Integrated Microfluidic Platform with Multiple Functions To Probe

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Integrated Microfluidic Platform with Multiple Functions to Probe the Tumor-endothelial Cell Interaction Ling Lin, Xuexia Lin, Luyao Lin, Qiang Feng, Takehiko Kitamori, Jin-Ming Lin, and Jiashu Sun Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02593 • Publication Date (Web): 18 Aug 2017 Downloaded from http://pubs.acs.org on August 19, 2017

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Integrated Microfluidic Platform with Multiple Functions to Probe the Tumor-endothelial Cell Interaction Ling Lin,†, // Xuexia Lin,¶ , ‡ Luyao Lin,‡ Qiang Feng,†, // Takehiko Kitamori,∆ Jin-Ming Lin,*, ‡ and Jiashu Sun*, †, // †

CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, P.R. China. ‡ Beijing Key Laboratory of Microanalytical Methods and Instrumentation, The Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084, P.R. China. // University of Chinese Academy of Sciences, Beijing 100049, P.R. China. ¶ College of Chemical Engineering, Huaqiao University, Xiamen 361021, P.R. China. ∆

Department of Applied Chemistry, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113– 8656, Japan. E-mail: [email protected]. Phone: +86 10 62792343; [email protected]. Phone: +86 10 82545621

ABSTRACT: Interaction between tumor and endothelial cells could affect tumor growth and progression, and induce drug resistance during cancer therapy. Investigation on tumor-endothelial cell interaction involves cell co-culture, protein detection, and analysis of drug metabolites, which are complicated and time-consuming. In this work, we present an integrated microfluidic device with three individual components (the cell co-culture component, the protein detection component, and the pre-treatment component for drug metabolites) to probe the interaction between tumor and endothelial cells. Co-cultured cervical carcinoma cells (CaSki cells) and human umbilical vein endothelial cells (HUVECs) show higher resistance to chemotherapeutic agents than single-cultured cells, indicated by the higher cell viability, increased expression of angiogenic proteins, and elevated level of paclitaxel metabolites under co-culture conditions. This integrated microfluidic platform with multiple functions facilitates the understanding of the interaction between tumor of endothelial cells, and may become a promising tool for drug screening within an engineered tumor microenvironment.

Tumor-endothelial cell interaction is critical in a variety of physiological and pathological processes, such as angiogenesis, cancer metastasis and colonization.1-4 Accumulating evidence suggests that the crosstalk between tumor cells (TCs) and endothelial cells (ECs) via the paracrine/juxtacrine action,5,6 could impact the tumor growth and progression, and induce drug resistance during the therapy of cancers.7-11 For better understanding of tumor-endothelial cell interaction, one prerequisite is the development of in vitro cell co-culture model to mimic the tumor microenvironment, in which the exchange of diffusible factors is enabled. Another prerequisite is the extended applications of the co-culture model, such as on-line detection of cell-secreted proteins for studying the mechanism of the tumor-endothelial cell interaction, and quantitative analysis of drug metabolites during anti-cancer drug screening. Conventional approaches to realize these functions require the intensive use of sophisticated instruments and complicated operations. For efficient investigation of tumor-endothelial cell interaction, the development of an integrated and versatile platform with multiple functions is urgent. Progress in microfluidic technology enables the precise construction of physiologically relevant microenvironment with high spatial and temporal resolution, thus facilitating the

study of cellular behavior inside the microfluidic devices in vitro. Through rational design of cell culture chambers and connection microchannels, microfluidic cell co-culture devices allow for the real-time exchange of diffusible factors, but not cells, between different cell culture chambers.12-15 This kind of microfluidic model has been applied to investigate the extravasation of TCs into ECs-conditioned microenvironment.16-18 Moreover, microfluidic cell co-culture device incorporated with the on-chip concentration gradient generator has been used for the study of TCs migration induced by oxygen, drug or growth factor gradients.19,20 The behaviors of TCs and ECs inside microfluidic in vitro models are generally observed under the confocal microscopy by staining the cells with different dyes.21 However, some critical information, such as protein signaling and drug efficacy, may be difficult to obtain by microscopic observation. To unveil the tumor-endothelial cell interaction, real-time detection of proteins and drug metabolites in co-cultured TCs and ECs is highly required. Beside the capability to engineer the cellular microenvironment, microfluidics also serves as the versatile platform for pre-treatment and detection of a variety of biomarkers such as proteins, nucleic acids, and drug metabolites, with unique characteristics including high

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integration, multiple biomarker detection, reduced sample/reagent consumption, and improved sensitivity.22 The microfluidic cell co-culture system combined with microfluidic pre-treatment/detection system could be an attractive approach for investigation of tumor-endothelial cell interaction. Yet few studies have developed an advanced microfluidic platform with multiple functions of cell co-culture, protein detection, and analysis of drug metabolites. In this work, we present an integrated microfluidic device with three individual components to investigate the tumor-endothelial cell interaction. The three components are: (1) the cell co-culture component, (2) the protein detection component, and (3) the pre-treatment component. Within the cell co-culture component, cell proliferation, drug-induced apoptosis, cell cycle, and the levels of reactive oxygen species (ROS) and glutathione (GSH) under different culturing conditions have been examined. Within the protein detection component, the expression of angiogenic proteins (VEGF165, and PDGF-BB) secreted by co-cultured cells is monitored, which are first captured by the pre-coated aptamer, followed by rolling circle amplification (RCA) to amplify the signal. The pre-treatment component is adopted to desalt and purify the drug (paclitaxel) metabolites from co-culture media before analyzed by mass spectrometry. This integrated microfluidic device has been proved as an attractive and powerful tool for co-culture and monitoring of cells, detection of cell-secreted biomarkers, and investigation of drug resistance within an engineered tumor microenvironment.

EXPERIMENTAL SECTION Martials. Recombinant human VEGF165 and PDGF-BB were purchased from ProSpec Protein Specialists (East Brunswick, USA). Cervical carcinoma cells (CaSki cells) and human umbilical vein endothelial cells (HUVECs) were purchased from Cancer Institute & Hospital (Chinese Academy of Medical Science, China). Phosphate buffer saline (PBS) and concanavalin A (Con. A) were purchased from Sigma Aldrich Chemical Co. (USA). Bovine serum albumin (BSA) and human immunoglobulin G (IgG) were from Dingguo Biotechnology Co., Ltd. (China). SYBR Gold was purchased from Invitrogen Corporation (Germany). A Live/Dead assay kit (Calcein-AM/EthD-1), and Hoechst 33342 were obtained from Invitrogen (USA). Dihydroethidium (DHE), Cell Counting Kit-8 (CCK-8), propidium iodide (PI) and RNase A were from Beyotime Biotechnology Company (China). All reagents for RCA such as Phi29 DNA polymerase, T4 DNA enzyme and its buffer were purchased from NEB Company (USA). TE buffer (pH 8.0，TE) was prepared with 10 mM Tris–HCl and 1 mM EDTA (Beijing Dingguo Changsheng, China). All reagents were of analytical reagent grade and used without further purification. Fabrication of Microfluidic Device. The microfluidic device consists of three sections for cell co-culture, protein detection, and pre-treatment by micro-solid phase extraction (micro-SPE) (Figure 1). The microfluidic devices were fabricated by standard soft lithography and replica molding techniques.17 In the cell culture section, each unit has two main channels (80 µm high, 1.5 mm wide and 13 mm long for each channel), and each main channel contains a cell culture chamber of 2.0 mm in diameter. The two main channels are connected by 19 connection channels (30 µm high, 80 µm wide, and 5.0 mm long of each connection channel). To

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fabricate the microchannels, negative photoresist SU-8 2050 (Microchem, USA) was spun onto a silica wafer at the speed of 2500 rpm for 50.0 s by a spin-coater. After baking at 65.0 °C for 10 min, UV light exposure and SU-8 developing were performed to produce the layer of connection channels (30 µm high). Another layer of SU-8 2050 photoresist was then coated onto the same wafer at the speed of 1800 rpm for 50.0 s. After baking at 65 °C for 10.0 min, UV light exposure and SU-8 developing were carried out to produce the layer of cell culture chambers and main channels (80 µm high). In protein detection section, each microstructure has a serpentine microchannel (40 µm high, 80 µm wide), a reaction chamber (2 mm in diameter), and a pair of fluid ports. The master mold was made by SU-8 2050 photoresist at the speed of 2200 rpm. In the pre-treatment section, each micro-SPE column is 80 µm high, 1.8 mm wide, and 7.0 mm length. The end part of the column is 10 µm high to trap the loaded C-18 particles (45 µm in diameter) inside. To fabricate the micro-SPE channel, we used SU-8 2007 at the spin speed of 1300 rpm to get the height about 10 µm, and then used SU-8 2050 at the spin speed of 1800 rpm to get the height about 80 µm. After complete these parts, silylation reagents and the silica master were put into vacuum kettle together to make the master hydrophobic. A premixed PDMS preploymer and curing agent (Dow Corning, Sylgard 184, Midland, MI, USA) were poured onto the mold and degassed under vacuum for 30.0 min. After curing at 80 °C for 2 h in an oven, the PDMS was peeled off and cut into the designed shape. The PDMS chip with embedded microchannels was sealed with glass slide by oxygen plasma. Cell culture in the cell co-culture component. CaSki cells or HUVECs were grown in DMEM supplemented with 10 % FBS, 100 U/mL penicillin, and 100 U/mL streptomycin inside the cell incubator with 5 % CO2 at 37 °C. Two kinds of cells were then trypsinized and removed from the Petri dishes, followed by centrifugation and resuspension to the density of 107/mL in cell culture medium. After introduction of CaSki cells and HUVECs into different main channels of the cell co-culture component, the cell culture medium was covered onto the inlet and outlet of microchip to allow for nutrition exchange within the channels. The inlet at the chamber containing HUVECs was connected to a syringe pump using polytetrafluoroethylene (PTFE) tubes for supplying cell culture medium continuously at the flow rate of 5 µL/h. The medium could flow into the chamber containing Caski cells through the connection channels, and flow out from the outlet at the Caski cell chamber. For cell culturing, the cell co-culture component was put into a Petri dish and placed in the cell incubator with 5 % CO2 at 37 °C for at least 6 h to enable the cells to adhere onto the glass substrate. Apparatus. Dlectrospray ionization quadruple time-of-flight mass spectrometry (ESI-Q-TOF MS) was conducted on the Bruker microTOF-Q mass spectrometer (Bruker Daltonics Inc., USA), and the mass spectra were obtained in the negative mode and analyzed using the data analysis software package provided by Bruker. A quadrupole ion trap mass spectrometer with ESI source (made in Germany, Bruker Company) was applied for paclitaxel detection. The cell images were taken by CCD camera on a fluorescence microscopy (Leica DMI 4000 B, Germany) with software of Leica Application Suite,

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Figure 1. Schematic of an integrated microfluidic device. Three individual components are designed for different functions: (1) The cell co-culture component for co-culture of tumor (CaSki cells) and endothelial cells (HUVECs), (2) The protein detection component for high-sensitive detection of angiogenic proteins (VEGF165, and PDGF-BB), and (3) The pre-treatment component for desalting and purification of paclitaxel metabolites before ESI-Q-TOF MS, respectively.

LAS V2.7. The fluorescence was measured by fluorescence spectrophotometer (Hitachi F-7000, Japan). The optical density was calculated by Photoshop CS4 and Origin 8.5. QCapture Pro (Version 5.1.1.14, Media Cybernetics, USA). Adobe Photoshop CS 5.0 was employed to analyze the intensity of intracellular ROS and GSH, cell viability, and cell apoptosis. Aptamer for Proteins Capture. For protein capture, aptamer was coated into the reaction chamber of the protein detection component by avidin-biotin binding. The reaction chamber was incubated with 100 µg/mL avidin at 37 °C for 30 min. After washing 5 times by PBS, various concentrations of biotin-modified aptamers were induced to the reaction chamber. To ensure that the capture aptamers could be bonded to the surface of reaction chamber, incubation time ranging from 0 to 60 min was investigated. After the aptamers were immobilized, 100 mM TE buffer was used to wash out the unbound aptamers for three times. After washing, the microchip filled with TE buffer was stored at 4 °C. RCA Reaction and Protein Detection by Functional Nucleic Acid. Different concentrations of VEGF165, and PDGF-BB were incubated inside the reaction chamber pre-coated with aptamers for 10 min, allowing for the specific aptamer-protein interaction. PBS buffer was then used to wash the reaction chamber at the flow rate of 1.0 µL/min for 10 min. After proteins were captured, the reaction chamber was filled with functional nucleic acid (FNA, the sequences in Table S1) and incubated for 10 min. After the FNA was bonded to protein, TE buffer with 200 mM MgCl2 was used to wash out the unbound FNA. 30 µM 5’-phosphorylated template DNA and 10 U/µL T4 DNA enzyme dissolved in 1 × ligase buffer were introduced into the reaction chamber. To ensure a complete binding of 5’-phosphorylated circle template DNA to the primer of FNA, the microchip was incubated at 37 °C for 30 min, and excess template DNA was washed off by TE buffer with 200 mM MgCl2. Subsequently, RCA reaction solution containing 1 × Phi29 DNA polymerase buffer, 10 mM dNTP, 0.01 mg/mL BSA and 10.00 U/µL of Phi29 DNA polymerase was introduced into the reaction chamber, followed by incubation at 37 °C for 1.5 h. The reaction was terminated by heating at 65 °C for 5 min, and washing by PBS for three times. After SYBR Gold labeling, the fluorescence

intensity was detected by fluorescence spectrophotometer. Fluorescence photographs were taken by a fluorescence microscopy. Cell Proliferation, Viability, Apoptosis, ROS and GSH Level Analysis. Cell proliferation was monitored by CCK-8, and the data were collected by microplate reader. Cell viability was determined by Calcein-AM/EthD-1 staining (live/dead kit). Live/dead solution was prepared by diluting 0.5 µL calcein AM and 2.0 µL ethidium homodimer into 1 mL PBS buffer. Incubation was carried out at 37 °C for 30 min after stained with the live/dead kit. Cell culture channels were then rinsed with PBS for three times. After washing, fluorescence images were taken by CCD camera on a fluorescence microscopy. For determination of cell apoptosis, ROS or GSH level, cells were incubated with 100 µM Hoechst 33342, 100 µM DHE (λex = 535 nm, λem = 610 nm), or 200 µM 2, 3-naphthalene dicarboxaldehyde (NDA, λex = 460 nm, λem = 530 nm) at 37 °C for 30 min, followed by washing with PBS for three times, before observation under the fluorescence microscopy. Analysis of Cell Cycle by Flow Cytometry. For cell cycle assay, paclitaxel-treated cells were trypsinized and collected into tubes. The control experiment was performed using cells without paclitaxel treatment. The collected cells were centrifuged at 1200 rpm/min for 3 min and washed three times by PBS. Cells were then fixed with 75 % ice-cold ethanol for 1 h at 4 °C and centrifuged again. The collected cells were washed and resuspended in 500 µL PBS containing 50 µg/mL propidium iodide (PI), 50 µg/ mL RNase A and 3.8 mM sodium citrate. The cells were incubated at 37 °C for 30 min, and then washed and resuspended in PBS. Finally, the labeled cells were processed in flow cytometry to analyze cell cycle. Chip-ESI-MS for Paclitaxel Detection. To study the influence of matrix, various concentrations of paclitaxel were spiked into cell medium and treated by micro-SPE before detection by ESI-Q-TOF MS. The micro-SPE was pre-conditioned by washing with 100 µL methanol followed by 100 µL water. After loading of cell medium containing paclitaxel, the

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Figure 2. Cytotoxicity of paclitaxel in co-cultured CaSki cells and HUVCEs. (a) Fluorescent images of co-cultured CaSki cells treated with paclitaxel (5.00 ng/mL) for 2-5 days and stained with Calcein-AM/EthD-1 (live/dead kit). The control group is the co-cultured CaSki cells without paclitaxel treatment. Green fluorescence indicates the live cells, and red fluorescence indicates the dead cells. Time-dependent viability of co-cultured CaSki cells (b) and HUVECs (c) treated with paclitaxel at different concentrations from 0 to 1000 ng/mL. (d) Long-term viability of CaSki cells treated with 5.00 ng/mL paclitaxel, with or without co-culture with HUVECs. The data are shown as Mean ± Standard Error Mean (SEM), n=3, **p