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Interdisciplinary approaches for molecular and cellular life sciences

www.rsc.org/ibiology Volume 5 | Number 9 | September 2013 | Pages 10891198

ISSN 1757-9694

REVIEW ARTICLE Edmond W. K. Young Cells, tissues, and organs on chips: challenges and opportunities for the cancer tumor microenvironment

Integrative Biology
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Cells, tissues, and organs on chips: challenges and opportunities for the cancer tumor microenvironment
Edmond W. K. Young*
The transition to increasingly sophisticated microfluidic systems has led to the emergence of organ-on-

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chip technology that can faithfully recapitulate organ-level function. Given the rapid progress at the interface between microfluidics and cell biology, there is need to provide a focused evaluation of the state-of-the-art in microfluidic systems for cancer research to advance development, accelerate discovery of novel insights, and facilitate cooperation between engineers, biologists and oncologists in the clinic. Here, we provide a focused review of microfluidics technology from cells- and tissues- to organs-on-chips with application toward studying the tumor microenvironment. Key aspects of the tumor microenvironment including angiogenesis, hypoxia, biochemical gradients, tumorstromal interactions, and the extracellular matrix are summarized for both solid tumors and non-solid hematologic malignancies. An overview of microfluidic systems designed specifically to answer questions related to different
Received 16th April 2013, Accepted 6th June 2013 DOI: 10.1039/c3ib40076j www.rsc.org/ibiology

aspects of the tumor microenvironment is provided, followed by an examination of how these systems offer new opportunities to study outstanding challenges related to the major cancer hallmarks. Challenges also remain for microfluidics engineers, but it is hoped that cooperation between engineers and biologists at the intersection of their respective fields will lead to significant impact on the utility of organs-on-chips in cancer research.

Insight, innovation, integration

Organ-on-chip technologies are attracting significant interest with existing reviews focusing mostly on technical aspects of the field. This review focuses instead on cells, tissues, and organs-on-chips specifically for studying cancer biology. By discussing the advancement of microfluidic cell-based systems from the standpoint of the disease and the key elements within the tumor microenvironment, this review oers a dierent perspective that may lead to new organ-on-chip innovations as well as unexpected insights on how we approach cancer research in the future.

1. Introduction
The integration of microfluidics and cell biology research has recently reached another significant milestone with development of organ-on-chip technologies. What began at the turn of the millennium as simple demonstrations of biological cells being transported and manipulated in microchannels for basic short-term analysis1,2 has now advanced to the point where we can engineer living cellular microsystems with controllable microenvironments that behave and function with organ-level complexity like their counterparts in vivo.35 While advancement from cell- to tissue- to organ-level function in vitro has been impressive from an engineering perspective, what is truly
Department of Mechanical & Industrial Engineering, University of Toronto, 5 Kings College Road, MC314B, Toronto, ON, M5S 3G8, Canada. E-mail: eyoung@mie.utoronto.ca; Fax: +1 (416) 978-7753; Tel: +1 (416) 978-1521

compelling is the potential impact this technology will have on the study of human diseases, and on clinical and therapeutic applications that may ultimately improve overall human health. Cancer research in particular has potential to reap immense benefits from the application of organ-on-chip technologies. A major challenge in cancer research has been the need to develop more accurate, more informative, and more predictive experimental models of human tumor development than those currently available. Traditional in vitro systems and in vivo animal models have both made major contributions to our current understanding of the disease, and to the discovery and practical application of oncotherapies, but these models also have shortcomings that limit their overall eectiveness toward predicting the success or failure of many candidate drug compounds during the screening process, as well as that of selected therapeutic strategies.6 Ineectiveness in the former case has led to a disappointingly high attrition rate of potential

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Review Article candidates late in the drug discovery process that is both costly and time consuming. Organ-on-chip technologies have potential to oer significant improvements in in vitro models of the disease, with better physiological mimicry of the tumor microenvironment than existing in vitro assays, and more species relevance than in vivo animal models (if primary human cells are employed). Importantly, the role of the tumor microenvironment in cancer initiation and progression, and the recognition that a tumor itself should be considered a complex organ,7 suggests that physiological microenvironments with improved relevance to humans and perhaps even organ-level complexity are integral to our understanding of the disease, and must be given more consideration in future methodologies of experimental research. The purpose of developing improved experimental models through organ-on-chip technologies is to accelerate progress in fundamental research, to increase eciency in drug discovery, and to advance the translation of new knowledge into clinical outcomes. To accelerate the potential impact of this technology on cancer research, it is important and necessary to review the pertinent literature to date, particularly work that has already shown the promise of microfluidic chip technologies in meeting the needs of cancer research. This review discusses the current state-of-the-art in cells-, tissues-, and organs-on-chips technology specifically for cancer microenvironments. While other recent reviews on this technology have provided more general overviews of technical advances in device functionality and complexity,8,9 this review focuses on cancer, and categorizes various microsystems based on key factors relevant to the tumor microenvironment. The potential for these microsystems to contribute to cancer research is proposed in relation to major cancer hallmarks, and a discussion of major challenges facing those working at the interface between microfluidics and cancer is provided. The combination of these dierent aspects distinguishes this review from other recent articles summarizing the application of microfluidics for cancer research.10,11 For convenience and completeness, a brief overview is provided on the essential elements of the tumor microenvironment for both solid tumors and nonsolid malignancies. Regarding nomenclature, where appropriate, dierent cellular microsystems are referred to in specific terms as cellson-chips, tissues-on-chips, or organs-on-chips, depending on biological complexity. However, for brevity, these microsystems are also collectively referred to here as either microfluidic systems or biochips to cover all three hierarchical levels.

Integrative Biology (although distant from the primary site in this case) may contribute to tumorigenicity, enabling metastatic lesions to thrive in an otherwise foreign and hostile environment.12,13 Since then, an extensive body of literature has provided strong support for the role of the tumor microenvironment,14,15 leading to major fields of research ranging from angiogenesis and hypoxia to dimensionality and cancer mechanobiology. The tumor microenvironment as a whole has thus emerged as a central player in cancer research, and as a consequence has become a major potential target for cancer therapy. In experimental biology, our inability to accurately model the human tumor microenvironment in experimental systems is one of the major and perhaps most important reason why many of our results in the lab do not translate directly to improved outcomes in the clinic. The question of whether current in vitro systems and in vivo animal models are suciently representative of human physiological conditions to be predictive is a major source of debate, and stems from the recognition that these models are limited in physiological context, either lacking important spatial and temporal cues in the case of simplistic planar (2D) in vitro models, or lacking the genotype specific to humans in the case of animal models. Thus, for decades, while both experimental paradigms provided significant contributions to our understanding of the disease, and resulted in novel discoveries in therapeutics, there remained an ongoing concern related to the limitations of our experimental repertoire. The potential for organ-on-chip technology to provide a realistic, unconventional and potentially disruptive alternative to existing in vitro and in vivo methods is therefore an exciting proposition. We now have the ability through microfabrication techniques and microengineered control to construct with varying levels of complexity living microsystems that recapitulate dierent aspects of the microenvironment using human cells, perhaps even those derived directly from diseased individuals. This level of physiological relevance to humans is not achievable via existing methodologies. Given such power to tailor the microenvironment as desired for each experiment or application, the question is no longer whether we can do it. Instead, the question is how we do it, and what we do with the technology to best serve the future of cancer research. To begin to answer these questions, we briefly examine the various aspects of the developing tumor microenvironment (Fig. 1), first in the context of an epithelial-derived carcinoma. Carcinomas represent over 80% of all cancers, including those of the breast, colon, liver, lung, pancreas, and prostate, among others, and are collectively considered the largest class of neoplasias. Subsequently, we will also examine aspects of the microenvironment for non-solid hematologic malignancies, a second major class of cancer that has unique features distinct from the microenvironment of solid tumors. For convenience, the discussion will proceed in sequence through the steps leading from normal health to diseased state. Solid tumors Carcinomas derived from epithelial tissue originate from single cell clones, which proliferate and aggregate into a mass of

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2. Essentials of the tumor microenvironment

It has become increasingly evident that cancer development, in its remarkable complexity, is not only a matter of individual tumor cells evolving as a result of multiple genetic mutations, but also involves the complex interactions between the tumor cells and the many physical, biological, and molecular factors of the surrounding tumor microenvironment. From a historical perspective, the well-known seed-soil hypothesis by Paget in 1889, as a proposed explanation for site-specific metastases, was one of the first signs of recognition that the local microenvironment

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Fig. 1 The cancer tumor microenvironment consists of a heterogeneous mix of cells in complex spatial arrangement. Cell types include: tumor cells (e.g., carcinoma), cancer stem cells, invasive phenotypes, inflammatory immune cells, assorted fibroblasts, pericytes, and endothelial cells of angiogenic blood vessels. The cells are supported by extracellular matrix. Regions far from blood supply are hypoxic and subject to oxygen gradients (dark shaded region). Invasive phenotypes may undergo epithelialmesenchymal transition (EMT), migrate away from the tumor core toward the margins (dotted line), and intravasate into neighboring blood vessels where they circulate to ectopic locations and form metastases. (a) Prostate cancer coculture spheroids in microchannels.33 (b) 3D side-by-side coculture of human mammary fibroblasts and ductal carcinoma in situ (DCIS) cells to study role of stromal interactions in transition to invasive ductal carcinoma.48 (c) Microfluidic chip for generating perfusable microvessel networks.58 HUVECs stained for nuclei (blue), f-actin (green), and CD31 (red). (d) Microfluidic system for creating oxygen gradient by oxygengenerating and oxygen-scavenging chemical reactions.63 For (a) to (d), images reproduced in part from ref. 33, 48, 58, and 63, respectively, with permission of The Royal Society of Chemistry. (e) Microfluidic system for studying cancer cell (HT1080, red) invasion and intravasation through endothelium (human microvascular endothelial cells, MVECs, green). Adapted from ref. 71 Copyright 2012 National Academy of Sciences, USA.

mutant cells that form the basis of a tumor. Because tumor cells continue to accumulate mutations during tumor progression, the population of cells becomes increasingly heterogeneous, with successive generations of mutant cells mixed with the initial clonal population. Accumulating evidence suggests that this amalgam also consists of tumor initiating cells, more popularly known as cancer stem cells, which are capable of spawning new tumors when injected into previously healthy mice.16 Tumor cells are surrounded by the stroma, which consists of the three-dimensional structural framework of extracellular matrix (ECM) components and the various other cell types that support the associated connective tissue. The ECM allows anchorage of tumor and stromal cells via cell surface integrins that transduce mechanical signals and mediate various mechanobiological responses. Non-tumor stromal cells make up an estimated 80% of the cells in the

tumor microenvironment, and are arranged in specific positions and in specific proportions, creating a complex network of heterotypic interactions that plays a central role in tumor progression.17 Notable cell types in the stroma include: the various types of fibroblasts (e.g., normal stromal fibroblasts, myofibroblasts, cancer-associated fibroblasts), which are closely related, yet display distinct phenotypic markers, and may originate from different precursors;18 immune cells such as macrophages, neutrophils, and T and B lymphocytes that sense pro-inflammatory cytokines and chemokines secreted by the tumor and infiltrate the microenvironment; adipocytes such as those found in the mammary gland;19 and endothelial cells and supporting pericytes, which are recruited into the microenvironment by the tumor-associated inflammatory cells that secrete soluble factors involved in inducing angiogenesis.20 Angiogenesis is the formation and extension of new blood

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Review Article vessels from existing vasculature that provides oxygen and nutrients to the tumor that are necessary for its growth. As the primary tumor grows and blood vessels infiltrate the tumor, internal oxygen and chemical gradients form in relation to the angiogenic network, creating regions of hypoxic and necrotic cells. The complex set of microenvironmental cues contribute to the differentiation of some tumor cells into more migratory and invasive phenotypes, possibly via epithelialmesenchymal transition (EMT), which gives rise to cells that escape the tumor mass via intravasation into nearby blood vessels. These circulating tumor cells (CTCs) then travel via the circulatory system to distant ectopic locations where they have the potential to reside, colonize, and form secondary tumors called metastases. Fig. 1 illustrates these major elements of the tumor microenvironment.
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Integrative Biology environmental factors. The important point to take from the summary above is that development of the tumor microenvironment progresses through a series of major events (e.g., tumor initiation; angiogenesis) that involve key features (tumor nodule formation; new blood vessels) with specific functional roles, and that the coordinated interplay of these features leads to tumor progression. Advances in microfluidic cell-based systems or biochips have led to a new class of in vitro tools with both demonstrated utility in recapitulating the various aspects of the developing tumor microenvironment (Fig. 1), and the potential to oer insight into unprecedented spatiotemporal dynamics of these events. Microfluidic systems have varying levels of complexity, ranging from simple 2D cell culture microchips to more physiologically relevant 3D tissue microchips. Importantly, microfluidic systems oer various advantages over traditional in vitro systems, including more complex geometries, better spatiotemporal control of microenvironmental factors and stimuli, added functionality, and increased throughput.24 In this section, recent research studies employing microfluidic systems to study aspects of the tumor microenvironment are reviewed, with an attempt to organize the studies in order of increasing physiologic complexity and more advanced stage of tumor development (Table 1). Tumor spheroids To model a tumor, a popular method is to employ multicellular tumor spheroids (MCTSs), which are derived from a collection of cells that aggregate under non-adherent culture conditions to form three-dimensional cellular masses. The morphology, growth kinetics, and cellcell and cellmatrix interactions within these spheroids resemble tumor nodules, making them excellent models of tumor initiation and growth, and a useful tool for testing drug delivery and efficacy.25 Various methods have been developed over decades to achieve efficient spheroid formation, including rotating cultures in roller tubes and spinner flasks, as well as stationary cultures in hanging drops, 96-well plates, and related organotypic cultures.26 Techniques involving 96-well plates have been adapted to enable highthroughput spheroid-based drug screens, with protocols becoming increasingly standardized.27 Although microtiter plates have many advantages, such as enabling testing and monitoring of spheroids in independent wells, and allowing considerable reduction in reagent consumption compared to rotating cultures, the static, diffusion-dominant conditions in wells lead to accelerated nutrient depletion and waste accumulation, which can affect spheroid formation and function, and potentially lead to false identification of drug candidates.27 Microfluidic systems have recently been shown to be effective at facilitating the formation of tumor spheroids on chip. Highthroughput arrays of hanging drops can be generated in wellplate format with the use of microfluidics to create uniform and independently addressable spheroids.2830 Microscale geometries can be designed to hydrodynamically trap cells to create spheroids with highly uniform size distributions, and can simultaneously allow continuous perfusion to maintain longterm spheroid culture.31,32 In addition, porous membranes

Liquid tumors A second major class of cancers is hematologic malignancies, or cancers related to the blood, bone marrow, and lymph nodes (i.e., liquid tumors).21 While certain elements of the microenvironment exist in both solid and liquid tumors (e.g., angiogenesis), elements unique to hematologic cancers are important enough to warrant mention here. For example, in terms of cell types, hematopoietic stem cells reside in the bone marrow and have been implicated in the pathogenesis and progression of hematologic malignancies,22 but have not been implicated in solid tumors. Other cell types such as mesenchymal stromal cells, bone marrow stromal cell (BMSCs), and monocyte-derived nurse-like cells belong to the hematopoietic lineage, and thus are also only found in the microenvironments of hematologic malignancies. The presence of lymphoma-associated macrophages (LAMs) suggests a similarly important role for inflammatory cells in liquid tumors as in solid tumors. Secondary lymphoid organs are characterized by presence of T cells responsible for assisting in antigen recognition. Naturally, the bone milieu also includes osteoblasts and osteoclasts that reside in the bone matrix. Whereas solid tumors can be described by the location of the primary site, hematologic cancers exist in the lymphatic and circulatory transport systems, and are thus characterized by their presence in circulation. Of importance is the notion that the role of the microenvironment in blood-related cancers is highly variable between cancer types, ranging from a very limited role in Burkitt lymphoma to a highly involved, coexisting role in multiple myeloma (MM) and chronic lymphocytic leukemia (CLL), which has implications on the likely responsiveness of a blood cancer type to therapeutic strategies that target the microenvironment.23

3. Microfluidic technology for tumor microenvironments

From a molecular and cell biology perspective, the brief overview of tumor microenvironments presented above is admittedly too simple to capture the inherent complexity of a multicellular system that can be influenced by so many

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Table 1

Summary of microfluidic technology for studying tumor microenvironments

Cancer model Continuous perfusion 3D cellmatrix and cellcell interactions Continuous perfusion Spheroid co-culture with 3 cell types Spheroid formation Continuous perfusion in adjacent 2D vs. 3D compartment separated by porous membrane Mono- vs. co-culture Flow-induced shear stress Static culture and continuous perfusion Long-term spheroid culture (7 d) Microfluidic well plate design (3D Biomatrix) Spheroid formation Spheroid formation via hanging drops Spheroid/spherical microtissue formation Microtissue staining and growth profiles Drug sensitivity Spheroid formation via hanging drops Spheroid formation Multicellular aggregate formation in 3D gel Cell viability Toh et al. (2007)34

Cell types

Notable microenvironment features

Focus of study

Ref.

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Tumor spheroid Breast cancer Liver cancer

Human breast cancer (MCF-7) Human liver carcinoma (HepG-2)

Breast cancer

Human breast cancer (MCF-7)

Wu et al. (2008)31 Hsiao et al. (2009)33

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Prostate cancer

Human prostate cancer (PC-3) Human umbilical cord endothelial (HUVEC) Mouse pre-osteoblast (MC3T3-E1)

Colon cancer

Human colon cancer (Colo205)

Agastin et al. (2011)32 Tung et al. (2011)29 Drewitz et al. (2011)28

Epidermoid cancer colorectal adenocarcinoma (HT-29, HCT-116) Automated workflow with microfluidic well liver carcinoma (HepG-2) plate design (InSphero) prostate cancer (DU-145) Long-term spheroid culture (14 d) kidney carcinoma (A-498) glioblastoma (SNB-19) Extra long-term spheroid culture (22 d) facilitated by micro-rings Modified microfluidic well plate design 2D co-cultures with detachable substrates

Human epidermoid carcinoma (A431.H9)

Colon cancer Liver cancer Prostate cancer Kidney cancer Glioblastoma

Human Human Human Human Human

Prostate cancer

Human prostate cancer (PC-3) Human umbilical cord endothelial (HUVEC) Mouse pre-osteoblast (MC3T3-E1)

Hsiao et al. (2012)30

Tumorstromal interaction 2D Cervical cancer Human cervical cancer (HeLa) Human umbilical cord endothelial (HUVEC) Mouse fibroblast (NIH-3T3)

Cell migration in co-culture Osteoclastogenesis by monocyte differentiation

Kaji et al. (2008)39

Prostate cancer

Human prostate cancer (PC-3) Mouse monocyte (RAW 264.7)

Diffusion dominant 2D co-cultures with microfluidic compartments Diffusion dominant 2D co-cultures with microfluidic compartments Diffusion dominant 2D co-cultures with microfluidic compartments Suspension cell culture enabled by low shear stress geometries 2D co-cultures with up to 3 cell types

Domenech et al. (2009)36 Domenech et al. (2012)38 Hedgehog signaling in myofibroblasts and effect of tumor growth NF-kB and STAT3 activation in MM cells

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Prostate cancer

Human prostate adenocarcinoma (LNCaP) Mouse prostate myofibroblasts (UGSM-2, UGli3/) Normal prostate fibroblasts (NPF, N2-1, N5-2)

Multiple myeloma Chronic lymphocytic leukemia (CLL) liver carcinoma (HepG-2) mucosa epithelia (GES-1) adenoid cystic carcinoma (ACC-2/M) embryonic lung fibroblast (HFL-1)

Human multiple myeloma (RPMI8226) Human bone marrow stromal (HS-5) Human primary CLL

Young et al. (2012)42

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Liver cancer Salivary gland adenoid cystic carcinoma

Human Human Human Human

Competitive cell migration in tri-culture

Ma et al. (2012)43

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1 (( continued Table 1 continued ))

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Cancer model 3D Type I collagen or Matrigel matrix 3D co-cultures with 2 cell types 3D Type I collagen+Matrigel mixed gel 3D co-cultures with 2 cell types Directional paracrine signaling via dynamic valve control 3D Type I collagen matrix VEGF gradient Horizontal 2D monolayer 3D Type I collagen matrix VEGF and ANG-1 gradients Vertical 2D monolayer 3D Type I collagen+Matrigel mixed gel VEGF gradients Circular 3D lumens 3D Type I collagen matrix Gravity-driven perfusion Circular 3D lumens 3D collagen/fibrin matrix 3D co-cultures between endothelial cells, stromal fibroblasts, pericytes and cancer cells Perfusion in microvessels 3D collagen/fibronectin matrix Controlled shear stress and interstitial flow VEGF gradients Horizontal and vertical 2D monolayers TGF-b signaling Myofibroblast activation Endothelial monolayer migration DCIS transition to invasive ductal carcinoma Cell migration in 3D gel ECM remodeling

Cell types

Notable microenvironment features

Focus of study

Ref. Huang et al. (2009)41 Sung et al. (2011)48

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Tumorstromal interaction 3D Breast cancer Human breast carcinoma (MB-MDA-231) Mouse macrophage (RAW 264.1)

Breast cancer

Human mammary gland epithelial (MCF-DCIS.com) Human mammary fibroblast (HMF)

Lung cancer

Human lung fibroblast (MRC-5) Human lung adenocarcinoma (CL1-0)

Hsu et al. (2011)37

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Angiogenesis Breast cancer, glioblastoma

Rat mammary adenocarcinoma (MTLn3) Human glioblastoma (U87MG) Human dermal microvascular endothelial (HMVEC) Mouse fibroblast (10T 1/2)

Chung et al. (2009)53

N/Aa

Human umbilical cord endothelial (HUVEC)

Flow-induced angiogenic sprouting

Song and Munn (2011)54

N/A

Human dermal microvascular endothelial (HMVEC)

Angiogenic sprouting and tip cell migration in multiple gradients Angiogenic sprouting in 3D co-culture Endothelial-pericyte interaction and microvascular formation Barrier permeability Angiogenic sprouting Perfusion of microbeads through patent microvessel network

Shin et al. (2011)55

N/A

Human umbilical cord endothelial (HUVEC) Mouse fibroblast (10T 1/2)

Bischel et al. (2012)56

N/A

Human umbilical cord endothelial (HUVEC) Human brain vascular pericytes

Zheng et al. (2012)57

Leukemia, glioblastoma

Human Human Human Human Human

leukemia cells (HL-60) glioblastoma cells (U87MG) umbilical cord endothelial (HUVEC) placenta pericytes lung fibroblasts (LF)

Kim et al. (2013)58

Oxygen Gradients N/A

Rat myofibroblasts (C2C12)

2D monoculture of myofibroblasts on microelectrodes Oxygen gradient by electrolysis 2D monoculture Continuous perfusion via dynamic valve control Oxygen gradient by passive consumption

Hyperoxia-induced apoptosis Oxygen consumption by myofibroblasts

Park et al. (2006)62

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Rat myofibroblasts (C2C12)

Mehta et al. (2007)61

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Table 1 1 ((continued continued ))

Cancer model 2D monoculture Oxygen gradient by oxygen-generating and oxygen-scavenging reactions Hyperoxia-induced apoptosis Hypoxia-induced cytotoxicity by tirapazamine

Cell types

Notable microenvironment features

Focus of study

Ref. Chen et al. (2011)63

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Lung cancer

Carcinomic human alveolar basal epithelial cells (A549)

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Invasion and Metastasis Breast cancer Human breast cancer (MCF-7; non-metastatic) Human breast carcinoma (MB-MDA-231) 2D monoculture 3D matrigel matrix coating in microgaps 2D monoculture 3D matrigel matrix coating in microgaps Subconfluent endothelial lining across microgaps 3D hydrogel matrix 3D cocultures between endothelial cells, monocytes and cancer cells Horizontal and vertical 2D monolayers Cell migration and invasion through microgaps Tumorendothelial interactions and 3D intravasation breast carcinoma (MDA-MB-435S) liver carcinoma (HepG-2) cervical cancer (HeLa) microvascular endothelial (HMEC)

Cell migration, invasion through microgaps

Chaw et al. (2007a)70 Chaw et al. (2007b)69

Breast cancer Liver cancer Cervical cancer

Human Human Human Human

Breast cancer Human breast carcinoma (MDA-231) Fibroblastic sarcoma Human fibrosarcoma (HT1080) Human umbilical cord endothelial (HUVEC) Human microvascular endothelial (MVEC) Mouse monocyte (RAW 264.7) Long-term spheroid culture (4 d) Diffused gradients of doxorubicin 3D matrix substitute (basement membrane extract) 3D spheroids in co-culture with neighboring CAFs 3D Matrigel matrix Constant media perfusion Induced hypoxia by CoCl2

Zervantonakis et al. (2012)71

Combination Colon cancer

Human colon carcinoma (LS174T)

Combined spheroids with gradients

Walsh et al. (2008)60 CAF-mediated tumor cell Liu et al. (2010)35 invasion Combined spheroids with stromal interaction Gao et al. (2011)40

Salivary gland adenoid cystic carcinoma

Human adenoid cystic carcinoma (ACC-M) Human embryonic lung fibroblast (HFL-1) Primary isolated carcinoma-associated fibroblasts (CAFs)

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Breast cancer

Mouse mammary tumor (4T1) Human dermal microvascular endothelial (HDMEC)

Hypoxia-dependent migration of tumor and endothelial cells Attempted model of combined hypoxia and angiogenesis

N/A not applicable or not specified.

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Review Article can be incorporated to create additional channel compartments that permit indirect perfusion, which has been applied to yield formation of coculture spheroids with as many as three cell types to model tumor heterogeneity within metastatic prostate cancer (Fig. 1a).33 Besides providing perfusion and physiological flow,32 microfluidic systems offer the potential for more advanced in vitro models of the microenvironment, where spheroids can be embedded within a 3D matrix,34 and can even support the culture of surrounding carcinoma-associated fibroblasts (CAFs) to allow studies on tumorstromal interactions.35 These examples offer evidence that we are beginning to witness more integration of tumor spheroid cultures into microfluidic systems. Tumorstromal interactions A large body of work within the area of microfluidic systems has focused on leveraging the high spatial resolution aorded by microfabrication techniques to create precise structures and arrangements for more complex in vitro coculture models to study tumorstromal interactions. Coculture models have traditionally been achieved by (i) placing one cell type in direct physical contact to the second cell type, with one forming a monolayer of underlying feeder cells, or (ii) using Transwell membrane inserts to compartmentalize two cell types while allowing soluble factor signaling through the membrane. While Transwell inserts are useful for decoupling paracrine and mechanical signals that are coupled in cultures having direct cellcell contacts, Transwell-based assays are static, are limited to two compartments, and the typical culture volumes and diffusion distances of these assays often lead to significant dilution of factors, and loss of effectiveness of factors with short half-lives. Microfluidic systems, even with the most basic designs, can achieve improved spatial organization, increased level of compartmentalization, and more controlled diffusion of factors than Transwell inserts, leading to improved sensitivity,36 and importantly, more control over the dynamics of the microenvironment, including fluid flow. Examples of microfluidic coculture models for tumorstromal interactions are quite varied, and include: lung cancer37 and prostate cancer cells38 cocultured with myofibroblasts; cervical cancer cells with fibroblasts;39 breast cancer cells with either endothelial cells40 or macrophages;41 and for hematologic cancers in the bone marrow microenvironment, multiple myeloma cells with bone marrow stromal cells.42 While these examples demonstrate improved compartmentalization, including a case with three separated cell types,43 as well as improved dynamic control via microvalves,37 the majority (except for ref. 41) have focused on paracrine signaling at the cellular level in 2D, neglecting the influence of dimensionality and 3D cellmatrix interactions at the tissue and organ levels. Nevertheless, microfluidic coculture systems offer an alternative to existing coculture assays with advantages that motivate continued interest in this area. ECM and three-dimensionality The addition of ECM components to create a 3D structural framework for cells in culture adds mechanical context and dimensionality to the in vitro microenvironment. While 2D
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Integrative Biology compartmentalized cocultures are advantageous because they are simple to use, have straightforward readouts, and are amenable to high-throughput applications, there is little doubt as to the importance of the third dimension in culture,26 and the role of mechanobiology in tumorigenesis.44,45 Tumor growth is associated with biomechanical alterations in the microenvironment, including increased solid stresses in the neighboring tissue, increased matrix stiness, and aberrant interstitial fluid flow.46 In addition, matrix remodeling and other mechanobiological cues aect the invasiveness and metastatic potential of tumor cells.47 Tumor spheroids, discussed above, is one approach to achieve three-dimensionality, but the morphology of spheroids does not necessarily apply to all tumor microenvironments, nor does it represent the optimal form for testing and experimentation. Various microfluidic systems have incorporated 3D matrix components and hydrogels into compartments, and thus advanced 2D cells-on-chip technology toward 3D tissues-on-chip. By incorporating collagen-based gels, cancer cell invasion can be monitored in 3D, and matrix remodeling can be visualized by second harmonic generation imaging of the changing collagen structure.41 This can now be achieved quite easily with pipette-based passive pumping into a simple Y-shaped microchannel (Fig. 1b).48 The gel has the capacity to support stromal cells such as mammary fibroblasts, and additional microchannel geometries can also be incorporated to modify diusion distances, allowing greater spatial control between cell types, even in 3D. Given the mounting evidence toward the importance of 3D culture, and the continued advancement of technology to facilitate its adoption, future microfluidic systems will likely shift even more toward 3D cultures. Angiogenesis One feature of the tumor microenvironment that requires specialized 3D tissue structure beyond the simple addition of a third dimension is the network of angiogenic blood vessels that supply oxygen and nutrients to the tumor. While stromal fibroblasts and immune cells can be simply embedded into 3D gels by re-suspending in the matrix solution prior to loading, endothelial cells must form confluent monolayers, with appropriate formation of cellcell junctions and basement membrane layers, in tubular lumen structures in order to resemble the actual structure of blood vessels. 2D endothelial monolayers have been popular for decades for studying flowinduced shear stress on vascular endothelial cells. This approach has been applied to enable shear stress studies on endothelial cells in microfluidic systems that promised to increase throughput, lower reagent consumption, and reduce sample quantities.49,50 Recently, microfluidic cell culture systems have advanced toward endothelial-lined blood vessel mimics that are potentially useful for cancer-related angiogenesis studies.51 Many of these microsystems employed simple 2D endothelial monolayers as before, but in a vertical orientation to allow visualization of horizontal angiogenic vessel growth into hydrogels.5255 One design in particular superposed VEGF gradients and shear flow over the vertical monolayers to study flow-induced angiogenic sprouting.54 To create more advanced

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Integrative Biology 3D lumens with cylindrical cross-section, a simple viscous fingering method via micropipetting was employed in one design,56 while an elaborate microfluidic assembly was constructed in another design to house a slab of collagen gel with molded microfeatures for the seeding and remodeling of endothelial-lined microvessels.57 Interestingly, by embedding the collagen gel with pericytes in this latter design, endothelial cells were able to recruit pericytes to the microvessel walls, and an appropriate basal lamina between pericytes and endothelial cells was allowed to form. This was also accompanied by angiogenic sprouting into the collagen gel slab. Altogether, this example demonstrates how complex interplay between distinct microenvironmental cues can lead to natural remodeling of a microvessel network that displays many in vivo-like characteristics, some of which can be applied to the engineering of a tumor microenvironment. Others have since been able to create full angiogenic microvessel networks in a 3D chip (Fig. 1c) that can be cocultured with different cell types and be perfused with microbeads,58 showing further advancement in complexity for models involving angiogenesis. Gradients In addition to aspects of the microenvironment related to cellular organization, dimensionality, ECM architecture, and morphological properties, microfluidic systems are also naturally well suited for generating and maintaining spatiotemporal gradients of biochemical and physicochemical components. Because of well-recognized laminar flow characteristics in low-Reynolds number microchannel flows, microfluidic gradient generators were one of the first and most popular types of systems developed in the field.59 Several microfluidic angiogenesis systems have employed gradients of VEGF to induce sprouting,53,54,56 including one in particular that used a combination of VEGF and ANG-1 gradients to examine cooperative eects between the two soluble factors.55 Furthermore, it is possible to superpose gradients on tumor spheroids to further add complexity to the spheroid model.60 Aside from biochemical gradients, the control of oxygen gradients have also been demonstrated as a way to model hypoxic conditions within the tumor. Oxygen gradients can either be produced passively by allowing oxygen consumption by cells cultured in the microchannels,61 or actively by controlling water electrolysis via patterned electrodes,62 or by oxygen-generating and oxygen-scavenging chemical reactions (Fig. 1d), the latter of which was used to investigate oxygen tension on alveolar basal carcinoma cells.63 The power of microfluidics will become even more evident as we continue to create more complex microenvironments through superposition of multiple gradients, as well as other microenvironment factors. Metastases While the focus of this section is to highlight advances specific to the tumor microenvironment, it should be noted that microfluidic systems have also been valuable for studies related to invasion, intravasation, extravasation, and metastasis, stages of tumor progression where the events have begun to dierentiate, or are dierentiated, from the primary tumor region.

Review Article Steps of the invasion-metastasis cascade have thus far been challenging to examine in detail with in vitro systems, with Transwell inserts again being a popular choice as an invasion assay.64,65 Metastasis studies have almost exclusively been conducted with in vivo mouse models because of the lack of in vitro methods.6668 Recently, a few microfluidic systems were developed to study specific dynamic events within the metastasis cascade, including migration through gels and transmigration through subconfluent endothelial linings,69,70 and real-time imaging of invasion and extravasation steps with insight into tumor cellendothelial cell interactions (Fig. 1e).71 Given the enormous challenges that remain in elucidating mechanisms of metastasis, these examples are likely just the beginning for microfluidic metastasis systems, with much research needed to further improve models. In summary, numerous studies of microfluidic systems, from cells-on-chips to tissues-on-chips, have been performed to date to tackle one specific aspect of the tumor microenvironment. Only a few systems have attempted to tackle more than one aspect in the same device (Table 1, Combination).35,40,60 Much research is necessary to further advance cells-, tissues-, and organs-on-chips toward even more complex microenvironments. Doing so will likely allow us to pursue our goal of acquiring more knowledge and answering more outstanding questions in cancer research.

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4. Cancer hallmarks: longstanding challenges meet new opportunities

Our understanding of the biology of cancer has been crystallizing gradually over the years with the recognition that amidst all its complexity, cancer can be described in terms of basic underlying principles that revolve around six essential acquired capabilities of cancer cells. These so-called hallmarks of cancer, namely (i) the self-suciency in growth signals, (ii) insensitivity to anti-growth signals, (iii) ability to evade apoptosis, (iv) sustained angiogenesis, (v) limitless replicative potential, and (vi) tissue invasion and metastasis, were first summarized in 2000,72 and then subsequently revisited a decade later in an updated review in 2011.15 The two landmark reviews have not only served as a guide to cancer research, but have also provided two mileposts on the cancer research timeline that oers a valuable perspective of the progress made in the intervening years. Since 2000, new insights on cancer stem cells have been discovered; the importance of tumor heterogeneity has been recognized; major progress has been made in understanding EMT, invasion, and metastasis; and two new emerging hallmarks and enabling characteristics have been identified.15 Of particular relevance to this review is the clearly emergent role of the tumor microenvironment, a topic which seemed to pervade the discussion across all the dierent hallmarks. The fact that these recognized hallmarks are essential for tumor development, yet remain inadequately understood, provides major challenges on one hand, but may also present new research opportunities on the other. To provide a glimpse of

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Review Article the types of opportunities that may be available, a brief journey is made through some outstanding problems associated with these well-known hallmarks. The ability of cancer cells to simultaneously (i) sustain their own growth and proliferation, (ii) evade anti-growth signals, and (iii) resist cell death, three distinct yet connected acquired hallmarks of cancer, demonstrates their commitment to break the delicate balance of cell population demographics that is crucial to homeostasis. Notably, such population demographics, which encompass dierent cell types, their numbers, and their locations, are important to the spatial and temporal patterns of regulatory signals governing proliferation, apoptosis and necrosis, and vice versa. The fact that our knowledge and understanding of the precise signals, their interactions, and the mechanisms regulating those interactions remains incomplete may be attributed to diculties in reconstructing and testing in vitro cellular microenvironments that capture the proper demographics found in vivo. Furthermore, it is suggested that the phenomenon of contact inhibition commonly observed in 2D culture may be related to mechanisms in vivo that control cell numbers. These problems may be dicult to address with conventional cultures, but the ability to use 3D cultures with improved spatial patterning of cell populations in microfluidic systems provides a method that can potentially resolve these complex interactions, and shed light on the importance of population demographics. The acquired capability of cancer cells to avoid senescence and eectively become immortalized with limitless replicative potential may involve molecular factors of the tumor milieu that have yet to be fully identified. Experimentally, it has also been suggested that culture conditions may be tuned in a manner that can influence the onset of cell senescence in vitro.15 Microscale systems can be applied either (i) to create tumor microenvironments suitable for testing dierent biochemical factors on normal cells in an accurate physiologic context and measuring their replicative potential, possibly by monitoring cell divisions within the engineered microenvironments, or (ii) to test various culture conditions with dierent microchannel dimensions, media components, serum and glucose levels, in a high-throughput, physiologically relevant manner to elucidate how culture conditions aect cell senescence. In both cases, the flexibility of microfluidic systems can be leveraged to help elucidate the role of the microenvironment, and the culture conditions within it. One cancer hallmark that has already received considerable attention in the microfluidics community is angiogenesis. Paradoxically, in vitro models of angiogenesis are becoming increasingly sophisticated in some cases,57 yet also becoming more routine and accessible in others.56 Both of these 3D coculture models have potential to elucidate important roles for stromal and perivascular cells in angiogenic sprouting. Given that the proper microvascular structure composed of precise apposition between endothelial cells and pericytes can be faithfully recapitulated in vitro as demonstrated, new questions can be posed regarding the eect of pericytes on endothelial barrier function, leakiness, and the susceptibility of the

Integrative Biology vessel wall to intravasation and extravasation. Real-time monitoring of engineered angiogenesis systems may allow us to trace the origin, progression, and/or transdierentiation of normal endothelial cells to tumor-associated endothelial cells, or track the interactions between endothelial tip and stalk cells within angiogenic sprouts. As more complex in vitro models become a reality, it may be possible to combine tumor spheroids and angiogenic microvessels on the same microchip, allowing the simultaneous monitoring of tumor growth and neovascular recruitment, and investigations into vascular normalization at the cellular level.73 While much progress has been made over the past decade in understanding invasion and metastasis, it is notable that many challenging questions remain unanswered.15 Appropriately, these questions present interesting opportunities for microfluidic systems to serve as novel experimental platforms that can oer fresh perspectives on the multistep metastatic process. For example, paracrine signals from stromal cells of the tumor microenvironment may be involved in activating a panel of transcription factors that coordinate EMT. Moreover, a spatial pattern exists where cancer cells that have undergone EMT appear more frequently near invasive margins of the tumor rather than near the tumor core, suggesting that spatial position and the local microenvironment are important factors of the EMT process. In both cases, spatial control oered by microfluidic systems may be exploited. Since dissemination of tumor cells from the primary site often occurs via the lymphatic system, research is also needed to create in vitro models of lymphatic vessels for potential application in studies of lymphatic metastasis. Additionally, a major phase within metastasis that is particularly fraught with experimental challenges is colonization, the development of micrometastases into secondary tumors. Interesting and unanswered questions related to dormancy and mechanisms of the seed-soil hypothesis must inherently involve both the seed (i.e., circulating tumor cell (CTC) originating from the primary tumor) and the soil (i.e., the tissue bed of the distant organ on which the CTC settles). While isolation and handling of CTCs may be the subject of other microfluidic manipulations,74,75 development of engineered tissue beds that serve as the soil in colonization experiments may be attainable with advances in organ-on-chip technologies. This work will likely be essential to the successful modeling of metastatic colonization in vitro. An important topic that has been identified as both an emerging hallmark and an enabling characteristic of cancer is inflammation.15 The incorporation of inflammatory cells into in vitro microfluidic coculture systems was reviewed for several examples involving macrophages (Table 1), providing a glimpse of the potential utility of biochips to understanding tumor stromal interactions involving other inflammatory cell types such as neutrophils and lymphocytes. In fact, other microfluidic systems developed to study neutrophil migration for other diseases76 can potentially be modified for cancer studies. Using these systems, the signals between tumor cells and immune cells can be monitored in real-time, and used to elucidate the temporal dynamics of the inflammatory response.

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Integrative Biology The other emerging hallmark is the ability for cancer cells to reprogram metabolism, and this is an area that can benefit significantly from the application of microfluidic systems. Much research is currently underway on the development of analytical methods for metabolomics in microfluidic systems, with the goal of being able to sample, detect, and measure quantities of metabolites in high throughput.7779 Within these systems, the front-end cultures are so far basic cell cultures without elaborate microenvironment features, but potential definitely exists to incorporate more sophisticated physiologic models that can then be sampled downstream for metabolites in the sample media. Since the most commonly used material for microfluidic devices is poly(dimethylsiloxane) (PDMS), and it is well-known to absorb hydrophobic molecules from the media,80 other device materials such as polystyrene (PS) and other thermoplastics may need to be considered for any study involving the measure of metabolites. In addition, recent research has shown that cells in microculture are under increased cell stress due to metabolic constraints,81 indicating that improved understanding of cellular metabolism in microfluidic cultures is fundamentally important to the future microenvironment studies on the effects of metabolism in cancer.

Review Article oer insights on how to potentially enable mass manufacturing capabilities for future commercialization. Besides selection of device material, other technical issues such as throughput, world-to-chip interfacing, and the cost-benefit ratio of integrating additional functionality are engineering challenges that must be considered for each specific application, and will no doubt be an important research focus if organs-on-chip platforms, particularly for cancer drug screening and personalized medicine, are to become a reality. While engineers will need to commit some eort to advancing device fabrication, they must also commit to designing for (i) user operability and (ii) endpoint measurements, aspects that are likely of greater concern to the biologists and clinicians who will actually be using the tools to acquire useful data. The majority of the microfluidic systems reviewed here were proofof-concept designs, many with primitive prototyping setups that do not translate immediately and conveniently into biology laboratories. Operability may not be a high priority during prototyping and proof-of-concept stages, but because it becomes critical during translation and adoption stages, it is prudent to design operability directly into the system as early as possible during development. Also, more research is needed toward more endpoint measurements. The majority of microsystems discussed so far have relied heavily on microscopy to acquire endpoint readouts. This is reasonable and desirable given that direct visualization of cells and their behavior, perhaps with dierent dyes and labels, oers the most reliable and direct proof that cells are viable and functioning as expected in these systems. Furthermore, functional readouts such as the number and length of angiogenic sprouts, or the migration rates of cells or monolayer fronts, can only be detected and measured via microscopy. However, much information at the molecular level that cannot be acquired by microscopy has yet to be uncovered in many microfluidic cell culture systems. Some microscale platforms allow cell lysis so that contents can be collected for qRT-PCR and western blotting o-chip;38,84,85 and a combination of immunocytochemical staining and protein localization analysis can be employed to quantify nuclear translocation as a substitute for electrophoretic mobility shift assays (EMSAs) used to study transcription factor activation;42 Moreover, other platforms have achieved single-cell resolution qPCR performed completely on-chip.86 However, it remains a challenge to harvest cells cultured from microfluidic systems with more complex 3D microenvironments. For example, it would be useful and powerful to have the ability to isolate and independently harvest the endothelial cells from angiogenic vessels without contaminating the sample with neighboring cocultured stromal cells, but this has yet to be demonstrated. In addition, an ongoing challenge has been to detect and quantify secreted factors from microcultures. While this has been shown as proof-of-concept with simple microcultures,87,88 more research is necessary to demonstrate this for complex tumor microenvironments. The interdisciplinary nature of our current discussion means inherently that the challenge not only exists from a technical perspective, but also from a pragmatic perspective.

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5. Remaining technical challenges

While many outstanding questions await cancer researchers, as outlined above, it should be emphasized that outstanding challenges also remain for microfluidic technologists. Some of these challenges are more technical, and apply to microfluidic technologies in general, while others are more pragmatic, and apply specifically to chips designed for cancer tumor microenvironments. Regardless, these challenges must first be overcome at the technology end, if biochips are to realize their full potential in providing a new and useful platform for research in cancer and the tumor microenvironment. The following discussion aims to synthesize the various challenges facing engineers, biologists, and those working at the interface, and importantly start the dialogue needed to further advance the application of biochip technologies. Starting from a technical standpoint, what remains at the core of engineered microsystems is microfabrication. Continued research in microfabrication methods is necessary for future advancements in biochips, and it simply cannot be neglected. The fact that soft lithography and PDMS oer many more pros than cons has been demonstrated time and again, but this fact should not limit us from pursuing innovative fabrication methods that may be faster, cheaper, more reliable, and/or more amenable to high volume manufacturing. Although polystyrene (PS) oers familiarity to biologists, and is gaining recognition in the microfluidics world,82,83 PS has its shortcomings as well, as its high modulus of elasticity precludes its use in applications that require deformations in the device structure, such as for a breathing lung-on-a-chip.4 Nevertheless, more research on how to fabricate microdevices in PS and other thermoplastics will still be valuable, both to serve as a practical alternative to PDMS in academic research, and to

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Review Article Even if the technical hurdles are overcome, many questions await regarding the application of the technology in cancer research. For example, assuming the technology has reached a level of maturity and robustness sucient for serious applications in biology beyond the proof-of-concept, what tumors and tumor microenvironments are the most interesting and immediately important to develop in vitro? Lung, breast, prostate, colon, and liver cancers appear to be excellent candidates to start given that they have the highest mortality rates, and also because lung and gut-on-a-chip devices have already been reported.4,5 The bone marrow microenvironment is also of interest because, as mentioned above, it is responsible for providing the niche within which the class of hematologic cancers are developed. Furthermore, the bone microenvironment also serves as a major site for metastases of the lung, breast, and prostate,89 and thus will be an important model for on-chip metastasis studies. Another question is how much complexity is required along the cell-, tissue-, and organ-level spectrum to yield useful, functional readouts that can oer new insights in human diseases like cancer. There is clearly a compromise between having more complex physiologic microenvironments with more convoluted interactions and less straightforward readouts versus less physiologic context, and thus less accuracy, in the microenvironment model, but more control over interactions and more basic but similarly useful readouts. This question can only be addressed by rigorous validation and comparison between in vitro systems oering varying levels of complexity, in addition to comparisons with in vivo animal models that serve as positive controls. Regardless of physiologic complexity, the basic units of any biochip are the basic units of life itself: the cells. While the majority of microdevices have demonstrated feasibility using immortalized cell lines or commercially available primary cells like HUVECs, the promise that organs-on-chips can significantly improve drug screens and provide a future involving personalized medicine depends on our ability to integrate into the chip technologies primary human cells obtained directly from individuals. This issue, and the others described before it, may not be specific to cancer research, but they should be emphasized for their importance to the successful technological advancement of these systems for cancer research, and other research areas.

Integrative Biology opportunity for significant impact in both areas, with potential for scientific breakthroughs. The impact of organs-on-chips on cancer research may not be apparent in the next few years, but if the current rate of progress is any indication, interesting developments are certainly on the horizon.

Acknowledgements
The author acknowledges financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC) through a Discovery Grant. The author also apologizes for any unintended omissions of pertinent literature in the current discussion.

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Summary
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Integrative Biology 81 X. Su, A. B. Theberge, C. T. January and D. J. Beebe, Anal. Chem., 2013, 85, 15621570. 82 E. W. K. Young, E. Berthier, D. J. Guckenberger, E. Sackmann, C. Lamers, I. Meyvantsson, A. Huttenocher and D. J. Beebe, Anal. Chem., 2011, 83, 14081417. 83 E. Berthier, E. W. K. Young and D. Beebe, Lab Chip, 2012, 12, 12241237. 84 X. Su, E. W. K. Young, H. A. S. Underkofler, T. J. Kamp, C. T. January and D. J. Beebe, J. Biomol. Screening, 2011, 16, 101111. 85 W. S. Hong, E. W. K. Young, W. H. Tepp, E. A. Johnson and D. J. Beebe, Toxicol. Sci., 2013, DOI: 10.1093/toxsci/kft082. 86 A. K. White, M. VanInsberghe, O. I. Petriv, M. Hamidi, D. Sikorski, M. A. Marra, J. Piret, S. Aparicio and C. L. Hansen, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 1399914004. 87 H. Zhu, G. Stybayeva, M. Macal, E. Ramanculov, M. D. George, S. Dandekar and A. Revzin, Lab Chip, 2008, 8, 21972205. 88 N.-T. Huang, W. Chen, B.-R. Oh, T. T. Cornell, T. P. Shanley, J. Fu and K. Kurabayashi, Lab Chip, 2012, 12, 40934101. 89 K. Hess, G. Varadhachary, S. Taylor, W. Wei, M. Raber, R. Lenzi and J. Abbruzzese, Cancer, 2006, 106, 16241633.

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The Royal Society of Chemistry 2013

Integr. Biol., 2013, 5, 1096--1109

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