Design and Construction of a Multi-Organ Microfluidic Chip Mimicking

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Design and construction of a multi-organ microfluidic chip mimicking the in vivo microenvironment of lung cancer metastasis Zhiyun Xu, Encheng Li, Zhe Guo, Ruofei Yu, Hualong Hao, Yitong Xu, Zhao Sun, Xiancheng Li, Jianxin Lyu, and Qi Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08746 • Publication Date (Web): 08 Sep 2016 Downloaded from http://pubs.acs.org on September 9, 2016

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ACS Applied Materials & Interfaces

Design and construction of a multi-organ microfluidic chip mimicking the in vivo microenvironment of lung cancer metastasis

Zhiyun Xu1, 2 #, Encheng Li1, #, Zhe Guo1, #, Ruofei Yu1, #, Hualong Hao1, Yitong Xu1, Zhao Sun1, Xiancheng Li3, Jianxin Lyu4, *, Qi Wang1,*

1

Department of Respiratory Medicine, the Second Affiliated Hospital of Dalian

Medical University, Dalian 116027, China 2

Department of Respiratology, Qilu Hospital Shandong University, Jinan 250012,

China 3

Department of Urology, the First Affiliated Hospital of Dalian Medical University,

Dalian 116027, China 4

Chinese Educational Ministry-designated Key Laboratory of Laboratory Medicine

and Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China

#

These authors contributed equally to this work.

*

Corresponding Authors: Qi Wang, Department of Respiratory Medicine, the Second

Affiliated Hospital, Dalian Medical University, Dalian 116027, China; Tel.: +86-41184671291; Fax: +86-411-84671291; E-mail: [email protected] Or Jianxin Lyu, The Chinese educational Ministry- designated Key Laboratory of Laboratory Medicine, Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China; Tel.: +86 577 86689805; fax: +86 577 8668 9771. E-mail: [email protected]

Keywords: multi-organ microfluidic chip; lung cancer; cancer metastasis; cancer microenvironment; in vitro

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Abstract Metastasis is a complex pathophysiological process.As the main cause of cancer mortality in humans it represents a serious challenge to both basic researchers and clinicians. Here we report the design and construction of a multi-organ microfluidic chip that closely mimics the in vivo microenvironment of lung cancer metastasis. This multi-organs-on-a-chip includes an upstream "lung" and three downstream "distant organs", with three polydimethylsiloxane (PDMS) layers and two thin PDMS microporous membranes bonded to form three parallel microchannels. Bronchial epithelial, lung cancer, microvascular endothelial, mononuclear and fibroblast cells were grown separated by the biomembrane in upstream"lung", while astrocytes, osteocytes, and hepatocytes were grown in distant chambers, to mimic lung cancer cell metastasis to the brain, bone, and liver. After culture in this system, lung cancer cells formed a "tumor mass", showed epithelial-mesenchymal transition (with altered expression of E-cadherin, N-cadherin, Snail1, and Snail2) and invasive capacity. A549 cells co-cultured with astrocytes overexpressed CXCR4 protein, indicating damage of astrocytes after cancer cell metastasis to the brain. Osteocytes overexpressed RANKL protein, indicating damage of osteocytes after cancer cell metastasis to the bone, and hepatocytes overexpressed AFP protein, indicating damage to hepatocytes after cancer cell metastasis to the liver. Finally, in vivo imaging of cancer growth and metastasis in a nude mice model validated the performance of metastasis in the organs-on-chip system. This system provides a useful tool to mimic the in vivo microenvironment of cancer metastasis and to investigate cell-cell interactions during metastasis.


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Introduction Metastasis is the main cause for most cancer mortality, including that related to lung cancer 1-2, and it represents a serious challenge to both basic researchers and clinicians. Cancer metastasis is a complex series of pathophysiological processes 3-4 that generally involves the growth of a primary tumor, detachment and transportation of the tumor cells, and growth into a metastatic tumor mass in a distant organ 5. In addtion, cancer metastasis is highly organ-selective and it involves numerous interactions between cancer cells and host organs. Clinically, lung cancer frequently metastasizes to the brain, bone, and liver, causing a shorter survival 6. However, the mechanism underlying organ-specific cancer metastasis is incompletely understood. Thus, there is an urgent need to develop a reliable and efficient in vitro culture model that closely mimics the in vivo microenvironment of lung cancer metastasis. A better understanding of lung cancer metastatic pathology requires a way to investigate the function of lung cancer cells and tissues in the lung and distant organs7. During lung cancer metastasis, epithelial-mesenchymal transition (EMT) involves cancer cells in primary site transitioning to metastatic cancer cells to spread through the blood stream, and forming secondary mass in distant organs by mesenchymal-epithelial transition (MET) 8. However, currently available animal models, which have been used to assess physiology and pathophysiological processes, are costly, slow, and associated with ethical concerns. Another limitation is that these models do not allow the manipulation of the location and tropism of metastatic lung cancer cells, and do not mimic the actual growth microenvironment of lung cancer cells. In vitro 3D cell culture models, which have been used to co-culture cancer cells with stromal cells in 3D gels 9, can offer well-controlled conditions for investigating the signaling effects



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between two different cell types in cancer metastasis. However, existing models are incapable of reconstituting the structural tissue–tissue interfaces, and dynamic microenvironments. The limitations of conventional cell culture and animal models have promoted the development of alternative in vitro models. As a result, biomimetic microfluidic organs-on-chips have been engineered to recapitulate the critical arrangements and complex function of organs. These biomimetic models were fabricated by polydimethylsiloxane (PDMS), which was a material with good biocompatibility. Recent works concerning newly developed materials also demonstrated that such nanodrugs may be used as an injectable material, showing enhanced antitumor efficacy10. Nevertheless, lung cancer and metastatic progression have not been researched with microfluidic culture models. Additionally, existing models rely on single-organtype-based assays do not capture multi-organ interactions. The single-lung-on-chip provide lung cell culture microenvironments, but do not reproduce critical transition, invasion and metastatic progression of lung cancer, and do not maintain viability of an integrated, multi-organ lung cancer pathogenesis system in vivo. To further improve this in vitro model, we developed a “multi-organs-on-a-chip” to recapitulate the tissue–tissue interfaces and complex function of lung and distant organs. We used this system to explore lung cancer metastasis to the brain, bone, and liver, and to analyze the cell physiology and cell–cell interactions in a more physiologically relevant context.

Materials and methods Cell lines and culture


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To mimic lung cancer metastasis to multiple distant organs, corresponding cell lines were used to reconstitute the in situ lung cancer and distant organ microenvironments in vitro. Human bronchial epithelial cells were cultured to mimic the bronchial epithelium and microvascular endothelial cells, human lung fibroblasts, and mononuclear cells were also introduced into the system. Astrocytes, osteoblasts, and hepatocytes were cultured to mimic the brain, bone, and liver, respectively. A human bronchial epithelial cell line, 16HBE, was obtained from the Chinese Academy of Medical Sciences (Beijing, China). The human non-small cell lung cancer cell line A549, microvascular endothelial cell line of human umbilical vein endothelial cells (HUVECs), human lung fibroblast cell line WI38, mononuclear cell line THP-1, astrocyte cell line HA-1800, osteoblast cell line Fob1.19, and hepatocyte cell line L-02 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in Roswell Park Memorial Institute medium-1640 (RPMI1640), Ham's F12 medium (F12K), Iscove’s Modified Dulbecco’s Medium (IMDM), Dulbecco’s modified Eagle's medium (DMEM), or DMEM/F12 (all of these reagents from Sigma-Aldrich, St Louis, MO, USA), supplemented with 10% fetal bovine serum (Gibco, Invitrogen, Inc, USA), 100 U/mL penicillin, and 100 U/mL streptomycin (Gibco, Invitrogen, Inc, USA) at 37°C in an incubator with 5% CO2 and 95% relative humidity.

Establishment of the organs-on-a-chip Design The multi-organ microfluidic chip was designed to reconstitute organ-level functions and provide a disease in vitro model that would mimic lung cancer



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formation and metastatic processes (Fig. 1A). The multi-organ-on-a-chip consisted of an upstream “lung organ” and three downstream “distant organs” (Fig. 1B). The upstream lung-on-a-chip was made of an optically transparent silicone elastomer that contained tightly apposed layers of human bronchial epithelial and stromal cells (microvascular endothelial cells, fibroblasts, and macrophages) separated by a microporous PDMS membrane coated with extracellular matrix (Fig. 1C–D). In the upper channel, the bronchial epithelial cells were exposed to air to mimic the bronchial air space. Culture medium flowed through the microvascular channel (Fig. 1E), and a cyclic vacuum was applied to the hollow side chambers to cyclically stretch the tissue layers (10% cyclic strain at 0.2 Hz 11) to mimic physiological breathing (Fig. 1F–G). In order to mimic lung cancer formation, A549 cells were co-cultured with 16HBE cells (Fig. 1H). The three downstream distant organs were made of astrocytes, osteoblasts, or hepatocyte cells that were induced to form 3D cultures in separate cell culture chambers (Fig. 1I). These were linked to the “lung organ” by side channels to mimic lung cancer metastasis to the brain, bone and liver. Cell culture medium was made to flow through the microvascular channel to mimic blood circulation using a syringe pump (Fig. 1J). When lung cancer cells grow into a cancer mass at a primary site, they invade to the surrounding stroma and spread to distant organs, normally the brain, bone, or liver. Construction The multi-organ microfluidic chip was fabricated from three PDMS layers and two thin microporous PDMS membranes (Fig. 2A). The upper, middle, and lower layers were produced by PDMS prepolymer, as previously described 12-13. The crosssectional sizes of the microchannels were 4 mm (width) × 10 mm (height) for the


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central culture channels, 2 mm (width) × 7 mm (height) for the side channels, and 1.5 mm (width) × 1.5 mm (height) for the downstream distant cell culture chambers (Fig. 2B–D). The microporous membrane was generated by PDMS prepolymer (15:1 w/w) at 3000 rpm for 1 min on a salinized SU8 substrate and bringing the spin-coated PDMS layer in conformal contact with a photolithographically prepared master that had an array of 20-mm-tall circular posts. The circular size of PDMS membrane was 10-µmthick (Fig. 2E–F). Before use, the microfluidic chip was ultraviolet -sterilized (Fig. 2G). The porous membranes embedded in the central culture channels were then coated with BME (R&D Systems, McKinley Place, MN, USA) and diluted 1:10 v/v in sterile water. Operation In the upstream lung-on-a-chip, 16HBE and A549 cells were 2D cultured on the upper membrane surface, and THP-1, WI38, and HUVEC cells were 2D cultured on the opposite side of the membrane. In the three downstream distant organs-on-a-chip, astrocytes, osteoblasts, and hepatocytes were 3D cultured in cell chambers. The microfluidic chip was inverted for seeding of THP-1 cells into the lower liquid channel at approximately 103 cells/cm2, and the THP-1 cells attached to the membrane surface before perfusion with phorbol myristate acetate (PMA) medium through inlet 1 (Fig. 1B) by a syringe pump at a volumetric flow rate of 24 mm/h. The excess was effused from outlet 1. The chip was then tilted to a declining position to allow the cells to attache to the side of the central culture channels. The THP-1 cells were stimulated to be M0 macrophages after 48 h, and the flow of PMA medium (100 ng/ml) was changed to regular culture medium. Next, WI38 were seeded into the same side of the chip as the macrophages at approximately 104 cells/cm2, and allowed



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to attach to the membrane surface for 4 h under static conditions. After that, the chip was reset to its original position and HUVEC cells were seeded to the lower liquid channel from inlet 1 at approximately 104 cells/cm2 and allowed to attach to the membrane surface for 4 h. Twenty-four hours later, the microfluidic chip was inverted for seeding of 16HBE cells into the upper channel through inlet 2 at about 104 cells/cm2, and the cells were allowed to attach to the upper membrane on the opposite side of the membrane. After cells on both membrane surfaces had grown to confluence, A549 cells were seeded into the upper channel at approximately 103 cells/cm2 and allowed to attach to the membrane surface for 4 h under static conditions. The astrocyte-, osteoblast-, and hepatocyte-BME mixtures were immediately seeded in the 3D cell chambers through inlets 3–5. To eliminate the influence of the position of the target organs on lung cancer metastasis, the inoculation site of distant target organ cells was stochastic. For the air–liquid interface culture, the culture medium was gently aspirated through outlet 2, and medium mixture was introduced through inlet 1. The “distant organ” astrocytes, osteoblasts, and hepatocytes were then perfused with culture medium into the upstream chambers through inlet 1. Simultaneously, outlet 1 was closed, and the excess was effused from inlets 3–5.

Evaluation of the organs-on-a-chip through characterization of the different cells cultured on the multi-organ microfluidic chip Lung-on-a-chip


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In the lung-on-a-chip, the morphology, proliferation, microenvironment, transition, and invasion of lung cancer cells were recorded using an inverted fluorescent microscope. To mimic in vivo microenvironment of lung cancer, the growth pattern and viability of human bronchial and microvascular endothelial cells in the lung-on-a-chip were observed. The growth patterns of cells were recorded using an inverted fluorescent microscope. Apoptosis was assessed by Hoechst/propidium iodide (PI) staining (Hoechst H33342, Invitrogen, Carlsbad, CA, USA; PI, Molecular Probes, Eugene, OR, USA) and image analysis (Image J, National Institutes of Health, Bethesda, MD, USA). Immunostaining of E-cadherin was used to prove the tightness of the epithelial and endothelial cell connections in the lung-on-a-chip. For E-cadherin immunostaining, epithelial and endothelial cells in the microfluidic chip were washed out with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 15 min. The cells were washed with PBS, blocked with 2% bovine serum albumin in PBS for 1 h, and then incubated with a mouse anti-E-cadherin antibody (Invitrogen) at 2 µg/mL for 2 h. Next, they were incubated with an Alexa 594conjugated secondary antibody for 1 h and further with DAPI (Sigma-Aldrich) before taking fluorescence images using an epifluorescence microscope (Olympus, Tokyo, Japan). Features of lung cancer cells, cancer-associated fibroblasts, and macrophages were examined using an inverted fluorescent microscope. Cell Tracker dye was used to label and detect lung cancer or stromal cells. Before lung cancer or stromal cells were seeded into the chip, we added pre-warmed Cell TrackerTM CM-Dil (Invitrogen) or C34554 working solution (10 µmol/L). Furthermore, the expression of the tumor marker carcino-embryonic antigen (CEA) in lung cancer cells was assessed using immunofluorescence. Similarly, we examined the expression of the fibroblast marker



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α-smooth muscle actin (α-SMA) 14and the M2 macrophage marker CD206 15in these cells. To label CEA, α-SMA, and CD206 proteins, we fixed cells obtained from the microfluidic chip, permeabilized them with 0.5% Triton X-100 in PBS for 20 min, blocked them, and incubated them with the following antibodies: polyclonal rabbit anti-human CEA (1:100, Abcam), polyclonal mouse anti-human α-SMA (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or polyclonal mouse anti-human CD206 (1:50) antibody. Then, they were incubated with an Alexa 488- or Alexa 594conjugated secondary antibody. In addition, we assessed the expression of EMT markers (e.g., E-cadherin, Ncadherin, Snail1, and Snail2 16) using immunofluorescence. The primary antibodies used were rabbit anti-E-cadherin (1:100, Proteintech, Wuhan, China) and anti-Ncadherin (5 µg/ml, Abcam), polyclonal mouse anti-Snail1 (1:50, Abcam), and rabbit monoclonal anti-Snail2 antibodies (1:400, Cell Signaling Technology, Boston, MA, USA). In order to perform the invasive ability of A549 cells in this system, we scanning A549 cells transferred to downside of the microporous PDMS membrane layer by layer by a confocal microscope. We patterned the mono-culture of A549 cells on the upper side of the membrane in this system as the control group, and the co-culture of A549 cells with cancer-associated fibroblasts (CAFs) and macrophages (CAMs) on the upper side of the membrane, and with HUVECs on the other side as experimental groups.After cells cultured 3 days, the numbers of lung cancer cells transferred to the down side of the membrane were counted. Brain-, bone-, and liver-on-a-chip To confirm the presence of metastatic lung cancer cells in the distant organs-onchips, we stained the cells with mesenchymal–epithelial transition (MET) markers (E-


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cadherin, N-cadherin, Snail1, and Snail2 16). To assess lung cancer cell invasion in these organs-on-chips, we immunofluorescently stained the cells with a rabbit polyclonal anti-CXCR4 antibody (1:100, Abcam) for metastasis to the brain 17, a rabbit polyclonal anti-RANKL antibody (1:50, Santa Cruz Biotechnology) for metastasis to the bone 18, 19, or a rabbit polyclonal anti-alpha-fetoprotein (AFP) antibody (1:100, Abcam) for metastasis to the liver 20.

Reliability of the organs-on-a-chip through a nude mice lung cancer model All experiments were approved by Dalian Medical University Licensing Committee. Lentiviral vectors containing a plasmid (pRNAT-U6.1/Neo) encoding green fluorescent protein (GFP) were used to infect A549 cells to be the highest GFP expression clone (LMB-A549), as previously described 21. Experimental group cells were trypsinized and collected from LMB-A549 cells after co-culture with stromal cells, as in the organs-on-a-chip systems, and the control group cells were the control LMB-A549 cells co-cultured in the organs-on-a-chip systems. Six-week old athymic nude mice were injected cancer cells, as previously described 21. After 2–3 weeks, in vivo imaging of cancer growth and metastasis was performed. The images of the cancer mass were autoly calculated by Living Image 3.1.0 software (Caliper Life Sciences, Hopkinton, MA, USA) and the fluorescence intensities were analyzed. In order to reduce occasional errors and obtain reliable data,



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at least three measurements were performed and the average was counted. Typical images of these nude mice cancer model were selected. To support the reproducibility of the developed system, all experiment operations (eg. chip construction, cell culture, Immunostaining, and mice experiment) were performed at least three times. There were no significant differences in the data obtained from three independent experiments.

Results Validation of the replication of lung cancer growth conditions in the lung-on-a-chip The lung-on-a-chip aimed to recapitulate the critical cell–cell interfaces, and dynamic microenvironments of living lung. Human macrophages and fibroblast cells were co-cultured on the membrane surface. As shown in Fig. 3A–B, after culture for 24 h, the fibroblast cells appeared fusiform and spherical junctions were present in macrophage cells, whereas several tight junctions were present between macrophages and fibroblast cells after 48 h, indicating that these stromal cells could be co-cultured effectively on the membrane surface. Human bronchial epithelial 16HBE and microvascular endothelial HUVEC cells appeared flat after 24 h, and rapidly integrated into flakes after 48 h, indicating that they were viable for prolonged time at an air-liquid interface (Fig. 3C–F). As shown in Fig. 3G, H, cell activity assays showed that the percentage of live cells was more than 95%. To investigate the function of the epithelial and endothelial cells in this system, E-cadherin of them were stained positive, indicating that into epithelial and endothelial cells formed intact monolayers, respectively (Fig. 3I, J). These observations confirmed that the lung-on-


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a-chip system recapitulated the situation in vivo and thus could be used for cell culture and further studies.

Reconstitution and characterization of lung cancer formation in the lung-on-a-chip We observed the formation of lung cancer mass and the interaction of cancer cells with stromal cells in this systerm. As shown in Fig. 4A–B, the morphological features of bronchial epithelial cells (16HBE) cultured alone or co-cultured with lung cancer cells (A549) were compared. They appeared flat with a grid shape. As shown in Fig. 4C–D, an immunofluorescence cell tracker expression indicated the localization of A549 cells co-cultured with 16HBE cells. Furthermore, expression of the tumor marker CEA confirmed the tumor cell phenotype of the lung cancer cells in the lung-on-a-chip (Fig. 4E, F). To evaluate the function of lung cancer cells in this system, we examined the expression levels of two lung-cancer-associated markers: fibroblast marker α-SMA and the M2 macrophage marker CD206. As shown in Fig. 4G–J, α-SMA was expressed in fibroblasts and CD206 was expressed in M2 macrophages after coculture with lung cancer cells, confirming that the stromal cells were cancer-cellassociated stromal cells and that they interacted with the lung cancer cells.

Recapitulation of lung cancer transition and invasion using the organs-on-a-chip The expression level of the EMT markers was used to evaluate the transition and invasion of lung cancer cells into “distant organs” in this system. As shown in Fig. 5A and B, the expression patterns of E-cadherin and N-cadherin were membrane, while



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Snail1 and Snail2 expressed at the nuclear. Compared with monocultured A549 cells, the fluorescence intensity of E-cadherin in A549 cells co-cultured with cancerassociated stromal cells was lower, while the expression of N-cadherin, Snail1, and Snail2 was higher. This suggests that, after co-culture of the A549 cells with cancerassociated stromal cells for a prolonged period of time, the lung cancer cells underwent EMT and metastatic transformation. Next, we assessed the invasive ability of A549 cells in this system. Specifically, the system enabled the co-culture of A549 cells with CAFs and CAMs on one side of the membrane, and with HUVECs on the other side (Fig. 5C). As shown in Fig. 5D, compared with the monocultured lung cancer cells of this system, greater numbers of invasive cells (lung cancer cells in the down side of membrane in the system) were found in the co-cultures, indicating that the A549 cells co-cultured with cancerassociated cells in our system underwent metastatic transformation to gain an invasive phenotype.

The organs-on-a-chip mimics lung cancer metastasis to the brain, bone, and liver When lung cancer cells grow into a cancer mass at a primary site, they invade to the surrounding stroma and spread to distant organs, normally the brain, bone, or liver. This system was designed to mimic this metastasis process in vitro by assessing the changes of cancer cells. To confirm the presence of metastatic lung cancer cells in the “distant organs” of our chip system, we stained the cells with the MET markers E-cadherin, N-cadherin, Snail1, and Snail2. Compared with A549 cells in the “lung organ” of the system, the expression of N-cadherin, Snail1, and Snail2 in A549 cells in “distant organs” was


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lower, whereas that of E-cadherin was higher. This indicated that lung cancer cells had metastasized from the lung-on-a-chip to the “distant organs” in our model system (Fig. 6A, B). To assess changes in the “distant organs” after lung cancer cell invasion, RANKL was used to verify lung cancer bone- specific metastasis, CXCR4 expression level was assayed to confirm lung cancer brain-specific metastasis, and AFP was used to verify liver cell damage with cancer metastasis. As shown in Fig. 6C, D, the expression of CXCR4 in the brain-on-a-chip, RANKL in the bone-on-a-chip, and AFP in the liver-on-a-chip were detected. Overexpression of CXCR4 protein indicates damage to astrocytes after cancer metastasis to the brain, overexpression of RANKL indicates damage to osteocytes after cancer metastasis to the bone, overexpression of AFP indicates damage to hepatocytes after cancer metastasis to the liver. We found that the changes observed were consistent with earlier studies 17-20.

Reliability validation of the organs-on-a-chip system a nude mice model Finally, to validate the reliability of the organs-on-chip, in vivo imaging of cancer growth and metastasis was performed in a nude mice model (Fig. 7A-B). “Experimental group” LMB-A549 cells were trypsinized and collected after coculture with stromal cells in the system, and “control group” cells were LMB-A549 cells monocultured in the system. As shown in Fig. 7C, in the lungs of nude mice, the epi-fluorescence of the experimental group was 31.77×109±10.03, which was clearly higher than with the control group (4.80×109±1.80), indicating that the proliferative ability of LMB-A549 cells co-cultured with stromal cells was superior to that of monocultured LMB-A549 cells. In livers of nude mice, the epi-fluorescence of the experimental group was 5.37×109±2.58, which was higher than the control group



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(1.75×109±1.20). In the brains of nude mice, the epi-fluorescence of the experimental group was 1.90×109±0.18, which was also higher than with the control group (1.28×109±0.36). These finding demonstrate that metastatic ability of co-cultured lung cancer cells in this system was greater than that of the monocultured lung cancer cells. All of the results described above demonstrate that LMB-A549 cells co-cultured with stromal cells differ from the monocultured LMB-A549 cells with regard to the expression of tumor cell markers and metastatic ability. These findings confirm that the organs-on-chip can effectively mimic lung cancer cell metastasis to distant organs such as the liver, bone and brain.

Discussion Lung cancer metastasis is a complex pathological process. The main steps of metastasis are: i) detachment of metastatic cells from their neighboring cells; ii) invasion to the surrounding stroma; iii) intravasation and survival in circulation; iv) arrest at the blood vessels in the target organ where they extravasate and invade the matrix; v) proliferation within parenchyma of the target organ 22–24. Lung cancer usually metastasizes to the bone, brain, and liver, contributing to a poor prognosis 25. The overall five-year survival rate is less than 15% for patients with, which has remained largely unchanged for the last three decades. However, lack of reliable, accurate models that can mimic the structure and function of lung, simulate the metastatic process, and reproduce the microenvironment of the target organ has meant that the lung cancer cells metastasis process remains poorly understood. To fully understand human pathophysiology, investigations of organs function are necessary. Human organs contain different types of tissues and cells arranged in


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3D with chemical gradients and mechanical forces. However, conventional cell culture technologies fail to actually reconstruct the organ microenvironment. Also, animal models were expensive, arguable ethically, and fail to predict body responses. Therefore, investigation of physiology and pathological process requires an alternative in vitro model. Based on situations such as these, multi-organ microfluidic chip could supply unparalleled platform to recapitulate the main tissue structure, and dynamic organ microenvironments 26-28. In the current study, we designed and tested a microfluidic chip that mimics the microenvironments of lung cancer metastasis to the utmost extent. This system included an upstream “lung” and three downstream “distant organs” to reproduce the growth, invasion, and metastasis of lung cancer cells. Cells of the “distant organs” (brain, bone, and liver) were cultured in 3D to mimic organ-level functions, and were linked to a lung-on-a-chip to mimic the physiological setting. This system was created by reconstituting lung cancer formation and co-cultured with cancerassociated stromal cells to induce lung cancer invasion, and by injecting solutions into microchannels to mimic hematogenous metastasis of lung cancer, and subsequently spreading three connected compartments of the liver, bone, and brain cells. The flow and distribution of lung cancer cells in this microfluidic chip and the inoculation site of distant target organ cells were random, and the difference (eg. tropism and location) of lung cancer cells metastasis to distant organs might be related to target organ microenvironment, such as excretion of cytokine and cell-cell interaction. However, distant target organ selectivity of lung cancer metastasis needs to be further explored. Indeed, this system provide particularly platform to analyze cell physiology and visualize complex cell behaviors different from existing in vitro models. This microdevice did show that changes occurred in the lung cancer cells when co-cultured



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together with other types of cells in this system. The rapid formation of a lung cancer “mass”, which migrated away from its natural margins, was followed by the attack of the adjacent components and the spread of cancer cells to “distant organs”. We also detected altered cell characteristics, such as the expression of epithelial and stromal cell markers. After culture in this system, lung cancer cells expressed EMT markers and gained an invasive capacity. Moreover, co-culture of A549 lung cancer cells with astrocytes resulted in overexpression of CXCR4, co-culture with osteocytes resulted in overexpression of RANKL, and co-culture with hepatocytes resulted in overexpression of AFP, indicating damage to these cell types after cancer metastasis to the brain-, bone- and liver-on-a-chip, respectively. Finally, to validate the reliability of the organs-on-chip, in vivo imaging of cancer growth and metastasis was performed in a nude mice model. These findings validate that the organs-on-chip can effectively mimic lung cancer cell metastasis to distant organs. Thus, this system may provide a useful platform to mimic the cancer microenvironment and to investigate cell–cell interactions during metastasis. Therefore, the combine of microfluidic chip and cell biology has promoted the multiorgans-on-chip. These systems reproduce the tissue structure and complex organ function, and research physiology and pathology in visualizing ways29. We will further test the reproducibility, reliability, and sensitivity of the developed system. Our current study is only proof-of-principle, and a number of improvements may be needed in addition to the analysis of alterations to gene expression and regulation using this system. Nonetheless, this versatile system may provide the direct observation and quantitative analysis of cell–cell interaction, cancer metastasis, and regulation in vitro.


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Conclusions In summary, we built a multi-organ microfluidic chip to study lung cancer metastasis. With this system, lung cancer growth, invasion and metastasis processes can be reproduced, direct versatile and quantitative analyzed. It might be possible to provide a biomimetic platform that integrated biological structures, chemical and mechanical microenvironment, and living organ functions.

Acknowledgements This work was supported in part by grants from the National Natural Science Foundation of China (#91129733 and #81330060) , the National High Technology Research and Development Program (863 Program Projects) of China (#2015AA020409), the Opening Project of Zhejiang Provincial Top Key Discipline of Clinical Medicine (No. LKFyc05), and the Science and Technology Plan Foundation of Liaoning Province (2014225003).

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Fig. 1. Design of the biologically inspired biomimetic multi-organ microfluidic chip system. (A) Illustration of lung cancer metastasis to multiple distant organs. (B) Schematic illustration of the multi-organ microfluidic chip, which includes an upstream “lung organ” and three downstream “distant organs”. (C) Three polydimethylsiloxane (PDMS) layers are aligned and irreversibly bonded to form two sets of three parallel microchannels separated by a PDMS membrane containing an array of through-holes with an effective diameter of 10 µm. (D) Human bronchial



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epithelial and stromal cells were cultured in two parallel microchannels separated by a membrane. (E) In the upper channel, the bronchial epithelial cell layer was exposed to air and culture medium was flowed through the microvascular channel. (F, G) Physiological expiration movements were recreated upon application of a vacuum to the side chambers, which caused mechanical stretching of the PDMS membrane. (H) Lung cancer cells were co-cultured with human bronchial epithelial cells. (I) Cells of multiple organs were cultured in 3D in the cell culture chambers. (J) The culture medium was flowed through the microvascular channel to mimic blood circulation in vivo.

Fig. 2. Configuration of the biomimetic multi-organ microfluidic chip. (A) The microfluidic chip was fabricated with three PDMS layers and two thin microporous PDMS membranes. (B) The upper PDMS layer. (C) The middle PDMS layer. (D) The lower PDMS layer. (E) Image of microporous PDMS membrane using Freehand. (F) Image of microporous PDMS membrane under a microscope. (G) Image of an actual biomimetic multi-organ microfluidic chip as viewed from above.


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Fig. 3. Characterization of cells cultured in a lung-on-a-chip. (A, B) Morphological features of fibroblast W138 and macrophage THP-1 cells cultured in the microfluidic chip after 24h (top panel) and after 48h (bottom panel). (C–F) Morphological features of bronchial epithelial 16HBE cells (top panel) and microvascular endothelial HUVEC cells (bottom panel) cultured in the microfluidic chip. (G, H) Cell viability was assessed by Hoechst/PI staining. (I–J) E-cadherin expression (red) and nuclear DAPI stain (blue).

Fig. 4. Formation of a lung cancer “mass” by cells cultured in the lung-on-a-chip. (A) Morphological features of 16HBE cells cultured alone (A) or co-cultured with A549 lung cancer cells (B). Morphology of 16HBE cells under an inverted fluorescent microscope cultured alone (C) or co-cultured with A549 cells (D) (tracked with



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CellTracker, red). Morphology of 16HBE cells under an inverted fluorescent microscope culture alone (E) or co-cultured with A549 cells (F) (stained with CEA, green). Human fibroblast cells (G) and cancer-associated fibroblasts (CAFs) (H) exhibiting SMA expression (red). Human macrophages (I) and CAMs (J) exhibiting CD206 expression (red). The cell nuclei were stained with DAPI stain (blue). Magnification, ×200.

Fig. 5. Lung cancer EMT and invasion in culture in a lung-on-a-chip. (A) A549 lung cancer cells expressed E-cadherin (green), N-cadherin (green), Snail1 (red), and Snail2 (green) after 1 day (top panel) or 4 days in culture (bottom panel) in the microfluidic chip. Cell nuclei were stained with DAPI (blue). (B) Analysis of EMT marker expression in A549 cells. *: p < 0.05 day 0 compared with day 4 (C) Diagram representing lung cancer cell invasion after 4 days of culture on the experimental side of the lung-on-a-chip. (D) Invasion of A549 cells (red) cultured alone or co-cultured with stromal cells. The first row: morphological features of the microporous membrane, the second row: morphological features of lung cancer cells and the


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membrane, the third row: lung cancer cells transferred to the down side of the membrane.



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Fig. 6. Characterization of lung cancer cell metastasis in the multi-organs-on-a-chip system. (A) Expression of MET markers in A549 cells in the lung-on-a-chip and metastatic lung cancer A549 cells in the brain-, bone-, and liver-on-a-chip. (B) Analysis of MET marker expression. * p < 0.05 compared to the controls, (C) Changes in the expression of CXCR4, RANKL and AFP in the “distant organs”, compared with A549 cells cultured in the lung-on-a-chip, after lung cancer cell invasion. (D) Analysis of expression of CXCR4, RANKL and AFP in the “distant organs”. * p < 0.05 compared to the A549 cells group, #p < 0.05 compared to the Fob cells group, ▲ p < 0.05 compared to the L-02 cells group.

Fig. 7. Performance validation of the organs-on-a-chip system with a nude mice lung cancer model. (A) Epi-fluorescence of the “control group” monocultured LMB-A549 cells from the lung-on-a-chip. (B) Epi-fluorescence of the “experimental group” LMB-A549 cells co-cultured with stromal cells in the system. The liver was drew a circle to differ from lung in every mouse. (C) Analysis of cell metastasis in the lung, liver or brain in mice model. *p < 0.05compared to the controls.


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TOC


Design and Construction of a Multi-Organ Microfluidic Chip Mimicking

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