Orthogonal Screening of Anticancer Drugs Using an Open-Access

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Orthogonal Screening of Anti-cancer Drugs using an Open-access Microfluidic Tissue Array System Dongguo Lin, Peiwen Li, Jinqiong Lin, Bowen Shu, Weixin Wang, Qiong Zhang, Na Yang, Dayu Liu, and Banglao Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02021 • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 23, 2017

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Analytical Chemistry

Orthogonal Screening of Anti-cancer Drugs using an Open-access Microfluidic Tissue Array System Dongguo Lin,1,2,3§ Peiwen Li,1§ Jinqiong Lin,1§ Bowen Shu,1,2,3 Weixin Wang,1 Qiong Zhang,1 Na Yang,1,2,3 Dayu Liu,1,2,3* Banglao Xu1,2,3 1.

Department of Laboratory Medicine, Guangzhou First People’s Hospital,

Guangzhou Medical University, Guangzhou 510180, China 2.

Department of Laboratory Medicine, The Second Affiliated Hospital of South China

University of Technology, Guangzhou 510180, China 3.

Clinical Molecular Medicine and Molecular Diagnosis Key Laboratory of

Guangdong Province, Guangzhou 510180, China

Author Contributions §Authors D. Lin., P. Li. and J. Lin. contributed equally and share the first authorship. * Corresponding author. 1, Panfu Road, 510180, Guangzhou, China. Fax: 86-20-81048058; Tel: 86-20-81048084; E-mail: [email protected] KEYWORDS: Microfluidic Chip, Nanoporous Membrane, Tissue Microarray, Microenvironment Reconstitution, Anti-cancer Drug Testing, Drug Combination 1

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Abstract Screening for potential drug combinations presents significant challenges to the current microfluidic cell culture systems, due to the requirement of flexibility in liquid handling. To overcome this limitation, we present here an open-access microfluidic tissue array system specifically designed for drug combination screening. The microfluidic chip features a key structure in which a nanoporous membrane is sandwiched by a cell culture chamber array layer and a corresponded media reservoir array layer. The microfluidic approach takes advantage of the characteristics of the nanoporous membrane: on one side, this membrane permits the flow of air but not liquid, thus acting as a flow-stop valve to enable automatic cell distribution; on the other side, it allows diffusion-based media exchange and thus, mimics the endothelial layer. In synergy with a liquid transferring platform, the open-access microfluidic system enables complex multi-step operations involving long-term cell culture, medium exchange, multi-step drug treatment, and cell viability testing. By using the microfluidic protocol, a 10×10 tissue array was constructed in 90 s, followed by schedule-dependent drug testing. Morphological and immunohistochemical assays indicated that the resultant tumor tissue was faithful to that in vivo. Drug testing assays showed that the incorporation of the nanoporous membrane further decreased killing efficacy of the anti-cancer agents, indicating its function as an endothelial layer. Robustness of the microfluidic system was demonstrated by implementing a 3-factor 3-level orthogonal screening of anti-cancer drug combinations, with which 67% of the testing (9 vs. 27) was saved. Experimental results demonstrated that the microfluidic tissue system presented herein is flexible and easy-to-use, thus providing an ideal tool for performing complex multi-step cell assays with high efficiencies.

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Introduction Combination chemotherapy, which was inspired in the 1960's and in the 1970's, was found to be more effective than the mono-drug strategy. The rationale for combination chemotherapy is to use drugs that work by different mechanisms, thereby decreasing the likelihood that drug-resistant cancer cells will develop1,2. When drugs with different effects are combined, each drug can be used at its optimal dose without causing intolerable side effects. In vitro cell culture assays are expected to play an important role in identifying potential effective drug combinations to achieve the maximum efficacy. In order to identify an appropriate sensitizer and find effective drug combinations, a large amount of experiments to screen variety of concentrations/combinations are required. Therefore, a highthroughput drug screening system is highly desirable for such applications. Microfluidic chip has been accepted as an advantage tool for cell biology research3-5. Compared to the conventional cell assaying systems, the emerging microfluidic chip technology offers great advantages in highthroughput drug screening6,7. Besides the common advantages of microfluidics, including highthroughput, lower reagent consumptions, and potential for integration, the most influential benefit of using microfluidics for cell-based drug testing assays is the ability to reconstitute cell microenvironment at microscales8. On one hand, microfluidic chip facilitates the transition from 2D to 3D cell culture, which is an important step in a trend towards better biomimetic models9-11; on the other hand, microfluidic technology allows precise control over fluids in micrometer-sized channels, thus has become a valuable tool to mimic the vascular vessel. The combination of 3D cell culture with microfluidic networks on a microchip offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system12-18. The majority of microfluidic cell culture systems employ perfusion systems19-28. The perfusion of fluid within microchannels with comparable size to the 3



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capillary system mimics the fluidic behavior in vivo. Although previously introduced perfusion microfluidics was very successful, there are still several challenges in culturing cells under flow conditions. First, it is difficult to precisely control and measure pH, oxygen, and nutrition levels at specific locations inside the micro-channel in real time; this often requires careful validation experiments and feeding schedule calibrations for every device; Second, these continuous perfusion cell culture systems often involve high reagent consumption and dead volume; Third, the majority of perfusion systems did not offer the flexibility to conduct complex multistep cell assays. In addition, perfusion microfluidic systems required multiple tubing connections and syringe pumps for operation which results in complexity in tubing and connection parts29-31. To these concerns, screening for potential drug combinations presents significant challenges to the current microfluidic cell culture systems. To solve the limitations mentioned above, open-access chip design for cell culture has been proposed. In this design, the cells are cultured in open-access reservoirs,

in

which

different

reagents

and

stimulus

can

be

added

simultaneously or sequentially, to provide a stable microenvironment. Thus, this design facilitates the media exchange in cell culture chambers and makes it possible to maintain and monitor cells continuously. Open-access chip uses static cell culture thus facilitates certain concentrations of chemical stimuli to be presented in certain time frames. As an example, Groot et al.32 presented a surface-tension driven open microfluidic platform for hanging droplet cell culture. Jowhar et al.33 developed an open microfluidic device to provide quantitative information on both the gradient and morphological changes that occurred as cells crawled through various microfabricated channels. Lo et al.34 reported the application of microfluidic gradient generation in an open-well culture model, in which a gradient of gas is delivered via diffusion through a gas permeable substrate that separates cells from the gas microchannels below. Similarly, Keenan et al.35 reported a method for studying 4


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gradient-induced neutrophil desensitization based on an open microfluidic chamber. Open microfluidic devices have also been applied for chemotaxis36 and cell migration37 studies. The use of open-well devices overcame problems that many current devices have been plagued with, such as complicated sample/reagents loading, media evaporation, and channel blockage by air bubbles. Nevertheless, as these chips are free of microfluidic networks, large-scale cell distribution is usually inconvenient. Therefore, analysis throughputs with open-access microfluidics were usually limited. To solve this issue, Zhu et al.38 recently developed a semi-open droplet array platform, in which multi-step operations involving picoliter-scale liquid handling, analysis, and screening were achieved by liquid transferring using a tapered capillary. This made the system capable of performing complex drug-testing assay in highthroughput format39. Inspired by the concept of open-access microfluidics, a novel drug screening protocol using open-access microfluidic tissue arrays was developed in the current study. The microfluidic chip contains a nanoporous membrane that separates the top layer with media reservoirs from the bottom layer with cell culture chamber arrays. The formation of cell culture arrays can be achieved simply by infusing cell suspension (in sodium alginate) into the cell culture chamber arrays. In synergy with the liquid transfer workstation, multi-steps involved with drug testing, including medium addition, hydrogel gelation, schedule-dependent drug dosage and stimulation, and cell viability testing can be implemented in a programmed manner (Fig 1). Within the on-chip cell culture scheme, the hydrogel-supported 3D cell culture mimics the solid tumor, while the nanoporous membrane mimics the endothelial layer (Fig 1c). Therefore, the presented open-access microfluidics is helpful to reconstitute a biomimetic microenvironment. Orthogonal screening of anti-cancer drugs was performed to demonstrate the flexibility of the open-access microfluidic tissue array system. Orthogonal design is an experimental design used to test the comparative effectiveness of multiple intervention components, which can be used to search for the best experimental 5



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conditions through calculating the response of variables. The critical advantage of orthogonal design (relative to typical controlled trials) is that it allows the researcher to test the effectiveness of many interventions simultaneously in a single experiment with far fewer experimental units than it would take to exhaust all of the possible intervention combinations. Therefore, the orthogonal experimental design is a powerful tool enabling a massive parallel interrogation of orthogonal agents to determine an optimal drug combination prescription. Orthogonal screening with the open-access microfluidic tissue-array system consisted of cell viability testing of breast cancer cells and hepatic cells, with the aim of selecting a drug combination to achieve the maximum killing efficacy to eliminate cancer cells with minimum unwanted off-target effects. Robustness of the open-access microfluidic tissue system was demonstrated by implementing the orthogonal screening. We demonstrated feasibility of the microfluidic approach for performing tissue-based screening with complex and multi-step operation procedures. Experimental Sections Microfluidic device fabrication and assembly The microfluidic device contains a microchip and a liquid transfer workstation (Fig.1a). The microchip was fabricated using the standard soft lithography protocol40. Microfluidic network layouts were designed using CorelDRAW software and they were printed on transparencies using a high-resolution printer. The molds were fabricated by spin-coating SU-8 3025 negative photoresist (Microchem Inc., USA) on clean glass wafers. The coated wafers were then soft-baked for 5 min at 65°C followed by 20 min at 95°C, before exposing the photoresist through the photomasks. After UV exposure, the substrates were post-baked for 1 min at 65°C followed by 10 min at 95°C, and then developed using the SU-8 developer (Microchem Inc.). The PDMS microchip was cast from the glass mold masters. A mixture containing the silicone elastomer and the curing agent (10:1 weight ratio) was poured onto the master and baked at 70°C for 2 h. After peeling them off of the glass masters, holes 6


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were punched on the PDMS replicas, which served as inlets and outlets of the microfluidic devices. As is shown in Fig.1b, the microfluidic chip contains three layers: a top PDMS layer with 10×10 via-hole arrays, a bottom PDMS layer with tandem microchamber arrays aligned to the via-hole arrays, and a middle layer of nanoporous polycarbonate (PC, 100-nm pore size, Whatman, UK). The PC membrane was bonded to the PDMS layers following a protocol reported by Kiana et al.41 (SI, experimental S1). The microfluidic chip has 10 modules, with 10 tandem cell culture units in each one (Fig. 1b). Each microfluidic module has an inlet for cell suspension introduction, and an outlet for waste disposal. Detail structure of the microchip is shown in Fig. S1.

a

b

c

Fig.1 a. The microfluidic system developed for anti-cancer drug combination screening. An open-access format microchip is installed on a liquid transferring platform equipped with a

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syringe pump connected to a capillary, which is responsible for liquid addition or removing. b. Construction of the open-access microchip, which contains three layers (see enlarged view): a top PDMS layer with via-hole arrays, a bottom PDMS layer with a microchamber array and linking channels, and a nanoporous PC membrane sandwiched between these two PDMS layers. c. Tumor microenvironment reconstitution on the microchip.

Liquid transfer workstation The microchip and the pipette tips were autoclaved, and dried in a laminar flow hood before the experiment. The microchip was mounted on the liquid transfer workstation while liquid transfer was needed. The precise control of liquid manipulations is accomplished using the liquid transfer workstation, which is equipped with a syringe pump connected to a pipette tip. The stage of the liquid transfer workstation has a linear speed between 0.50 and 50 mm/s over the moving range of 800 mm (L) × 600 mm (W) × 80 mm (H), with a positioning resolution of 0.20 mm and precision of liquid volume of 0.1 µL. With the function of x−y−z stage movement controlled by software, liquid of certain volume can be deposited in or aspirated from a certain reservoir of the microchip. After the operation, the microchip was enclosed in a box and put back into the incubator for long-term culture (Fig.1a). Cell seeding and cell culture Human breast cancer cells (MCF-7) and hepatic cells (LO 2 ) were maintained in DMEM medium (Sigma-Aldrich, USA) and 10% FCS (Gibco, Invitrogen Corp, USA). Cells were grown to ~85% confluence in cell culture flasks, and then harvested using 0.25% Trypsin EDTA (Gibco, Invitrogen Corp.) for the microchip experiments. The process of cell culture and the drug combination screening experiment are illustrated in Fig.2. The harvested cells were resuspended in DMEM media containing 2% sodium alginate (Sangon, Shanghai, China) to a final density of 3×106 cell·mL-1. Suspension of MCF-7 and LO 2 cell was introduced into the microchip from the inlet driven by the syringe pump (Longer, Baoding, China) at a flow rate of 30 µL·min-1,

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respectively. The cell suspensions were sequentially allocated into the cell culture chambers through the flow channel. After all of the chambers were filled, the excessive liquid was expelled from the outlet. After cell distribution, calcium solution (40 mmol·L-1) was added to the top reservoirs and kept there for 15 min. Then, the calcium solution was replaced by 10 μL of DMEM medium. Finally, the microchip was transferred to a CO 2 incubator. The cell culture medium in the top reservoirs was replenished every day.

Fig.2 Schematic diagrams showing operation of the open-access microfluidic chip. a. Cell distribution. (I) Cell suspension (3×106 cell·mL-1) is perfused into the microchip; (II) the microchambers are sequentially filled with cell suspension; (III) all of the microchambers are filled with cell suspension; and (IV) excessive fluid is expelled from the main channel. b. Multi-step operations for cell culture and drug stimulation. (I) Calcium is added to the top wells and alginate calcium hydrogel is formed in the bottom chamber upon contacting calcium; (II) cell culture medium (with or without anti-cancer agents) is added as designed; (III) live/dead dyes are added to the top wells; and (IV) the microchip is mounted on a microscope for cell viability testing.

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Construction of tissue arrays Upon contacting Ca2+, alginate calcium hydrogel was formed through polymerization and served as a scaffold to support the 3D cell culture. Within the microchip culture scheme, the cells cultured in 3D mimic the tumor parenchyma; the hydrogel supports, nourishes, and protects the cells, mimicking the mesenchyma; and the PC membrane, which has a pore size similar to the endothelial cell gap of the tumor vascular, mimics the endothelial layer (Fig. 1c). Therefore, artificial tissue arrays are constructed on the open-access microfluidic chip. Drug treatment After visual confirmation of tumor spheroid formation on the third day, the drug-free culture media was replaced with 10 μL of DMEM medium containing an anti-cancer agent. The drug concentrations used for the stimulation of single drugs and drug combinations are listed in Table 1 and Table 2, respectively. Table 1 Serial drug concentrations used for single-drug testing

Concentrations (µmol•L-1) Drug 1

2

3

4

5

6

Doxorubicin

10

5

2.5

1.25

0.625

0.312

Paclitaxel

10

5

2.5

1.25

0.625

0.312

Cisplatin

50

25

12.5

6.25

3.125

1.56

Drug combination screening using the open-access microfluidic chip included several steps: (1) the old medium in the reservoirs was replaced by fresh medium containing different concentrations of the first drug. The old media was gently aspirated from the reservoir and disposal into the waste reservoir through the pipette tip was manipulated by the liquid transfer workstation. Then, the fresh media with the first drug was aspirated and added into a designated reservoir by the liquid transfer workstation as well. The tissue array chip with the first drug dosage was placed in the incubator for 24 h. (2) after the stimulation of the first drug, the reservoir was washed twice with 3

10


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μL of PBS solution to avoid carryover of the former drug. Then, fresh medium containing the second drug was added to the reservoirs. (3) after a 24-h incubation and stimulation with the second drug, the drug media in the reservoir was replaced with fresh culture medium containing the third drug, and the chip was incubated for another 24 h. Drug testing with 2D and traditional 3D cells cultures were also conducted as comparisons. In drug testing with 2D cell cultures, MCF-7 and LO 2 cells (1×106∙mL-1)

grown on 96-well plate after 24 h were exposed to anti-cancer agents. In drug testing with traditional 3D cell cultures, the harvested cells were resuspended in DMEM media containing 2% sodium alginate to a final cell density of 3×106 cell·mL-1.

Suspension of MCF-7 and LO 2 cells was added into the microwell, then 40 mmol∙L-1 CaCl 2 was added to form alginate calcium hydrogel. Cells were exposed to anti-cancer agents after three days of cell culture. During cell culture, the old medium in the microwells was replaced by fresh medium every day. Multicellular tumor spheroid imaging To validate the formation of the multicellular tumor spheroid, the on-chip cultured cells were imaged by confocal laser scanning on the third day of culture, and tissue sections were prepared from the harvested cell aggregates for hematoxylin/eosin (HE) staining and fluorescent immunohistochemistry assaying. After three days of cell culture, cell culture media in the top reservoirs were replaced with calcein AM/EthD-1 dye cocktail (4 µmol•L-1 EthD-1 and 2.5 µmol•L-1 calcein AM in PBS) and held there for 10 min. Then, the fluorescence dyes were removed and the microchip was mounted on a confocal microscope (IX80, Olympus, Japan). At the same time, alginate calcium hydrogel were taken from the microchip and transferred to an EDTA-Na 2 containing vial, where the alginate calcium hydrogel dissolved upon contact with the Ca2+ chelating agent EDTA. The released cell aggregates were quickly washed with cold PBS and fixed in paraformaldehyde

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solution (3.7% in PBS) for 15 min. The collected cell aggregates were embedded in agarose following the method described by Koga et al

42

. The agarose was then

embedded in a paraffin block, from which tissue sections were prepared. The tissue sections were ready for HE and immunofluorescence staining using standard protocols after deparaffinizing (SI, experimental S2). Cell imaging and cell viability assessment Cell viability was indicated with live/dead stains. The dyes were diffused through the nanoporous membrane and alginate gels to stain the cells. Calcein AM (Ex 495 nm, Em 515 nm) was retained within the live cells, which appears green fluorescence by turning the non-fluorescent cell-permeant calcein AM into fluorescent calcein, whereas EthD-1 (Ex 495 nm, Em 635 nm) can only stain dead cells. The ethidium homodimer has high affinity to nucleic acids and appears red fluorescence. Therefore, the living cells show green fluorescence and the dead cells show red fluorescence. After staining, the tissue array was imaged using fluorescence microscopy. The imaging system consisted of a fluorescent microscope (IX71, Olympus, Japan) and a cooled, color CCD camera (DP80, Olympus, Japan). The number of living cells N G was calculated by counting the number of pixels in the green channel and normalizing for the size of one cell. The number of dead cells N R was similarly calculated using the red pixels. The survival rate was calculated as N G /(N G +N R ) × 100%, showing the proportion and distribution of live and dead cells inside the alginate beads. In each assay, the cell viability of the breast cancer cells was compared to that of the hepatic cells. Results Cell array formation As is shown in Fig. 3, while perfusing the alginate solution with suspended cells in the main channel, the liquid preferentially filled the microchambers. Therefore, alginate solution was sequentially trapped in a series of microchambers. It took about 12


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90 seconds to form the cell culture array (SI, Mov S1). The excessive liquid was then expelled from the main channel, thus individual microchambers were insulated by air. Upon contact with Ca2+, the sodium alginate solution gelled into calcium alginate hydrogel, serving as the scaffold to support the 3D cell culture. Fluorescence density in cell culture chambers were detected after cell seeding as an indicator of cell number. RSD of fluorescence density in different cell culture chambers (n=10) was determined to be 9.04 %, indicating the consistency of cell number.

a

b

Fig.3 Consecutive bright-field (a) and fluorescence photos (b) showing cell suspension loading into the microchambers. (I) Sodium alginate solution with a color indicator was introduced into the main channel; (II-III) microchambers were sequentially filled with the liquid; and (IV) excessive fluid was expelled from the main channel. Cells were stained with calcein AM and suspended in 2% alginate sodium/PBS. (Scale bar for Fig.3b is 100 µm.)

Long-term 3D cell culture After seeding, the cells were randomly distributed throughout the microchambers. Fluorescent images showed that dispersed individual tumor cells maintained intact cell membranes after loading into the microchamber. Cells kept proliferating to form large cell aggregates. The cell density increased by ~100% within 3 days of the cell culture (Fig.4a-d) and the cell viability was maintained over 90%. Confocal laser 13



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scanning showed that breast cancer cells formed small aggregates within the microchambers (Fig. 4e). The HE-stained tissue section showed glandular tube structures with incomplete polarity inside the spheroids (Fig.4f).

a

b

c

d

e

f

Fig.4 (a-d) Fluorescence images showing consecutive cell culture on the microchip for 3 days (Day 0 to Day 3, Calcein-AM/EthD-1staining, scale bar: 200 μm); (e) confocal laser scanning of the microchamber on Day 3 (stained with Calcein-AM/EthD-1); and (f) a paraffin-embedded tissue section prepared from cell aggregates harvested on Day 3 (HE staining, scale bar: 20 μm).

Cellular response to anti-cancer drugs As shown in Fig.5, the results showed a dose-dependent increase in the mortality of the MCF-7 cells and a decrease in the survival rate of the LO 2 cells. Cells cultured in different environments differed in their cellular responses. Cells cultured under 2D showed obvious dose-related effects upon anti-cancer drug stimulation, whereas 3D cultured cells were less sensitive to these drugs. So far as the IC 50 is concerned, the values increased in 2D→3D→3D Chip order in both the MCF-7 and the LO 2 cell cultures. To balance anti-cancer efficacy and side toxicity, the optimal 14


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drug concentration was set as the drug concentration corresponding to the intersection of the viability and the mortality line. It can be found that this optimal concentration increased in the order of 2D→3D→3D Chip, too. This tendency was especially apparent in the cellular responses to paclitaxel.

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Fig.5 Comparison of cellular responses to anti-cancer agents in 2D, 3D, and 3D Chip environments. In each figure, the solid line indicates the viability of the MCF-7 cells, while the dash line indicates the mortality of the LO 2 cells.

Orthogonal Screening Three anti-cancer agents were exerted on microfluidic tissues using an orthogonal design. The orthogonal experiment used a 3-level factorial design achieved by matching each level of each factor with an equal number of each level of the other factors. The 3 factors, including doxorubicin, paclitaxel, and cisplatin, contributed to a serial chemotherapy drug combination strategy for breast cancer. The drug screening assay consisted of cell viability testing of breast cancer cells and hepatic cells, with the aim of selecting a drug combination to achieve the maximum killing efficacy to eliminate cancer cells with minimum unwanted off-target effects. Results of the orthogonal experiment are shown in Table 2. The killing effect of the anti-cancer drug combinations was reflected by the death rate of the MCF-7 cells, while additive toxicity was reflected by the survival rate of the LO 2 cells (Fig. 6). To leverage the tumor-killing effect and additive toxicity, the drug combination with an outcome of LO 2 survival rate

Orthogonal Screening of Anticancer Drugs Using an Open-Access

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