguchi, S. Hyperproduction of Hyaluronan in Neu-Induced Mammary Tumor Accelerates Angiogenesis Through Stromal Cell RecruitmentPossible Involvement of Versican/PG-M. Am. J. Pathol. 2007, 170, 1086−1099. (108) West, D. C.; Hampson, I. N.; Arnold, F.; Kumar, S. Angiogenesis Induced by Degradation Products of Hyaluronic Acid. Science 1985, 228, 1324−1326. (109) Gobaa, S.; Hoehnel, S.; Roccio, M.; Negro, A.; Kobel, S.; Lutolf, M. P. Artificial Niche Microarrays for Probing Single Stem Cell Fate in High Throughput. Nat. Methods 2011, 8, 949−955. (110) Nur-E-Kamal, A.; Ahmed, I.; Kamal, J.; Schindler, M.; Meiners, S. Three-Dimensional Nanofibrillar Surfaces Promote Self-Renewal in Mouse Embryonic Stem Cells. Stem Cells 2006, 24, 426−433.

(111) Higuchi, A.; Ling, Q.-D.; Ko, Y.-A.; Chang, Y.; Umezawa, A. Biomaterials for the Feeder-Free Culture of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells. Chem. Rev. 2011, 111, 3021−3035. (112) Hongisto, V.; Jernström, S.; Fey, V.; Mpindi, J.-P.; Kleivi Sahlberg, K.; Kallioniemi, O.; Perälä, M. High-Throughput 3D Screening Reveals Differences in Drug Sensitivities Between Culture Models of JIMT1 Breast Cancer Cells. PLoS One 2013, 8, e77232. (113) Weiss, M. S.; Bernabé, B. P.; Shikanov, A.; Bluver, D. A.; Mui, M. D.; Shin, S.; Broadbelt, L. J.; Shea, L. D. Biomaterials. Biomaterials 2012, 33, 3548−3559. (114) Beck, J. N.; Singh, A.; Rothenberg, A. R.; Elisseeff, J. H.; Ewald, A. J. Biomaterials. Biomaterials 2013, 34, 9486−9495. (115) Lutolf, M. P.; Lauer-Fields, J. L.; Schmoekel, H. G.; Metters, A. T.; Weber, F. E.; Fields, G. B.; Hubbell, J. A. Synthetic Matrix Metalloproteinase-Sensitive Hydrogels for the Conduction of Tissue Regeneration: Engineering Cell-Invasion Characteristics. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5413−5418. (116) Paszek, M. J.; Zahir, N.; Johnson, K. R.; Lakins, J. N.; Rozenberg, G. I.; Gefen, A.; Reinhart-King, C. A.; Margulies, S. S.; Dembo, M.; Boettiger, D.; Hammer, D. A.; Weaver, V. M. Tensional Homeostasis and the Malignant Phenotype. Cancer Cell 2005, 8, 241− 254. (117) Barcus, C. E.; Keely, P. J.; Eliceiri, K. W.; Schuler, L. A. Stiff Collagen Matrices Increase Tumorigenic Prolactin Signaling in Breast Cancer Cells. J. Biol. Chem. 2013, 288, 12722−12732. (118) Kumar, S.; Weaver, V. M. Mechanics, Malignancy, and Metastasis: The Force Journey of a Tumor Cell. Cancer Metastasis Rev. 2009, 28, 113−127. (119) Lance, A.; Yang, C. C.; Swamydas, M.; Dean, D.; Deitch, S.; Burg, K. J. L.; Dréau, D. Increased Extracellular Matrix Density Decreases MCF10A Breast Cell Acinus Formation in 3D Culture Conditions. J. Tissue Eng. Regener. Med. 2013, DOI: 10.1002/ term.1675. (120) Boyd, N. F.; Martin, L. J.; Bronskill, M.; Yaffe, M. J.; Duric, N.; Minkin, S. Breast Tissue Composition and Susceptibility to Breast Cancer. JNCI 2010, 102, 1224−1237. (121) Barker, H. E.; Chang, J.; Cox, T. R.; Lang, G.; Bird, D.; Nicolau, M.; Evans, H. R.; Gartland, A.; Erler, J. T. LOXL2-Mediated Matrix Remodeling in Metastasis and Mammary Gland Involution. Cancer Res. 2011, 71, 1561−1572. (122) Levental, K. R.; Yu, H.; Kass, L.; Lakins, J. N.; Egeblad, M.; Erler, J. T.; Fong, S. F. T.; Csiszar, K.; Giaccia, A.; Weninger, W.; Yamauchi, M.; Gasser, D. L.; Weaver, V. M. Matrix Crosslinking Forces Tumor Progression by Enhancing Integrin Signaling. Cell 2009, 139, 891−906. (123) Provenzano, P. P.; Inman, D. R.; Eliceiri, K. W.; Keely, P. J. Matrix Density-Induced Mechanoregulation of Breast Cell Phenotype, Signaling and Gene Expression through a FAK-ERK Linkage. Oncogene 2009, 28, 4326−4343. (124) Wang, N.; Butler, J. P.; Ingber, D. E. Mechanotransduction across the Cell Surface and through the Cytoskeleton. Science 1993, 260, 1124−1127. (125) Baker, E. L.; Srivastava, J.; Yu, D.; Bonnecaze, R. T.; Zaman, M. H. Cancer Cell Migration: Integrated Roles of Matrix Mechanics and Transforming Potential. PLoS One 2011, 6, e20355. (126) Lee, D. A.; Knight, M. M.; Campbell, J. J.; Bader, D. L. Stem Cell Mechanobiology. J. Cell Biochem. 2011, 112, 1−9. (127) Li, B.; Wang, J. H. C. Application of Sensing Techniques to Cellular Force Measurement. Sensors 2010, 10, 9948−9962. (128) Saez, A.; Anon, E.; Ghibaudo, M.; du Roure, O.; Di Meglio, J.M.; Hersen, P.; Silberzan, P.; Buguin, A.; Ladoux, B. Traction Forces Exerted by Epithelial Cell Sheets. J. Phys.: Condens. Matter 2010, 22, 194119. (129) Pirone, D. M.; Chen, C. S. Strategies for Engineering the Adhesive Microenvironment. J. Mammary Gland Biol. 2004, 9, 405− 417. J



Review

(130) Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Geometric Control of Cell Life and Death. Science 1997, 276, 1425−1428. (131) Mori, H.; Gjorevski, N.; Inman, J. L.; Bissell, M. J.; Nelson, C. M. Self-Organization of Engineered Epithelial Tubules by Differential Cellular Motility. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 14890− 14895. (132) Boghaert, E.; Gleghorn, J. P.; Lee, K.; Gjorevski, N.; Radisky, D. C.; Nelson, C. M. Host Epithelial Geometry Regulates Breast Cancer Cell Invasiveness. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 19632−19637. (133) Brownfield, D. G.; Venugopalan, G.; Lo, A.; Mori, H.; Tanner, K.; Fletcher, D. A.; Bissell, M. J. Patterned Collagen Fibers Orient Branching Mammary Epithelium Through Distinct Signaling Modules. Curr. Biol. 2013, 23, 703−709. (134) Betts, J. I.; Baganz, F. Miniature Bioreactors: Current Practices and Future Opportunities. Microb. Cell Fact. 2006, 5, 21. (135) Burdick, J.; Khademhosseini, A.; Langer, R. Fabrication of Gradient Hydrogels Using a Microfluidics/Photopolymerization Process. Langmuir 2004, 20, 5153−5156. (136) Markov, D. A.; Lu, J. Q.; Samson, P. C.; Wikswo, J. P.; McCawley, L. J. Thick-Tissue Bioreactor as a Platform for Long-Term Organotypic Culture and Drug Delivery. Lab Chip 2012, 12, 4560. (137) Nyga, A.; Loizidou, M.; Emberton, M.; Cheema, U. A Novel Tissue Engineered Three-Dimensional in Vitro Colorectal Cancer Model. Acta Biomater. 2013, 9, 7917−7926. (138) Szot, C. S.; Buchanan, C. F.; Freeman, J. W.; Rylander, M. N. 3D in Vitro Bioengineered Tumors Based on Collagen I Hydrogels. Biomaterials 2011, 32, 7905−7912. (139) Derda, R.; Laromaine, A.; Mammoto, A.; Tang, S. K. Y.; Mammoto, T.; Ingber, D. E.; Whitesides, G. M. Paper-Supported 3D Cell Culture for Tissue-Based Bioassays. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 18457−18462. (140) Evans, C. L.; Abu-Yousif, A. O.; Park, Y. J.; Klein, O. J.; Celli, J. P.; Rizvi, I.; Zheng, X.; Hasan, T. Killing Hypoxic Cell Populations in a 3D Tumor Model with EtNBS-PDT. PLoS One 2011, 6, e23434. (141) Willard, J. J.; Drexler, J. W.; Das, A.; Roy, S.; Shilo, S.; Shoseyov, O.; Powell, H. M. Plant-Derived Human Collagen Scaffolds for Skin Tissue Engineering. Tissue Eng., Part A 2013, 19, 1507−1518. (142) Rodríguez-Seguí, S. A.; Ortuño, M. J.; Ventura, F.; Martínez, E.; Samitier, J. Simplified Microenvironments and Reduced Cell Culture Size Influence the Cell Differentiation Outcome in Cellular Microarrays. J. Mater. Sci. Mater. Med. 2013, 24, 189−198. (143) Simon, R.; Sauter, G. Tissue Microarrays for Miniaturized High-Throughput Molecular Profiling of Tumors. Exp. Hematol. 2002, 30, 1365−1372.

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