An integrated in vitro model of perfused tumor and cardiac tissue

Cancer and cardiovascular disease remain the two leading causes of death in the United States. Progress in treatment to reduce morbidity and mortality will include the development of new drugs. Recent advances in induced pluripotent stem cell technology, tissue engineering, and microfabrication techniques have created a unique opportunity to develop three-dimensional (3D) microphysiological systems that more accurately reflect in vivo human biology when compared with two-dimensional flat systems or animal models. Our group is working to develop 3D microphysiological systems using induced pluripotent stem cell technology that simulates the microcirculation, the cardiac muscle, and the solid tumor, and then to combine these systems into an integrated microphysiological system that simulates perfused cardiac muscle and solid tumor on a single platform. The platform will be initially validated to predict anti-cancer efficacy while minimizing cardiac muscle toxicity. A critical feature will be blood flow through a human microcirculation (capillaries and larger microvessels), which is necessary to overcome diffusion limitations of nutrients and waste products in realistic 3D cultures, and serves to integrate multiple organ systems. This is a necessary and critical feature of any platform that seeks to simulate integrated human organ systems. The results of our project should produce a new paradigm for efficient and accurate drug and toxicity screening, initially for anti-cancer drugs with minimal cardiac side effects, and a platform technology that can be eventually used to integrate multiple major organ systems of the human body.

network by convection, or the bulk movement of fl uid to meet the dynamic and complex needs of metabolic tissues. Current models to investigate the micro circulation include human studies that are costly and have limited potential for mechanistic intervention, in vivo animal studies that require extrapolation to human biology, or static in vitro models that employ purifi ed one-component ECM materials (for example, collagen, fi brin), which do not adequately refl ect the diversity of ECM composition between tissues.
Developing dynamic 3D vasculature-fed organ-specifi c in vitro human microtissues has the potential to provide whole new opportunities for discovery while reducing the use of animals in research. Current in vitro models that lack interstitial fl ow and a perfused capillary network off er a limited mimicry of the tissue microenvironment. ECs experience fl uid shear forces throughout the vascular tree that can impact their function. Interstitial fl ow not only provides important biomechanical cues in the microenvironment, but can also markedly impact extracellular gradients of solutes or small molecules. Additionally, the microcirculation is the major conduit for drug delivery to tissues.
Recently, microfabrication technology has led to the creation of precise microchannels on nonbiological substrates (for example, silicon or polydimethylsiloxane) [5] or within purifi ed single-component substrates such as collagen [6]. While these approaches introduce convection as a mechanism of transport, even when endothelialized, the channels are not human capillaries and thus lack the fl exibility to adapt to changing metabolic needs.
To create patient-specifi c vascular networks, our group will expand our previous work in engineering vasculature [7][8][9] in vitro by developing vessel networks derived from Figure 1. The project will create three separate microphysiological systems that can then be incorporated. Three separate microphysiological systems will simulate: (a) a perfused network of human capillaries derived from human induced pluripotent stem cells in a porcine cardiac extracellular matrix, (b) a network of human capillaries in a the presence of cardiac muscle spheroids derived from the human induced pluripotent stem cells, and (c) a network of human capillaries in the presence of solid tumor spheroids. As depicted, the arteriole and venule are microfl uidic channels of high pressure/high oxygen and low pressure/low oxygen, respectively. (d) Any of these individual microphysiological systems can be incorporated into a modular high-throughput platform that includes a row of perfused microphysiological systems (perfused tumor spheroids shown as an example). The platform can include modular connectors to facilitate addition of other organ systems. (e) The fi nal phase is to combine the individual microphysiological systems into a single integrated system of perfused tumor spheroid and cardiac muscle. SC, stromal cells; PC, pericyte-like cell; TS, tumor spheroid; CS, cardiomyocyte spheroid.
human iPSC-derived ECs in a cardiac-derived ECM. Over the past 2 years, we have developed a novel microfl uidic-based system that supports a metabolically active stroma with culture medium-perfused human capillaries [10]. In the original iteration of the design, endothelial colony-forming cell (ECFC)-derived ECs harvested from cord blood were used to form vessels with the support of stromal cells, normal human lung fi broblasts, in a fi brin matrix. More recently, we demonstrated the versatility of this platform to form robust capillary networks using various matrices such as type I collagen and porcine cardiac-derived ECM [11] blended with fi brin (Figure 2a). Using ECFC-derived ECs, we achieved perfused microvessels in the cardiac-derived ECM blend (Figure 2b). Current work is focused on creating a patient-specifi c 3D perfused vascular network using human iPSC-derived ECs.

Perfused cardiac muscle
Accurately predicting adverse cardiac side eff ects of new pharmaceutical drugs currently relies heavily on animal models or 2D and relatively simple 3D in vitro models [12,13]. Although animal models allow insight into pharmaco kinetics and whole organ drug response, some drugs have been shown to only eff ect cardiomyocytes of human origin [14]. Th is has lead to unexpected and undesirable cardiotoxicity in human clinical trials that were not predicted in preclinical animal testing. Alternatively, human iPSC-derived cardiomyocytes off er many advantages over animal models, including human origin, culture adaptation, and ability to create patientspecifi c cell lines. Furthermore, human iPSC-derived cardio myocyte 2D monolayers exhibit predictable responses to known cardioactive drugs [15,16]. Nonetheless, in order to fully mimic the human response, human iPSC-derived cardiomyocyte drug screening platforms should be multicellular (for example, contain cardiomyocytes, stromal cells, ECs) [17], should be 3D [18,19], and should have nutrients and drugs delivered physiologically through the microcirculation.
We demonstrate feasibility for using our proposed system to create a vascularized cardiac microtissue by fi rst diff erentiating human iPSCs (generous gift from Professor Bruce Conklin, Gladstone Institutes, San Francisco, CA, USA) into cardiomyocytes following a matrix sandwich method [20]. Briefl y, human iPSCs are cultured as a monolayer on matrigel and then overlaid with matrigel while sequentially exposed to activin A and bone morphogenic protein-4. Th en 3D contracting cardiac organoids with physiologically relevant cell density of 10 8 to 10 9 cells/cm 3 are formed using AggreWell™ plates (STEMCELL Technologies Inc., Vancouver, BC, Canada). We then combine the 3D cardiac organoids with ECFCderived ECs and normal human lung fi broblasts in a microfl uidic platform [10]. Human iPSC-derived cardiomyocytes survive and continue to contract within the device for up to 28 days while a surrounding vessel network develops (Figure 2c).
Our polydimethylsiloxane-based microdevice is transparent, enabling the use of non-invasive and nondestructive optical techniques to probe and characterize cardiomyocyte function. Changes in beat frequency and force are tracked using brightfi eld microscopy while the electrophysiology of cardiomyocytes is visualized using voltage-sensitive dyes. Drug-induced cardiotoxicity can be monitored using the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. Finally, the metabolism will be tracked by measuring the ratio of protein bound:free NADH using fl uorescent lifetime imaging [21].
Future research will focus on creating capillary perfusion within our 3D model of vascularized cardiac tissue and then validating the cardiac response with a panel of drugs with known mechanism (for example, epinephrine). Current 3D in vitro models have yet to incorporate vasculature necessary for physiological convective transport of nutrients, waste removal, and drug delivery to human iPSC-derived cardiac tissue -or any other functional human tissue, for that matter.

Perfused solid tumor
Th e study of the tumor microenvironment relies heavily on animal models [22,23] or 2D and relatively simple 3D in vitro models [24][25][26]. In addition, multicellular tumor spheroids have been employed, but do not feature a perfused capillary network [27][28][29]. Animal models are capable of simulating the aggregate response of the tumor and host, but suff er from limitations in the response of a species, and are severely limited in their ability to screen large libraries of potential anti-cancer drugs. In vitro models have focused on the response of isolated tumor cells to a soluble factor(s) [30] or neighboring cells (for example, fi broblasts) [31]. Th ese models have provided a wealth of information regarding intercellular signaling pathways in tumor cells. Unfortunately, most tumor cells are programmed with redundant and dynamically changing pathways that control diff erentiation and migration, and also respond to multiple factors within the microenvironment. Finally, it is unrealistic to create 3D models of all NCI60 tumor cell lines; hence, because anti-colon cancer drugs have known cardiac toxicity, our focus is on two colon cancer cells lines that are part of the NCI60 (SW620 and HCT116). Th is focus provides the opportunity to compare the response of our 3D system with a large body of data collected in simpler 2D systems, while also focusing our eff orts on a model system that has the potential to distinguish the effi cacy of new chemotherapeutic agents on epithelial-mesenchymal transition and early metastatic events. Our premise is that improved 3D models of the tumor microenvironment will signifi cantly improve the effi ciency of anti-cancer drug screening.
Our initial experiments demonstrate that our proposed model is appropriate to develop as a platform for the 3D tumor microenvironment. SW620 colorectal cancer cells, transduced with a Wnt-regulated GFP reporter cassette (generous gift from Professor Marian Waterman, University of California, Irvine, CA, USA), demonstrate signifi cant growth between days 10 and 20 within the polydimethylsiloxane microdevice in the presence of fi broblasts and ECFC-derived ECs (Figure 2f ). In addition, a vessel network develops rapidly over the course of the initial 10 days (Figure 2f ).
A critical barrier to developing new anti-cancer drugs is the creation of a realistic in vitro model of the tumor microenvironment that has the potential to simulate key features such as the leaky and tortuous microcirculation. Th ese features of the tumor microcirculation probably Figure 2. The microfl uidic platform is conducive to the development of human microvessel networks. (a) Perfusable three-dimensional microvessels are generated using an optically clear polydimethsiloxane microfl uidic-based platform, (b) consisting of two fl uid-fi lled microfl uidic channels on either side of 12 mm diamond-shaped tissue microchambers. Scale bar = 500 μm. The fl uidic channels loop down and connect with each diamond through a single 30 μm diameter pore that represents the only port for transport of nutrients and waste. A coculture of endothelial colony-forming-derived endothelial cells and normal human lung fi broblasts are mixed with fi brin matrix or another blend of extracellular matrix and microinjected into the central tissue chamber and allowed to gel. By 14 to 21 days, a robust network of microvessels develops. (c) Fluorescent microscopy of CD31-stained (green) microtissues at 18 days depicts an interconnected network of vessel in a porcine cardiac-derived extracellular matrix blend. Scale bar = 200 μm. (d) Vessel patency and perfusion is verifi ed by introducing microspheres (red, white arrows) into the fl uidic channels and observing their movement through the network. Scale bar = 200 μm. (e) A third cell type, such as tumor or cardiomyocyte spheroids, can also be added to the tissue chamber to create specifi c microorgan systems. Cardiomyocyte spheroids (cTnT, red) remain viable over 29 days in the microfl uidic device as the vessel network (CD31, green) develops in the surrounding tissue. Scale bar = 100 μm. (f) Tumor spheroids (black arrows) from colorectal cancer cell line SW620 (transduced with Wnt-regulated GFP reporter cassette) proliferate and increase signifi cantly in total mass at the same time as the continuous vessel network develops, especially between day 10 (inset) and day 20. Scale bar = 500 μm.
play an important role in early metastatic events such as intravasation. Our optically clear platform is ideal to view these events with high spatial and temporal resolution. Th e next steps in the development of the perfused solid tumor will be creating an environment in which rapid and reproducible anastomosis between the vessel network and the microfl uidic channels occurs, and validating the response of the system to a panel of wellcharacterized anti-tumor drugs.

Conclusion
Cardiovascular disease and cancer remain the two leading causes of death in the United States, and innovative solutions to create new therapeutic interventions are needed. Our laboratory has spent the past decade developing 3D microphysiological systems [7][8][9][10][32][33][34][35][36], including recent results that demonstrate perfusion of living, dynamic human microvessels [10]. Our group is thus poised to develop microphysiological systems of cardiac muscle and solid tumor perfused by a living dynamic microcirculation on a single integrated platform. Th e results should produce a new paradigm for effi cient and accurate drug and toxicity screening.