Modeling of cancer metastasis and drug resistance via biomimetic nano-cilia and microfluidics

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Biomaterials 35 (2014) 1562e1571

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Modeling of cancer metastasis and drug resistance via biomimetic nano-cilia and microfluidics Ching-Te Kuo a, Chi-Ling Chiang b, Chi-Hao Chang c, Hao-Kai Liu a, Guan-Syuan Huang a, Ruby Yun-Ju Huang d, e, Hsinyu Lee c, *, Chiun-Sheng Huang f, *, Andrew M. Wo a, * a

Institute of Applied Mechanics, National Taiwan University, Taipei, Taiwan, ROC School of Biomedical Science, Ohio State University, Columbus, USA Department of Life Science, National Taiwan University, Taipei, Taiwan, ROC d Department of Obstetrics & Gynaecology, National University Hospital, Singapore e Cancer Science Institute of Singapore, National University of Singapore, Singapore f Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan, ROC b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 October 2013 Accepted 2 November 2013 Available online 22 November 2013

Three-dimensional (3D) tissue culture platforms that are capable of mimicking in vivo microenvironments to replicate physiological conditions are vital tools in a wide range of cellular and clinical studies. Here, learning from the nature of cilia in lungs e clearing mucus and pathogens from the airway e we develop a 3D culture approach via flexible and kinetic copolymer-based chains (nano-cilia) for diminishing cell-to-substrate adhesion. Multicellular spheroids or colonies were tested for 3e7 days in a microenvironment consisting of generated cells with properties of putative cancer stem cells (CSCs). The dynamic and reversible regulation of epithelialemesenchymal transition (EMT) was examined in spheroids passaged and cultured in copolymer-coated dishes. The expression of CSC markers, including CD44, CD133, and ABCG2, and hypoxia signature, HIF-1a, was significantly upregulated compared to that without the nano-cilia. In addition, these spheroids exhibited chemotherapeutic resistance in vitro and acquired enhanced metastatic propensity, as verified from microfluidic chemotaxis assay designed to replicate in vivo-like metastasis. The biomimetic nano-cilia approach and microfluidic device may offer new opportunities to establish a rapid and cost-effective platform for the study of anti-cancer therapeutics and CSCs. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Metastasis Drug resistance EMT CSC Biomimetic Microfluidics

1. Introduction 3D tissue cultures have emerged as invaluable cancer models for a wide range of clinical issues that exhibit microenvironmental heterogeneity as well as for tumors in vivo [1,2]. Compared with traditional two-dimensional (2D) cell cultures, cells cultured in a 3D manner differ considerably in cellular morphology, mass transport properties, and complex cellematrix and cellecell interactions [3,4]. Moreover, the malignant phenotypes of cancer cells in 2D dramatically diminish and the effects of chemotherapeutic drugs or selective inhibitors employed on cellecell

* Corresponding authors. E-mail addresses: [email protected] (H. (C.-S. Huang), [email protected] (A.M. Wo).


[email protected]

0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.

communication, EMT and CSCs are also reduced, whereas the behavior of cells cultured in 3D will respond more closely to in vivo conditions [5e9]. Therefore, 3D culture platforms have served as versatile tools to explore fundamental cell biology, tissue engineering, and drug development, thereby increasingly becoming a potential pre-clinical model between traditional cell cultures and in vivo experiments e allowing a reduction in whole-animal testing, thereby saving cost [1,10e12]. Various techniques of 3D culture platforms have been established, including the use of plastic plates coated with organic (such as agarose [13] and collagen [14]) or inorganic (such as poly-HEMA [15] and hydrogel [16]) matrices, active control of cell suspension by physical forces [17,18], and layer-by-layer assemblies [19]. These platforms provide a suitable environment for 3D cultures, however, challenges still remain. For example, the matrix coating might interfere with the microscopic imaging and fluorometric assays [20], thus questioning the feasibility of integration with other

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Fig. 1. 3D spheroid culture with triblock copolymer (nano-cilia)-based locomotion. (a) Illustrations show the configuration of the triblock copolymers (nano-cilia) system utilized for 3D multicellular spheroid cultures, in which x and y indicate the monomer units. Through the hydrophobicehydrophobic interaction, the hydrophobic PPO chains will bind to the PS surface and beat the hydrophilic PEO chains freely in medium, thereby diminishing the cell-to-substrate adhesion and directing cellecell interactions to organize a cellular spheroid. (b) DI water drop with blue dye on PS surface before and after treatment with copolymers (1% Pluronic F108). A contact angle change of 13 was observed. (c) Morphology of MCF7 cells cultured in conventional 2D monolayers and 3D spheroids, as shown by phase contrast (top panel) and immunofluorescence (bottom panel; cell adhesion molecule stained with EpCAM, nuclear with Hoechst) images. Scale bar, 100 mm.

technologies, such as micro- and nano-fluidics. In addition, complexity arising from the use of external systems is less attractive than more simplistic approaches. Other challenges include the use of organic matrices or engineered nanoparticles for 3D cultures, which might have certain effects on the cells, both biochemically and physiologically. Cancer metastasis and drug resistance are important malignant phenotypes of tumors in vivo that cause roughly 90% mortality in human-associated cancers [21]. During cancer invasion and metastasis, EMT plays a crucial role and purportedly generates cells with properties of CSCs that reveal self-renewal, tumorigenesis, and drug resistance in malignant tumors [22]. Recent studies have demonstrated that EMT and putative stemness may be induced in cellular spheroids cultured in serum-free medium supplemented with adequate mitogens e such as the basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) e using low-attachment culture dishes [5,23], but this approach might be costly and ineffective. In addition, the population of CSCs can be enriched via the selective cell-surface markers, such as CD44þ/CD24/low and CD133 [24,25]. Nevertheless, other studies present that non-stem-like cancer cells can be triggered forcedly or can be stochastically reversed to progenitor/stem-like cancer cells [26,27], suggesting that there is a dynamic regulation between stem and non-stem conditions and further indicating that existing methods based on CSC markers may be unreliable. Consequently, new methods need to be developed, particularly for the study of tumor microenvironment and heterogeneity.

Our recent work has demonstrated that triblock copolymers can direct human ovarian epithelial cancer cell reprogramming and EMT independent of soluble factors [28]. The copolymer mainly contains two separate and hydrophilic PEO chains (approximately 45 nm in length) that kinetically swing themselves freely in solutions and sterically repulse proteins, indicating that this is a biomimetic version resembling the way in which cilia function on lung mucosal epithelia and clear mucus/pathogens out of the airways. Therefore, triblock copolymers may be called biomimetic “nano-cilia” or “molecule cilia” [29]. However, the time and cost by using this method might not be economic. Here, to address the drawbacks and limitations mentioned above, we present a cost-effective and purpose-tailored 3D spheroid culture platform to identify dynamic EMT/MET process and further effectively enrich stem-cell-like/drug resistance cells from a pool of cancer cells. Furthermore, we determine the phenotypic characterization altered by the hypoxia microenvironment in nanocilia and perform the primary evaluation of feasibility for the study of tumor cell biology. 2. Materials and methods 2.1. Cell lines Human ovarian cancer cell line SKOV3 (HTB-77, ATCC) was maintained in Dulbecco’s modified Eagle medium (DMEM, 31600-034, GIBCO, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS, SV30014, Hyclone, South Logan, UT, USA), 1% penicillin/streptomycin (P/S, 15140, GIBCO), and 1.5 g/L D-(þ)-glucose (G5400-250G, SIGMA, St. Louis, MO, USA). Human breast cancer cell line MCF7 (HTB22, ATCC) was maintained in DMEM/F12 (12400-024, GIBCO), supplemented with 10% FBS and 1% P/S. Panc 02.03B human pancreatic cancer cells (provided from


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AbGenomics International Inc., Taipei, Taiwan) were maintained in RPMI-1640 medium (23400-021, GIBCO), supplemented with 15% FBS, 1% P/S, and 1 mM sodium pyruvate (11360-070, GIBCO). DLD-1 (CCL-221, ATCC) and SW480 (CCL-228, ATCC) human colon cancer cells were maintained in RPMI-1640 medium, supplemented with 10% FBS, 1% P/S, and 3 mM L-glutamine (03-020-1B, Biological Industries, Kibbutz Beit Haemek, Israel). All cells were cultured in a humidified 5% CO2 incubator at 37  C.

2.3. Scratch wound assays Scratch wounds were made with a p1000 pipette tip on confluent cells, derived from 2D or 3D cultures, in 3.5 cm cell culture dishes (11035, SPL Lifescience). Wound closure was imaged by microscopy at 10 magnification at 0 h and 8 h. Three independent migration assays were performed for each condition. The size of the remaining wound and the migration rate were determined using imaging software (MetaMorph, Molecular Devices Corp., CA).

2.2. Preparation of triblock copolymers for 3D spheroid cultures

2.4. Immunofluorescence

Triblock copolymers (Pluronic F108) were prepared as described in our earlier work [28]. Briefly, plastic Petri dishes of 6-cm diameter were coated with 1% F108 for 1 h at room temperature prior to cell loading. After washing with PBS twice, human cancer cell lines were seeded by 2  105 cells with culture medium and incubated in a 5% CO2 incubator at 37  C. Cellular spheroids were observed by microscopy every day, and the medium was replaced with fresh medium every week. Prolonged spheroid cultures were achieved by weekly collection of the performed spheroids that were sieved via a 40-mm cell strainer (352340, BD, Franklin Lakes, New Jersey, USA) to yield spheroids larger than 40 mm in diameter. Afterwards, the collected spheroids were dissociated enzymatically (5 min in 0.25% trypsin) and mechanically into single cells then reseeded into copolymer-coated dishes at 2  105 cells/dish. Each generation was designated by the week of cell cultures (i.e. SP1 and SP2).

SKOV3 and MCF7 cancer cells used for 2D cell cultures were prepared by plating w1  104 cells in a 3.5 cm cell culture dish and were incubated overnight to enable cell attachment. 3D cellular spheroids from copolymer-coated dishes were collected, trypsinized into single cells, and plated in a cell culture dish. For epithelial cell adhesion molecule (EpCAM) (anti-EpCAM-PE, 130-091-253, MACS; 1:100 dilution) and CD44 (anti-human CD44-FITC, 555478, BD; 1:200 dilution) staining, cells were subsequently washed with PBS twice and fixed in 4% paraformaldehyde in PBS for 20 min at room temperature. For vimentin (anti-vimentin (V9), sc-6260 AF488, Santa Cruz Biotechnology; 1:100 dilution) staining, cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X100 in PBS for 30 min. Blocking was carried out with PBS containing 1% bovine serum albumin (BSA) for 30 min, followed by PBS washing twice. The cells then were incubated with antibodies in PBS containing 1% BSA for 1 h, and the resulting nuclei were

Fig. 2. Characteristics of triblock copolymers in steric repulsion. (a) SKOV3 monolayer was cultured in collagen gel (100 mg/ml)-coated PS surface (control), whereas multicellular aggregates were generated after one day culture in triblock copolymers of different concentrations (experiment). Scale bar, 100 mm. (b) Comparison of contact angles on native and copolymer-coated PS surfaces. Colors in blue and red represent the surfaces without any treatment and by dipping in cell culture medium for 1 h, respectively. Contact angles were measured by dripping of a 1 mL droplet of DI water onto the PS surfaces. The data are presented as mean  SD from three independent experiments (***p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Phenotypic analyses of tumor spheroids cultured from triblock copolymer-based platform. (a) Schematic showing the phenotypic analysis procedure. First, parental cancer cells were plated in a 1% F108-coated dish to form primary tumor spheroids (SP1) for one week. Afterwards, the spheroids were dissociated into single cells either for the following phenotypic analysis or for being replated in Pluronic copolymers to form secondary spheroids (SP2) for additional analysis. (b) Comparison of cellular morphology with parental MCF7 monolayers and that derived from spheroids (SP1 and SP2). Scale bar, 100 mm. (c) Scratch-wound migration assay. The data are presented as mean  SEM, n ¼ 3. (d) Immunofluorescence detection of EpCAM, vimentin, and CD44 on the three cell monolayers. Total area of signal intensities in the cell monolayers were measured by MetaMorph image software. Each bar represents the mean  SEM of arbitrary units obtained from randomly-captured images (n ¼ 9). (e) Relative expression of the mRNAs encoding EMT markers (including E-cadherin, N-cadherin, vimentin, and fibronectin), stemness markers (including CD44, CD133, and ABCG2), and hypoxia marker (HIF-1a) in the three cells from (a), as evaluated by real-time RT-PCR. GAPDH was used as an internal control and to normalize the variability in sample loading. The data are presented as mean  SD from three independent experiments. Similar to breast MCF7 cancer cells, (fei) show the results of ovarian SKOV3 cancer cells. All quantitative and statistical comparisons were relative to the parental cells, except for the particular ones (*p < 0.05, **p < 0.01, ***p < 0.001).


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Fig. 4. Acquisition of drug resistance in MCF7 and SKOV3 spheroid cells cultured in triblock copolymers. (a) and (b) show the dose responses of breast MCF7 and ovarian SKOV3 cancer cells, respectively, treated with cisplatin or paclitaxel of various concentrations. Different drug concentrations were added to the culture medium of the cell monolayers e from the 7-day culture in the Pluronic copolymers (SP1 and SP2) or conventional 2D rigid dishes (parental) following the procedure illustrated in Fig. 3a e during the 24 h treatment. The detection of viability was conducted using MTT assays and the cell viability with different drug concentrations was normalized against that of untreated cells. The data was presented as mean  SEM, n ¼ 3 (*p < 0.05, **p < 0.01, ***p < 0.001). stained with Hoechst (33342, Invitrogen) in PBS for 10 min. The dishes were extensively washed with PBS and mounted on the stage of fluorescent microscopy (DM IL, Leica). Bright-field and fluorescent images of the cells were captured using a charge-coupled device camera (DP-70, Olympus, Shinjuku-ku, Tokyo, Japan). Image acquisition and analysis were controlled by MetaMorph software using identical exposure times.

400 rpm for 10 min. Then, a plate reader (MQX 200, BioTek) was used to determine the absorbance of DMSO at 570 nm, which was used for interpreting the quantity of live cells. The relative cell viability, obtained from the absorbance values, in different drug concentrations was normalized against that of untreated cells. 2.7. In vitro microfluidic chemotaxis assay

2.5. mRNA expression analysis Total RNA of cells from parental cells and different passages of spheroids were individually extracted and isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary (c)DNA was synthesized from 1 mg total RNA with a reversetranscription polymerase chain reaction (RT-PCR). A real-time PCR with the mixture reagent SYBR-Green I (Thermo Scientific, San Diego, CA, USA) was performed in an iCycler iQ real-time detection system (Bio-Rad, Hercules, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), E-cadherin, N-cadherin, vimentin, fibronectin, CD44, CD133, breast cancer resistance protein (BCRP1/ABCG2), and hypoxiainducible factor (HIF)-1 alpha mRNA expressions were examined. The primer sequences are shown in Table S1. The specificity of the primers was confirmed from a single peak of the melting curve. Each target mRNA level was evaluated from the real-time threshold cycle and compared to the GAPDH amount as an internal control. 2.6. Chemosensitivity assay Chemosensitivity assay was conducted using MTT (3-[4,5-dimethylthiazol-2y1]-2,5-diphenyl tetrazolium bromide) assay in 96-well plates, in which the wells were pre-coated with 100 mg ml1 collagen solution (Type I, BD Biosciences, Franklin Lakes, NJ, USA). Then, cells were plated at a density of 4000 cells/well in 100 ml medium per well and incubated for 5 h. Next, the cell culture medium was exchanged with drug-contained medium of 1e100 mg ml1 paclitaxel or cisplatin (Bristol-Myers Squibb, Park Avenue, NY, USA), and cells were kept in the medium for 24 h culture. Afterwards, the medium was added with 12 mM MTT (M5655, SIGMA; 20 ml/well) and cells were incubated at 37  C for 4 h. Since MTT assay is based on the cleavage of the yellow tetrazolium salt MTT to purple formazan crystals by dehydrogenases and reductases in live cells [30], the formazan crystals then were solubilized by replacing the MTT solution with dimethyl sulfoxide (DMSO, D4540, SIGMA; 100 ml/well) under vibration at

We followed the protocols of the microfluidic chip published previously [28] and made improvements to fulfill the chemotaxis assay in vitro. 2.7.1. Fabrication of the microfluidic device The microfluidic device was prepared as described [28]. Briefly, two fabricated polydimethylsiloxane (PDMS; Sylgard 184, Dow Corning, Midland, MI, CA, USA) microchannels and a perforated membrane with a diameter of 10 mm were aligned and then permanently bonded together. The dimensional size was 600 mm width  85 mm height for the top channel and 8000 mm width  40 mm height for the bottom channel. The thickness of the membrane was fabricated as 10 mm to fulfill the standard porous membrane of boyden chamber [31]. Polymethylmethacrylate (PMMA) nuts with an inner volume of 100 ml were used as solution reservoirs and adhered onto the inlet/outlet of the device. 2.7.2. Operational procedures for in vitro chemotaxis assay in the device To create an in vivo-like model, type I collagen gel (BD Biosciences, Franklin Lakes, NJ, USA) was chosen to simulate extracellular matrix (ECM) and EGF (E9644, SIGMA) as the stimulated signal from stromal cells in the surrounding tumor microenvironment, which are essential components of tumor dissemination during metastasis [32]. Before loading of cells, the microfluidic device was first sterilized using UV light in a laminar-flow hood for 1 h, followed by the wash with phosphatebuffered saline (PBS) twice. Afterwards, the device was stored in a sterilized dish by sealing with parafilm at 4  C until needed. Loading of collagen was performed by fluid height difference along the bottom channel. First, collagen of 100 mg/ml in PBS was loaded into the bottom channel and placed in a humidified incubator at 37  C overnight to allow and enhance binding to the surface in the bottom channel. Subsequently, 3D collagen scaffold was performed by loading a higher EGF concentration of 1 mg/ml at 37  C for 2 h, followed by washing away PBS with cell culture medium. 100 ml of MCF7 spheroids cultured from triblock copolymers (approximately 10 spheroids) in culture medium was loaded from the top channel

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Fig. 5. Design of a microfluidic device for in vitro chemotaxis assay. (a) Illustration of the overall configuration with top and bottom channels sandwiching a cell-supporting membrane with fabricated through-holes. Photography in the right panel shows the fabricated device, in which four wells were used as reservoirs to control the flow rates by selectively tuning the solution levels. Green and blue colors represented the top and bottom channels, respectively. Green wells were served as the cell inlet/outlet for the top channel, and the blue ones served as the EGF source and no-EGF sink, respectively. The enlarged image shows the cross-sectional view of the observation zone for chemotaxis assay. Scale bar, 100 mm (b) and (c) show the computational simulations of EGF concentration distributed in the microdevice under the experimental conditions (please see Materials and Methods for the details). Results show that a stable and uniform concentration profile may be generated in the bottom channel (b), thereby creating a linear concentration gradient of 20 ng/ml/mm in the top channel to stimulate tumor spheroid spreading and migrating (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

into the device and, then, EGF of 500 ng/ml was loaded into the source well at day 1. The stable and uniform concentration gradient of EGF in collagen matrix would be performed as followed by Abhyankar, V. V. et al. reported, in which the long-lasting linear concentration profiles were maintained within a 3D matrix by daily replacement of the fresh source concentration as calculated [33]. The cellular migration and invasion stimulated by the EGF gradient were observed by microscopy (DM IL, Leica, Wetzlar, Germany) daily. In addition, immunofluorescence detection of EpCAM and live cell staining were performed and captured using a charge-coupled device camera (DP-70, Olympus, Shinjuku-ku, Tokyo, Japan) on an inverted microscopy at day 8. 2.8. Statistical analysis Student’s t test was used to compare data from two groups of data, and p < 0.05 was considered statistical significance.

3. Results and discussion The characteristics of nano-cilia (Pluronic F108) in enabling steric repulsion of proteins and small hydrophobic molecules, formation of 3D multicellular spheroids, subsequent viability, and their phenotypic examinations either in vitro or on the microfluidic device will be presented below. Additional materials are also available in the Supplementary data.

3.1. Biomimetic nano-cilia generate multicellular tumor spheroids Fig. 1a presents the methodology of 3D multicellular spheroid cultures that rely on engineered nano-cilia and cellecell interactions, which is based on the steric repulsion of cell-tosubstrate adhesion and subsequent locomotion and selfaggregation of cells into a tumor spheroid. In this study, we adopted triblock copolymer monolayer e Pluronic F108 e as a candidate of such biomimetic nano-cilia. Pluronic F108 is known for its nontoxicity and biocompatibility for a wide range of cellular studies [28,34,35]. It has two separated hydrophilic PEO chain lengths of 128 monomer units and a hydrophobic PPO chain length of 54 units, in which the fully extended PEO chain length in solution would be approximately 45 nm [36], thereby making it easily adjustable to typical cell culture platforms and micro-scale technology without interfering with the microscopy observation. In addition, we recently have demonstrated that Pluronic copolymers may be applied for deterministic 2D/3D cell patterning by microfluidics [28]. Nevertheless, the combination of biomimetic nano-cilia-mediated repulsion, locomotion, and selfaggregation to achieve 3D spheroid cultures, as well as the


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Fig. 6. In vitro microfluidic chemotaxis assay of MCF7 spheroid cells. (a) Schematic showing the microfluidic procedure for the chemotaxis assay of tumor spheroids from nanocilia. The microdevice contains a porous membrane sandwiched between a top and a bottom channel. The formed spheroid was introduced into the top channel and then the chemokine or growth factor was loaded from the bottom channel to generate a concentration gradient. Collagen scaffolds were performed in the bottom channel to simulate the stromal tissue. Afterwards, spheroid migration and invasion induced by the gradient would be observed by daily microscopy. Migration/invasion of the spheroid cells through the porous membrane could serve as a standard assay of spheroid-based boyden chamber [31]. (b) Top-view photographs of cell migration/invasion in the microdevice over 5 days.

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subsequent phenotypic characteristics from the resulting spheroids, has not yet been studied systematically. To first evaluate and then confirm the feasibility of triblock copolymers e coated on conventional polystyrene (PS) dishes/plates e for spheroid cultures, we applied several methods, including multiple cell line testing for spheroid generation, contact-angle measurement, protein repulsion, immunofluorescence detection, and long-term viability testing (Figs. 1b,c and 2, see also Supplementary Figs. S1eS4). Fig. 1b presents the contact angle images (top panel) and the proposed schematics of the chain alignment (bottom panel). The hydrophobic PPO chains were bound to the PS surface due to hydrophobicehydrophobic interaction, and the flexible and hydrophilic PEO chains then were swung freely (with a beating frequency of approximately 10 GHz [29]) in the hydrated layer of solutions, suggesting that this resulted in a decreased contact angle of 64 compared to that of native PS of 77. MCF7 breast cancer cells cultured in Pluronic copolymers (1% F108) formed multicellular spheroids similar to those found in mammospheres, except no supplementary mitogen was used in this study, whereas cells cultured on traditional tissue culture dishes formed typical 2D monolayers (Fig. 1c, see also Supplementary Fig. S1 for SKOV3 ovarian cancer cells and Supplementary Fig. S2 for long-term observation of spheroid growth). In addition, we prepared Pluronic F108 of different concentrations coated onto PS dishes and determined that 1% F108 was the optimal polymer for multicellular aggregate cultures and steric repulsion of hydrophobic molecules/proteins (Fig. 2, see also Supplementary Fig. S3). Notably, results from Fig. 2b indicate that PS’s hydrophobic nature tends to absorb small hydrophobic molecules in culture medium and might decrease the water contact angle. Similar results were found in the polymer coatings of 0.01% and 0.1% F108 as well. In contrast, 1% F108-coated PS wound efficiently prevents the absorption of molecules and proteins; therefore, it has the capacity for 3D spheroid cultures to diminish cell-tosubstrate adhesion. Similar to MCF7 and SKOV3 cells, human pancreatic cancer cell line (Panc 02.03B) and two human colon cancer cell lines (DLD-1 and SW480) all formed multicellular spheroids in 1% F108 (Supplementary Fig. S4). Thus, the biomimetic nano-cilia by Pluronic F108 has the potential to achieve modeling of tumor spheroids in vitro. 3.2. Phenotypic characteristics of breast MCF7 and ovarian SKOV3 tumor spheroids To explore the particular biological attributes afforded by 3D cultures, we followed the procedure illustrated in Fig. 3a to examine the phenotypic characteristics of spheroids cultured in triblock copolymers (Pluronic F108). Results showed that (1) the cellular monolayers derived from the spheroids (SP1 and SP2) cultured in copolymers reduced cellecell interaction but acquired fibroblast-like mesenchymal morphology compared to parental cells (Fig. 3b for MCF7 and Fig. 3f for SKOV3); (2) the spheroid cells (SP1 and SP2) acquired an increased ability of migration, as compared to parental cells (Fig. 3c and g, see also Supplementary


Fig. S5); and (3) the differentiated expressions of EpCAM (broadly expressed on cells of epithelial origin and on epithelium-derived tumor cells [37]), vimentin (often used as a marker of mesenchymally derived cells [23]), and CD44 (a surrogate CSC marker [22]) in the three cells (parental, SP1, and SP2 cells; Fig. 3d and h for MCF7 and SKOV3 cells, respectively; see also Supplementary Fig. S6). It is noteworthy that the gains in mesenchymal-like and stem-cell-like properties were evident by a marked increase in vimentin and CD44 protein expressions e two classic features of EMT [22] e for 3D cultures compared to that obtained from 2D rigid cultures. Moreover, a reduction in EpCAM expression appeared in SKOV3 spheroid cells; however, there was no significant difference in EpCAM expression between MCF7 parental and spheroid cells. Subsequently, Fig. 3e and i show the examination of mRNAs e encoding EMT markers (E-cadherin, N-cadherin, vimentin, and fibronectin), stemness markers (CD44, CD133, and ABCG2), and a hypoxia marker (HIF-1a) e in MCF7 and SKOV3 cells, respectively. Notably, several apparent differentiations were found between the two cell lines. First, the expressions of EMT markers were all significantly upregulated in MCF7-SP2 cells (Ecad with an 8.6-fold increase, N-cad with a 16.4-fold increase, Vim with a 4.4-fold increase, and FN1 with a 9.2-fold increase), whereas MCF7-SP1 cells only achieved a downregulated expression of E-cadherin with a 0.6-fold decrease. For SKOV3 cells, only fibronectin expression was significantly upregulated with a 2.9fold increase in the primary spheroid cells; however, this expression in SKOV3-SP2 cells was downregulated compared to SKOV3-SP1. Second, only the putative stemness of CD133 expression was significantly upregulated with an 18.5-fold increase in MCF7-SP2 cells. In contrast, the CD133 expression was significantly downregulated in SKOV3 spheroid cells (with 0.4fold and 0.5-fold decreases for SP1 and SP2 cells, respectively). Additionally, the CD44 and ABCG2 expressions displayed significant increases compared to parental cells, in which the increments of CD44 expression were 2.9-fold and 3.9-fold, and that of ABCG2 were 2.0-fold and 3.9-fold in SKOV3-SP1 and SKOV3SP2 cells, respectively. Finally, for the examination of hypoxia marker (HIF-1a), we found that both SKOV3-SP1 and SKOV3-SP2 cells displayed significant upregulations with 2.2-fold and 3.1fold increases, respectively. MCF7-SP2 cells were significantly upregulated with a 3.4-fold increase as well; however, MCF7-SP1 cells were downregulated with a 0.5-fold decrease, compared to parental cells. To assess the chemosensitivity of the parental cells and spheroid cells cultured in triblock copolymers, we treated these cells with two commonly-used chemotherapeutic drugs e cisplatin and paclitaxel e of different concentrations up to 100 mg/ ml in order to evaluate cell viability, which was measured using MTT assays after 24 h of drug treatment (Fig. 4a and b for MCF7 and SKOV3 cells, respectively; see also Supplementary Fig. S7). Several results are noteworthy. First, spheroid cells from the two cell lines were more drug resistant than the parental cells, respectively, implying that the copolymers may promote selection of drugresistant cells from a pool of cancer cells. Second, the

The spheroid cells started to spread at Day 1, and these cells were then attracted and followed with a directional migration/invasion caused by the EGF gradient (20 ng/ml/mm) from Day 3 to Day 6. The white dashed lines illustrate the location of the formed spheroid/spreading cells at each day. In contrast, (c) shows that parental cells were not attracted but resulted in a symmetric spreading. (d) Time sequence of the top-viewed photographs showing 4 breast carcinoma cells (highlighted by purple, red, green and yellow colors, respectively) in the process of invasion in the presence of an EGF gradient (20 ng/ml/mm). Immunofluorescence detection of epithelial marker (EpCAM, red), live cell staining (green), and Hoechst nuclear staining (blue) was applied in the device at day 8. The dashed line illustrates the top channel boundary. (e) Comparison of the number of invasive cells into the bottom channel with parental and spheroid cells. The data are presented as mean  SD, n ¼ 3 (**p < 0.01). (f) Total area of EpCAM signal intensities in cells (primary cells in the top channel and invasive cells in the bottom channel) were measured by MetaMorph image software. Each bar represents the mean  SD (n ¼ 5), ***p < 0.001 compared to the primary cells. Scale bars, 100 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)


C.-T. Kuo et al. / Biomaterials 35 (2014) 1562e1571

differentiation of dose responses between MCF7-SP1 and SKOV3SP1 cells correlated with the mRNA analysis of putative stemness (Fig. 3e and i), in which SKOV3-SP1 cells exhibited upregulated CD44 and ABCG2 but there was no significant regulation in MCF7SP1, indicating that the drug resistance might correlate with the stemness expression. The above in vitro tumor formation analyses suggest that cells within the spheroids formed in triblock copolymers may acquire features of a stem cell. It is known that “cancer stem cells” would have the capacity to self-renew and to generate the heterogeneous lineages of a tumor as well as to resist the chemotherapeutic druginduced apoptosis [2,5,22]. Through 3D spheroid culture, MCF7 and SKOV3 cells acquired stemness to various degrees. The above phenotypic differentiations might be attributed to the different cell characteristics and the diverse microenvironments formed in MCF7 and SKOV3 spheroids. In line with the expression of putative CSC markers, 3D MCF7 and SKOV3 cells were more resistant to apoptosis compared to those from conventional rigid dishes (Fig. 3e and i and Fig. 4). In addition, upregulation of vimentin and CD44 in protein expression was further confirmed with real-time RT-PCR results, although the increment of mRNA in CD44 for MCF7 cells and vimentin for SKOV3 cells was not significant (Fig. 3d, e, h, and i). Another key feature of the results is that dynamic regulation of EMT appeared in the formed spheroids. It is known that the in vivolike hypoxia niche could induce EMT and promote expansion of CSCs [38,39]. Our platform may also provide the hypoxia microenvironment (see HIF-1a expression in Fig. 3e and i) and facilitate selection of drug-resistant or tumorigenic cells (Fig. 4, see also supplementary Fig. S8), along with further modeling of tumor heterogeneity (Supplementary Fig. S9). Although previous studies have revealed that EMT may be induced through 3D cultures in vitro, to the best of our knowledge, none of them mentioned the spatiotemporal and regulatory properties between EMT and a transition reverted from mesenchymal to epithelial (MET), indicating that the phenotypic plasticity exhibited during EMT and MET is nonetheless difficult to capture [9,13,23,40]. Our data herein have partially explained that this examination is possible in vitro (Fig. 3e and i), in which we postulated that the “reversible EMT model” [41,42] dominates in tumor metastasis (Supplementary Fig. S10). Together, these findings suggest that triblock copolymers are unique in promoting multicellular tumor spheroid formation and growth, further providing new insights and a newly applicable in vitro model for EMT and CSC research (Table S2).

channel) and a uniform profile (along with BeB0 cross-section) beneath the top channel, thereby creating a linear concentration gradient of 20 ng/ml/mm in the top channel (Fig. 5c) to guide tumor spheroid migration, indicating that the auxiliary simulations are beneficial for explaining and validating the following experimental results. Fig. 6bef shows the main results, including that (1) spheroid cells spread and migrated to the higher epidermal growth factor (EGF) gradient, whereas parental cells remained and spread symmetrically (Fig. 6b and c, see also Supplementary Fig. S11), suggesting that the spheroids acquired an upregulated EGFR expression (in line with other reports on 3D cultures [45]), (2) the spheroid cells acquired a higher invasive ability than parental cells (Fig. 6d and e), and notably, (3) the invasive cells exhibited a significant downregulation of epithelial characteristic (EpCAM) by measuring the fluorescent intensity (Fig. 6f), suggesting that these disseminated cells from the primary tumor spheroids (i.e., from the top channel to the bottom channel) may have undergone EMT to form metastases, as the hypothesis reported [21,41,42]. 4. Conclusions In summary, we have presented a tumor spheroid culture platform by biomimetic nano-cilia (Pluronic copolymers) that can control differentiation behaviors and epigenetic reprogramming of tumor cell subsets. This platform is valuable in modeling of cancer and in selection of drug-resistant and cancer-stem-like cells, independent of CSC markers. Key results demonstrated that dynamic EMT and MET regulations in vitro could be examined in this platform, and the in vivo-like metastatic behaviors could be also examined by the developed microfluidic device. Thus, the present approach not only provides a useful platform for the study of tumorigenicity and cancer metastatic cascade in vitro, but potentially achieves a long-term goal of realizing “organotypic cultures” that represent tumor microenvironments for personalized drug testing. Acknowledgements We are grateful to Prof. Jiahn-Chun Wu for providing the SKOV3 cell line. Financial support from the National Science Council, Taiwan, under grants NSC 100-2120-M-002-001 and 101-2221-E002 -085, is gratefully acknowledged.

3.3. In vitro microfluidic chemotaxis assay of 3D spheroids Appendix A. Supplementary data We wondered whether the 3D spheroids formed in triblock copolymers acquired more efficient metastatic ability than those cultured on conventional 2D rigid dishes. Nevertheless, the assay tools remained poor because of the challenges of probing cellecell interactions in vivo and the lack of physiologically related in vitro models [43]. To this end, we developed an in vivo-like environment by microfluidics for the in vitro chemotaxis assay (Figs. 5a and 6a), in which MCF7 spheroids were examined in this proof-ofconceptual assay. To assess the concentration profile of EGF within the device, a commercial finite-element package (COMSOL 4.2) was utilized for simulating and analyzing the profiles along the bottom channel (3D matrix) and top channel (observation zone for the chemotaxis assay). Constant concentration boundary conditions were defined at the source well with 500 ng/ml and sink conditions at the sink well in the bottom channel. The diffusion coefficient of EGF within the ECM was set as 6  1011 m2/s [43,44]. Fig. 5b shows that the concentration profile performed in the bottom channel exhibited a linear gradient near the observation zone (i.e., near the top

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