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Simultaneous Assay of Oxygen-Dependent Cytotoxicity and Genotoxicity of Anticancer Drugs on an Integrated Microchip Lili Li, Yaqiong Li, Zixing Shao, Guoan Luo, Ming-Yu Ding, and Qionglin Liang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02070 • Publication Date (Web): 31 Aug 2018 Downloaded from http://pubs.acs.org on September 3, 2018
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Analytical Chemistry
Simultaneous Assay of Oxygen-Dependent Cytotoxicity and Genotoxicity of Anticancer Drugs on an Integrated Microchip Lili Li1,2, Yaqiong Li1, Zixing Shao1, Guoan Luo1, Mingyu Ding1, Qionglin Liang1,* 1. MOE Key Laboratory Bioorganic Phosphorous Chemistry & Chemical Biology, Beijing Key Lab of Microanalytical Methods & Instrumentation, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China 2. Department of Pharmacy, Beijing Pharmaceutical University of Staff and Workers, Beijing 100079, P. R. China ABSTRACT: Oxygen deprivation is a common feature in a variety of cancer tissues and associated with tumor progression, acquisition of anti-apoptotic potential and clinical therapeutic resistance. Thus, it has been aroused much interest to develop new platform or approaches of activity assays to impact on the hypoxic microenvironment and oxygen-dependent drug responses in order to improve the productivity of new drug discovery. In this study, an integrated microsystem is established to combine the cytotoxic and genotoxic tests together for continuous multiple measurements under mimicking hypoxic tumor microenvironment. We fabricated a double-layer chip device by combining single-cell-arrayed agarose layer with microfluidics-based oxygen gradientgenerating layer using a PDMS membrane. Using tirapazamine (TPZ) and blemycin (BLM) as model anticancer drugs, we demonstrated its application and performance in single cells loading, cell cultivation and subsequent drug treatment as well as in situ analysis of oxygen-dependent cytotoxicity and genotoxicity of anticancer drugs. The results demonstrated the opposite oxygendependent toxicity of TPZ and BLM, which also indicated that the formation of DNA breaks is related with cell apoptosis. Compared with the traditional assays, this device takes advantage of microfluidic phenomena to generate various oxygen concentrations while exhibiting the combinatorial diversities achieved by the single cell microarray, offering a powerful tool to study single cell behaviors and responses under different oxygen conditions with desired high-content and high-throughput capabilities.
It is known that in vitro drug research contributes to the evaluation of anticancer efficiency, cellular resistance and malignant potential in clinical practice.1,2 Cell based assays in early stages form fundamental practices in drug screening process. The investigation of cellular and molecular events is of special importance not only in providing pharmaceutical information, but also economizing work force and costs for further clinical trials. Recently, cell-to-cell heterogeneity in tumor tissue has been reported as a major driver in cancer evolution, progression, and emergence of drug resistance.3,4 The increasing evidence of heterogeneous responses obtained from single cells arouses the interests of new approaches capable of revealing effective information at both individual and population level. Although in vitro assays are widely used for screening purposes to provide meaningful parameters in biological and biomedical research, the conventional studies are often found to be tedious and costly.5,6 Additionally, cellular analysis is typically performed by using image-based techniques. However, assays on single cells are hard to perform due to the overlap of cells as well as low throughput. Some other approaches are limited by its impossibility in performing in situ analysis and dynamic monitoring of cells. Most recently, the rapid progress in biochip technologies, in combination with the basic in vitro quantifiable assays, showed prominent feasibility for performing single cell analysis with high throughput and sensitivity.7-10 For example, Toriello et al have developed an integrated microfluidic system which combine several steps such as single cell selection and capture, enzymatic reaction and quantitative detection all on a single chip.11 Brouzes et al develop a fully integrated droplet-based micro-
fluidic platform for performing a mammalian cell cytotoxicity analysis at a very high throughput.12 Novak et al have realized the genetic analysis process of single cells including DNA extraction from encapsulated cells as well as subsequent DNA amplification and detection in a microfluidic chip.13 Previous work of our group have demonstrated that microarray technique can be used to construct 3D single cell culture and perform in situ cytotoxic and genotoxic analysis of crosslinking agents in one time experiment.14 It is obviously noted that the high degree of on chip integration revolutionize our ability to perform multi-step manipulations of single cells in a largescale, fast and easy-to-use device with high flexibility.15,16 Moreover, statistically meaningful data can be collected from multiple individual cells by using biochip technique, therefore high throughput analysis is achieved. The advances in microfluidic cell-based systems have also led to a new class of in vitro tool for spatiotemporal control of microenvironmental factors and stimuli.17,18 Hypoxia is a dominant state in tumor tissues, which contain a significant fraction of microregions which are chronically or transiently hypoxic.19,20 The low overall oxygen levels was demonstrated to be exist in a variety of human solid tumors, including those of the brain, head and neck, breast and cervix tumors.21 The vasculature of solid tumors is often found to be highly irregular and tortuous, making the oxygen diffusion and nutrients transportation much less efficient to cells reside at distances from vessels.22,23 The adaptation to enable cell survival was occurred following the decrease in oxygen availability. The anticancer activities of some chemotherapeutic agents, such as cisplatin, 5-fluorouracil and melphalan, have already been
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found to be reduced under relative hypoxic conditions.24 Despite this fact, oxygen content is still an overlooked impact in constructing physiologically realistic environment for drug analysis and screening. Therefore, rational in vitro microfluidic systems aimed to clarify hypoxia-related toxic effects and cell resistance are required. Funamoto et al has introduced a microfluidic device which could produce an oxygen gradient by flowing predefined gas mixtures into channels to monitor the cellular behavior in a three-dimensional (3D) gel scaffold.25 Another microfluidic chip developed by Tung and his colleagues has been demonstrated to have the ability in generating oxygen gradients by using a pair of chemical reactions in adjacent chemical reaction channels.26 However, the relevant cell-based microfluidic assays only showed the cytotoxicity results from a population of cells in chips. Substantial evidence shows that the survival chance of cells can be directly influenced by cellular heterogeneity due to different gene expression profiling at single cell levels.27 Methods which use average responses from a population often mask the difference between individual cells. Furthermore, there has been none of report on the assay of oxygen-dependent genotoxicity of anticancer drugs. The oxygen-dependent single cell cytotoxicity and genotoxicity should be both taken into consideration to get a better understanding of the antitumor mechanism and cell resistance. In this paper, we constructed a biochip device by using microfluidic and microarray technique to simulate the hypoxic microenvironment in tumor tissue and investigate the oxygendependent cytotoxicity and genotoxicity of chemotherapeutic drugs. We demonstrated its application in single cells loading, cell cultivation and subsequent drug treatment as well as in situ analysis of oxygen-dependent cytotoxicity and genotoxicity using tirapazamine (TPZ) and blemycin (BLM) as model drugs. The combinatorial diversities achieved from single cell microarray provided direct evidence that the exploited device could be used as a powerful tool in performing in situ drug screening and analysis at both individual and population levels with desired high-content and high-throughput capabilities. Moreover, the corresponding results offered valuable information in clarifying the hypoxia-related molecular mechanism and the cellular heterogeneity.
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purchased from the Cell Center of Peking Union Medical College. All other chemicals used in this study such as Na2EDTA, NaOH, NaCl, K2HPO4, KH2PO4 were obtained from Beijing Chemical Works (Beijing, China). Deionized water was from Milli-Q purification system of Millipore Corporation (Bedford, MA, USA). Cell Culture and Chemical Treatment SF767 cells were grown in MEM medium containing 10% v/v FBS and 1% v/v antibiotic-antimycotic. A549 cells were cultured in RPMI 1640 medium supplemented with 10% v/v FBS and 1% v/v antibiotic-antimycotic. The stocks were both cultured in T25 cell culture flasks at 37°C in 5% CO2 atmosphere and passaged by dissociation with 0.25% trypsin-EDTA. Cells were treated with two different anticancer drugs, TPZ and BLM (Toronto Research Chemicals Inc., Ontario, Canada). TPZ was prepared in DMSO at a concentration of 20 mM. BLM was dissolved in PBS solution at a stock concentration of 10 mM. The stock solutions were both stored at -20°C until immediately prior to use. Microfluidic Device Fabrication Figure 1A displayed the double-layer PDMS structure of the developed microfluidic device. The top layer was the cell culture reservoir and the bottom layer was the chemical reaction channel. The PDMS membrane with the thickness of 100 µm was sandwiched between two layers to prevent cells directly contacting the chemical reactants and maintained excellent oxygen permeability. A piece of glass coverslip was placed on the top layer to prevent oxygen exchange between the atmosphere and the cell culture medium. Briefly, the mixture of PDMS precursor and curing agent (10:1, v/v) was first degassed and poured onto blank masters for curing (70°C for 3 h) to form the top layer with the thickness of 5 mm. The length and width of the cell reservoir in top layer was set as 1.5 cm and 1 cm. The bottom layer with gradient-generating microfluidic channels were designed using AutoCAD software (Autodesk Inc., San Rafael, CA, USA). Then standard soft lithography methods and rapid prototyping techniques were performed to obtain the features of 100 µm height.
EXPERIMENTAL SECTION Materials and Reagents Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) dTris (4,7-diphenyl-1,10 -phenanthroline) ruthenium dichloride (II) complexes, sodium lauryl sarcosinate, dimethyl sulfoxide (DMSO), trichloro-(1H, 1H, 2H, 2H-perfluorooctyl) silane, normal melting point agarose (NMA) and low melting point agarose (LMA) plus all electrophoresis reagents and buffers were purchased from Sigma Aldrich (St. Louis, MO, USA). GelBond film was purchased from Lonza (Walkersville, MD, USA). Polydimethylsiloxane (PDMS) Sylgard 184 was purchased from Dow Corning (Midland, MI, USA). Phosphate buffered saline solution, eagle’s minimum essential medium (MEM), RPMI medium 1640, fetal bovine serum (FBS), penicillin-streptomycin, 0.25% trypsin-ethylene diaminetetra acetic acid (EDTA) solution and Calcein-AM/EthD-1 (Live/Dead Viability/Cytotoxicity Kit) were supplied by Invitrogen Corporation (Carlsbad, CA, USA). Cell counting kit-8 (CCK-8) was from Dojindo Corporation (Kumamoto, Japan). Human SF767 glioma cells and human A549 lung cancer cells were
Figure 1. The structure of the microchip. (A) The schematic of the fabricated microchip. (B) The cross sectional view. (C) Photo of the integrated microchip device, a microarray agarose chip was embedded in the cell culture reservoir of the top layer.
Figure 1B displayed the cross section view of the developed microfluidic chip. A microarrayed agarose chip with single cell embedded was settled in the cell culture well. The pattern in microarray chip was created by using the silicon stamp con-
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Analytical Chemistry
sisting of micrometer-scale posts. The width and depth of the microposts could be varied to optimally accommodate different cell types. In order to perform long-term cell cultivation, the fibronectin solution with the concentration of 50 µg/mL was used in the per-treatment of agarose chips for surface functionalization.28,29 Figure 1C illustrated the integrated microchip device. After gel solidification, the agarose chips with microarrayed single cells was taken into the cell reservoir for drug exposure followed by next cytotoxicity and genotoxicity tests under different oxygen tensions. Oxygen Gradient Generation and Characterization The investigation of generating oxygen gradients in the fabricated device was performed by using a spatially confined chemical reaction to achieve feasible control of oxygen contents in cell culture reservoir. Firstly, pyrogallol (benzene1,2,3-triol, C6H6O3) (Alfa Aesar, Ward Hill, MA, USA) and sodium hydroxide were introduced into chemical reaction channels from two inlets at the concentration of 200 mg/mL and 1 mol/L. As a strong reducing agent, pyrogallol absorbed oxygen from the ambient environment in the presence of alkaline solution. The high gas permeability of PDMS allowed oxygen to penetrate from the top layer into the microfluidic flow, thereby forming an oxygen gradient along the cell culture chamber. To calibrate the oxygen gradients, an oxygen sensitive fluorescent dye, tris(4,7-diphenyl 1,10phenanthroline) ruthenium dichloride (II) complexes (TCI, Tokyo, Japan) was exploited in this experiment.30 The oxygen gradients could be characterized by observing the changes of fluorescence intensity in cell culture well after scavenging reactants were introduced into the chemical reaction channels. 31 The fluorescence measurement was performed by using an inverted fluorescence microscope (IX81, Olympus, Japan) with the excitation light passing through a 470 ± 20 nm optical filter. Images were analyzed by using the MetaMorph 7.7 software (Universal Imaging Corp., West Chester, PA, USA). The excitation wavelength for the fluorescent indicator was set at 455 nm and the exposure time for fluorescence imaging was 30 ms. The oxygen gradient-dependent fluorescence intensity was measured by recording the fluorescence images at different positions of the cell culture chamber. A series of flow rate tests were performed to estimate if the flow rates might influence the oxygen gradient dynamics. The fluorescence intensity was converted to an oxygen tension based on the SternVolmer equation: ࡵ ࡵ
= + ࡷ ሾࡻ ሿ
(1)
where I and I0 refer to the fluorescence intensities in oxic and hypoxic atmospheres, respectively. Kq is the quenching coefficient which can be calculated as follows: ࡷ =
ࡵ ࡵ
−
(2)
where I100 refers to the fluorescence intensity with saturated oxygen contents ([O2]=100%). The fluorescence intensities of I100 and I0 in the cell culture channel were measured and used for calculating Kq.32 The validation experiment was performed three times to check the reproducibility by three independent microfluidic chips. The oxygen concentration was estimated based on the measured fluorescence intensities and calculated Kq. Thereafter, the oxygen gradient profiles were constructed. Besides, numerical simulation was also performed to estimate the device performance in generating oxygen gradient across the cell culture chamber. Finite element analysis (FEA) was
conducted using the COMSOL Multiphysics 5.3a software (COMSOL Inc., Burlington, MA, USA) for comparison. Cytotoxicity and Genotoxicity Testing on Agarose Chips The procedures of cytotoxicity and genotoxicity testing on agarose chips were performed by following the prior work with some modifications.14 Cell viability assessment was performed based on the Live/Dead staining protocol. Firstly, the inlets of microfluidic channels were injected with continuous NaOH and pyrogallol solution after the assembling of single cell agarose chip into the cell culture reservoir. The microarrayed SF767 and A549 cells were exposed to cell culture medium containing TPZ and BLM for 6 h with the drug concentration of 10, 20, 50 and 100 µM, respectively. The control groups without drugs were set for comparison. After removing the medium and washing with PBS, 1 mL of the Live/Dead staining solution containing 1 µL Calcein AM and 4 µL EthD1 were introduced into each cell culture reservoir. Then the single cell arrayed chips were immersed in the stain for 15 min and rinsed three times with PBS solution. Viable cells were stained with Calcein-AM, which could be converted into green fluorescent Calcein by intracellular esterase, while the dead cells took up the EthD-1 dye and resulted in red. Fluorescence images was collected from an inverted fluorescence microscope (IX81, Olympus, Japan) with MetaMorph 7.7 imaging software (Universal Imaging Corp., West Chester, PA, USA). The excitation/emission of 495/515 nm was set for Calcein and 525/610 nm for EthD-1. The scanned images were analyzed with the use of ImageJ software (National Institute of Health, Bethesda, MD, USA) to threshold and count live or dead cells. Multiple images were acquired automatically by using the scan slide module and stitched seamlessly together. After the cell survival measurement, the chips were immediately immersed into cell lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% N-lauroyl-sarcosine, pH=10; supplemented before use with 1% Triton and 10% DMSO) and lysed overnight at 4°C for further genotoxicity testing. The procedures were performed as standard comet assay. Fluorescent images of stained single cells were digitally captured on a fluorescence microscope system (IX81, Olympus, Japan) with a barrier filter of 590 nm and subsequently analyzed by using Comet Assay IV (Perceptive Instrument, UK). Multiple images were acquired automatically and stitched seamlessly together. The background image was taken from the blank agarose chip and subtracted from the image of comets. The comet macro automatically sets a threshold value to distinguish the comet from the background. The level of DNA single-strand breaks were quantitated as DNA in tail (tail DNA %). For every region with different oxygen content, at least 100 cells were scored to analyze the oxygen-dependent genotoxicity. All the experiments were repeated three times and the error bars represented the standard deviation of three trials. Traditional Cytotoxicity and Genotoxicity Testing The cytotoxicity of TPZ and BLM were estimated in SF767 and A549 cells by traditional CCK-8 assay. Cells were digested with 0.25% trypsin-EDTA from flasks and resuspended, and aliquots (100 µL) were seeded (1×104cells) into 96-well clear flat-bottomed microplates. After incubating for 24 h, the cell culture medium was replaced by freshly prepared drugcontaining medium of TPZ or BLM with different drug concentrations (10, 20, 50 and 100 µM). Then the 96-well plates were maintained in a humidified atmosphere containing 5% CO2 under normoxia condition for 6 h. The growth media
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without drugs was set as control group. Hypoxic treatment was carried out by using drug-containing oxygen free medium in an incubator with 1% O2, 5% CO2 and 94% N2. Afterwards, cells were washed with PBS, and CCK-8 solutions diluted in FBS-free medium were added immediately. The templates were placed in an incubator at 37°C for 1 h. Absorbance was monitored at 450 nm with a reference absorbance of 630 nm by using a microplate reader (BioTek Instruments Inc., Winooski, VT, USA). Each experiment was repeated three times. The results were expressed as the percent ratio of viable cells. The genotoxicity was investigated by using alkaline comet assay. SF767 and A549 cells were dissociated from flasks and seeded in 6-well plates at a density of 5×104 cells/mL, respectively. The stock solutions of TPZ and BLM were directly added into cell culture medium to achieve the final concentrations of 10, 20, 50 and 100 µM. Cells in the 6-well plates were then treated under corresponding normoxia (21% O2) or hypoxia (1% O2) condition for 6 h in the humidified atmosphere of 5% CO2 at 37°C. Negative controls were treated with serum free medium under same conditions. After drug treatment, cells were dissociated and isolated by centrifugation for 5 min at 1000 rpm. Traditional slides were prepared as previously described.14 For each condition, pictures of 100 randomly selected comets from two slides were captured using a fluorescence microscope. All the experiments were independently replicated three times. Data analysis was carried out by Comet Assay IV (Perceptive Instrument, UK). Statistics Analysis All the statistical analysis was performed by using SPSS Statistics version 17.0 (SPSS Inc., Chicago, IL, USA) and Excel 2013 (Microsoft Corp., Redmond, WA, USA). The data are presented as the mean values with the standard deviation (mean±SD). Comparisons were performed by using Student’s t-tests. Corrected P
