Restriction of Cancer Metastatic Potential Using Embryonic Stem Cells

Jun 2, 2017 - *Phone: 518-956-7381. Fax: 518-956-8687. E-mail: [email protected]. ... expression following coculture with ESC-microstrands. Future app...
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Article pubs.acs.org/journal/abseba

Restriction of Cancer Metastatic Potential Using Embryonic Stem Cells Encapsulated in Alginate Hydrogel Microstrands Bridget Mooney, Nurazhani Abdul-Raof, Yangzi Isabel Tian, and Yubing Xie* Nanobioscience, Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, New York 12203, United States S Supporting Information *

ABSTRACT: Current treatments focused on eradicating metastatic tumors have proven unsuccessful due to cancer’s ability to quickly undergo epithelial-to-mesenchymal transition (EMT) and metastasize to secondary sites. Using human triple negative breast cancer cells (BCCs) as a model system, this work establishes a platform for the study of aggressive cancer phenotypes by demonstrating the inhibition of human metastatic cancer cells with 3D cultured embryonic stem cells (ESCs) encapsulated in alginate microstrands (ESC-microstrands), which mimic the embryonic microenvironment and recapitulate pluripotent signaling. Coculture with ESCmicrostrands significantly decreases triple negative BCC proliferation and survival and reverses abnormal cancer metabolism. In particular, coculture with ESC-microstrands markedly restricts the metastatic potential of highly aggressive cancer cells, demonstrated as decreased migration and invasion, and reversed EMT marker expression. This indicates that pluripotent signaling from 3D ESC-microstrands could restrict cancer metastasis through restriction and reversion of EMT. Furthermore, two soluble factors associated with dysregulated oncogenic signaling were identified which display altered relative mRNA expression following coculture with ESC-microstrands. Future application of this model to mechanistic studies will enable a better understanding of cancer metastasis and the discovery of therapeutic targets for metastatic diseases. KEYWORDS: breast cancer restriction, embryonic stem cell (ESC), 3D coculture, alginate, epithelial-to-mesenchymal transition (EMT), metastasis

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INTRODUCTION According to the American Cancer Society, cancer is responsible for almost as many deaths as heart disease, the number one killer of Americans, and is on track to surpass it as the leading cause of death within the next decade.1 Metastasis is the greatest contributor to cancer deaths,2 accounting for 90% of cancer mortalities.3 Current treatments for cancer metastases primarily rely on some variation of chemotherapy.4 Although many types of cancers are initially sensitive to chemotherapy, over time these cancer cells can develop drug resistance causing relapse in an alarmingly high percentage of cancer patients,5,6 eventually leading to uncontrolled metastatic diseases and low survival rate.7 In order to successfully combat this trend, it is critical to address underlying causes that challenge current cancer treatment, such as drug resistance and lack of targeted therapy.8,9 This requires a more thorough understanding of how to mitigate the aggressive tumor phenotype, which can be achieved by constructing a model that restricts or reverses malignant transformation. The importance of embryonic microenvironments in restricting cancer progression and reprogramming cancer cells to a less aggressive phenotype has been demonstrated in zebrafish, chick, and mouse embryo models.10−18 More than a century ago, scientists demonstrated that injection of embryonal tissue into mice causes transplanted tumor rejection. This effect © XXXX American Chemical Society

was attributed to antibodies formed in response to the embryonic tissue that were also capable of recognizing and attacking cancer cells because of their shared antigens, known as oncofetal antigens.19 Along the same lines of using embryonal tissue to restrict cancer in vivo, it is possible to take advantage of soluble factors secreted by embryonic stem cells (ESCs) to inhibit cancer in vitro since embryonic and tumorigenic signaling pathways are convergent.20−22 For example, it has been demonstrated that cancer cell growth could be inhibited by human ESCconditioned Matrigel (for melanoma cells),23,24 human ESCconditioned media (for ovarian, papillary, prostate, and breast adenocarcinoma cells),25 and human ESC-derived exosomes (for breast and colorectal adenocarcinoma cells).26 These studies prompt further investigation to understand the influence of ESC soluble factors on the metastatic potential of aggressive cancer cells. Triple negative breast cancer was chosen as a metastatic cancer cell model in this study, considering its extremely aggressive properties, such as its ability to evade immune system detection, programmed cell death, cell-cycle regulation, and chemoReceived: April 14, 2017 Accepted: June 2, 2017 Published: June 2, 2017 A

DOI: 10.1021/acsbiomaterials.7b00237 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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glutamine. 3T3 fibroblasts were obtained from the National Institutes of Health (NIH) and were cultured in growth medium containing DMEM supplemented with 15% (v/v) FBS, 100 U/mL penicillin, and 100 μg/ mL streptomycin. Coculture of Metastatic MDA-MB-231 BCCs with ESC-Microstrands. ESCs suspended in a 1.5% sodium alginate solution at a cell density of 1 × 106 cells/mL were driven into a 50 mM CaCl2 solution at a rate of 100 μL/min with a New Era (NE)-1000 syringe pump utilizing a 200 μm microfluidic tip that yielded alginate hydrogel microstrands with a 200 ± 23 μm diameter. MDA-MB-231 BCCs were seeded at a cell density of 2 × 104 cells/mL in 0.5 mL of BCC media in each well of the 48-well plate. ESC-microstrands were formed in another dish and placed in ESC maintenance media. Twenty-four hours later, BCC media were replaced with ESC maintenance media. These ESC-microstrands were measured, cut to a length of 17.5 mm, and cocultured with the BCCs. After 24, 48, and 72 h, effects that exposure to the ESC microenvironment had on BCCs were analyzed using various biological assays. Noncocultured BCCs and empty alginate hydrogel microstrands were used as controls. The noncocultured control consisted of BCCs in ESC maintenance media, and the empty microstrand control included BCCs in ESC maintenance media cocultured with empty microstrands cut to a length of 17.5 mm per 1 × 104 BCCs. The length of 17.5 mm was chosen as the microstrand unit in order to make it comparable to our previous study using alginate hydrogel microbeads.37 Microstrands of 17.5 mm in length (200 μm in diameter) have the same volume as five microbeads (600 μm in diameter), which showed the greatest degree of inhibition of cancer cell growth in our previous study. Immunocytochemistry. Immunocytochemistry was performed as described previously.37 To examine pluripotent marker expression, ESC-microstrands were incubated with primary antibodies against Oct4 (Sigma-Aldrich), SSEA-1, and alkaline phosphatase (ALP) (Santa Cruz Biotechnology, Dallas, TX). For EMT marker expression, metastatic MDA-MB-231 cells grown on glass coverslips with or without exposure to ESC-microstrands were incubated with antibodies against E-cadherin (Santa Cruz Biotechnology, Dallas, TX) and vimentin (Abcam, Cambridge, MA). Nutrient Depletion Analysis. The YSI 7100 Biochemical Analyzer (Yellow Spring Instruments) was used to determine the concentrations of glucose and lactate within the media following 24, 48, and 72 h of coculture with ESC-microstrands. Cell Viability, Cell Proliferation, and Apoptosis/Necrosis Assays. Cell viability was assessed with a Live/Dead Cell Double Staining Kit (Sigma-Aldrich, St. Louis, MO), cell proliferation was measured using a Premixed WST-1 Cell Proliferation Reagent (Clontech, Mountain View, CA), and cell apoptosis/necrosis was measured using an Annexin V-FITC Apoptosis Detection Kit (SigmaAldrich, MO), all per the manufacturer’s instructions. Cell Cycle Analysis. GFP-expressing MDA-MB-231 BCCs were stained with DRAQ5 nuclear stain followed by flow cytometry imaging (Amnis Image StreamX Mark II flow cytometer, Amnis Corporation, Seattle, WA). High-speed multispectral automated image acquisition and analysis enabled the separation of cells based on DRAQ5 nuclear stain intensity into bins representative of different phases of the cell cycle. Specific phases of the cell cycle were identified and gated on plots of intensity vs normalized frequency. The quality of the DNA histograms was assessed by calculating the coefficient of variation (CV) of the G1 peak as follows:

therapeutic drug treatment.27 Specifically, the ability to avoid cell cycle checkpoints coupled with an increased reliance on glycolysis allows for substantial tumor growth through enhanced proliferation. Subsequently, metastatic cancer cells undergo epithelial-to-mesenchymal (EMT) transition, invade into surrounding tissue(s) and vasculature, and migrate to distant areas of the body.28,29 During EMT, cancer cells become highly aggressive and invasive via the breakdown of cell polarity and intercellular contacts normally present in epithelial cells, which is accompanied by cytoskeletal remodeling, a decrease in the tumor suppressor E-cadherin expression, and an increase in vimentin levels.30,31 Targeting EMT could restrict cancer metastasis and overcome drug resistance in cancer.32 Earlier in vivo works have revealed that the pluripotent phenotype of ESCs is essential to render cancer cells less aggressive in their presence.33−35 ESCs depend on an environment that reduces surface contact to promote pluripotent signaling and soluble factor secretion.36 We hypothesized that ESCs encapsulated in alginate microstrands (≤200 μm in diameter) could restrict EMT, leading to reduced metastatic potential of human triple negative breast cancer cells (BCCs). In this study, we encapsulated ESCs in alginate microstrands (ESCmicrostrands) to mimic in vivo embryonic signaling and maximize pluripotent soluble factor secretion. We cocultured human triple negative MDA-MB-231 BCCs with 3D ESCmicrostrands and examined cancer cell survival using a WST-1 Cell Proliferation Assay, Annexin V-FITC Apoptosis Detection, and cell cycle analysis via flow cytometry imaging. We measured cancer cell metabolism using a Seahorse Bioscience XFe24 Extracellular Flux Analyzer and evaluated BCC metastatic potential using an invasion assay, migration analysis, and immunocytochemistry and Western blot analysis of EMT markers (E-cadherin and vimentin). The findings demonstrated that 3D ESC-microstrands restricted human triple negative BCC growth, and in particular, metastatic potential through the reversal of EMT, creating a cancer restriction model for mechanism studies and future therapeutic target discovery. For example, mechanistic studies identified two soluble factors involved in the epidermal growth factor receptor (EGFR) and canonical Wnt/β-catenin signaling pathways, which are both hyperactivated in triple negative breast cancer, that displayed reversed relative mRNA expression following coculture with ESC-microstrands. Future work with this model will include a more intensive investigation into secreted ESC soluble factors responsible for BCC inhibition, as well as, a more in-depth analysis of effects of coculture with ESC-microstrands on the EGFR and canonical Wnt/β-catenin signaling pathways.

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MATERIALS AND METHODS

Cell Lines and Cell Culture. Mouse CCE ESCs from StemCell Technologies (Vancouver, Canada) were cultured in 0.1% gelatincoated flasks and maintained in maintenance medium consisting of Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L D-glucose supplemented with 15% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 0.1 mM MEM nonessential amino acids, 10 ng/mL murine recombinant leukemia inhibitory factor (StemCell Technologies, Vancouver, Canada), 0.1 mM monothioglycerol, 2 mM L-glutamine, and 1 mM sodium pyruvate. Human metastatic MDA-MB-231 and nonaggressive MCF7 BCCs were purchased from American Type Culture Collections (ATCC, Manassas, VA), and GFP-expressing MDA-MB-231 BCCs were obtained from Mount Sinai School of Medicine. These cells were cultured in BCC growth medium consisting of DMEM supplemented with 15% (v/v) FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-

⎡ standard deviation ⎤ CV = ⎢ ⎥ × 100% ⎣ G1 peak channel intensity ⎦ Mitochondrial Stress Test. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cocultured and noncocultured MDA-MB-231 BCCs were measured using the Seahorse Bioscience XFe24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA). MDA-MB-231 BCCs were seeded in XFe24 plates at a cell density of 40,000 cells/well and cocultured with ESCmicrostrands the next day. After 24 h, the sensor cartridge was hydrated in calibration buffer (Seahorse Bioscience, North Billerica, MA) at 37 °C in a CO2-free incubator. Twenty-four hours later, these BCCs were B

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Figure 1. ESCs encapsulated in alginate hydrogel microstrands (ESC-microstrands). (a) Schematics of microfluidic synthesis of alginate hydrogel microstrands. (b) The effect of the syringe tip size on the diameter of alginate microstrands formed by microfluidic synthesis at a flow rate of 100 μL/min over time. (c) Optical image of ESCs encapsulated in alginate microstrands. (d) Confocal images of ESCs within the microstrands, retaining high cell viability (green cells in left panel) and exhibiting expression of the pluripotent markers, Oct-4, SSEA-1, and ALP (right panels). (e) Optical image of ESC-microstrands cocultured with BCCs in ESC maintenance media 24 h after formation. Cell Migration Analysis. Cell migration of MDA-MB-231 BCCs was analyzed as described previously.37 Leibovitz 15 (L15) media (Sigma-Aldrich, St. Louis, MO) supplemented with L-glutamine and 0.08% (v/v) BSA were added, and a 4 h video of the cells was compiled at 37 °C using a Nikon TS100-F inverted microscope programmed to capture the image in 2 min intervals. Cells were individually tracked using a Mastracker plugin specifically designed for ImageJ software to calculate total displacement, total path length, and velocity of cell migration. Western Blot Analysis. Total protein was assessed using a BCA kit, per the manufacturer’s instructions. The boiled protein samples were loaded into each well (15 μg) of a NuPage Novex 4−12% Bis-Tris Protein Gel (Thermo Fisher Scientific, Waltham, MA) for separation through electrophoresis followed by wet transfer onto a nitrocellulose membrane. The nitrocellulose membrane was incubated for 1 h with a primary antibody against β-actin (housekeeping) (Santa Cruz Biotechnology, Dallas, TX), E-cadherin, or vimentin in Tris-buffered saline and Tween 20 (TBST), followed by a secondary antibody diluted in TBST. Chemiluminescence was induced with Supersignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA) and detected in a FluorChem E system (Protein Simple,

washed with PBS and XF assay media (Seahorse Bioscience, North Billerica, MA) supplemented with 25 mM glucose and 1 μM sodium pyruvate was added to each well, and the plate was incubated for 30 min at 37 °C in a CO2-free incubator. Drugs for the mitochondrial stress test were loaded into the injection ports as follows: 10 μM oligomycin, 7.5 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and 10 μM antimycin A. The sensor plate was placed into the machine and allowed to calibrate for 30 min, at which time the cell plate was added, and these drugs were injected sequentially followed by three 2 min interval measurements separated by mixing. The experimental conditions and control were performed in replicates of 10. ECAR and OCR values were scaled to the second basal point and normalized to total protein content, measured using a bicinchoninic cell assay (BCA) (Pierce Chemical, Rockford, IL), per the manufacturer’s instructions. Matrigel Invasion Assay. MDA-MB-231 BCC invasive ability was assessed using Corning Biocoat Matrigel Invasion Chambers (BD Biosciences, Bedford, MA). Invading BCCs were incubated with 4′,6diamidino-2-phenylindole (DAPI) for 15 min at 37 °C and counted under a Nikon TS100-F inverted fluorescent microscope in four different fields of view. C

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Figure 2. ESC-microstrands significantly reduced triple negative BCC proliferation but had no effect on proliferation in less aggressive MCF7 BCCs or normal 3T3 fibroblasts. (a) The effect of initial ESC density and alginate microstrand volume on proliferation of MDA-MB-231 BCCs following coculture with ESC-microstrands with empty and noncocultured microstrands as controls (standard deviation for the noncocultured MDA-MB-231 BCC control = 1.00 ± 0.08). (b) Reduced cell viability following coculture with ESC-microstrands was unique to triple negative MDA-MB-231 BCCs. (c) Changes in glucose and lactate concentrations following 1, 2, and 3 days of coculture with ESC-microstrands were comparable to the empty microstrand and noncocultured controls for metastatic MDA-MB-231 BCCs and 3T3 fibroblasts, indicating that any changes following coculture are not the result of nutrient depletion. * = p < 0.05. San Jose, CA). Protein expression was analyzed by densitometry using ImageJ and normalized to the housekeeping β-actin. qRT-PCR Analysis. Total RNA of MDA-MB-231 BCCs was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA), per the manufacturer’s instructions. The RNA was of high purity with 260/ 280 values close to 2.0. cDNA was synthesized using a first-strand cDNA synthesis kit (Invitrogen, Grand Island, NY). qRT-PCR was executed using a StepOnePlus Real Time PCR System (Applied Biosystem, Foster City, CA) using forward and reverse primers for TGF-α, NKD2, and β-actin as shown in Table S1. Each sample was performed in triplicate with a negative control. The ΔΔCt method was applied for quantification using β-actin as an internal control. Statistical Analysis. All samples were run in duplicate or triplicate and repeated two or three times. Each data point was represented as a mean ± SD. Differences between two groups were assessed by unpaired t tests. Significance levels were indicated by p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), and p < 0.0001 (****).

diameter microstrands because they were easier to handle for coculture. Following encapsulation, the ESCs maintained viability, and the pluripotent markers, Oct-4, SSEA-1, and ALP, were retained (Figure 1d). ESC-microstrands and BCCs were cocultured 24 h after formation and seeding, respectively (Figure 1e). Experimental wells and controls received ESC maintenance media, and microstrands were placed in the well with the BCCs. Empty alginate microstrands served as a control, and all values were normalized to a noncocultured BCC control. The necessary ESC density and microstrand volume for human metastatic triple negative MDA-MB-231 BCC restriction were identified based on a cell proliferation assay. There was a significant decrease in proliferation compared to both noncocultured and empty microstrand controls when BCCs were cocultured with ESCmicrostrands with a length of 17.5 mm (volume = 5.5 × 10−4 mL) at an ESC density of 1 × 106 cells/mL (Figure 2a). Subsequent experiments were executed using these parameters. The ability of the ESC-microstrands to decrease proliferation was unique to highly aggressive MDA-MB-231 BCCs as there were no detectable changes in proliferation of nonaggressive MCF7 BCCs or 3T3 fibroblasts when cocultured with ESC-microstrands (Figure 2b). Finally, BCC restriction was not caused by nutrient depletion in the media because coculture with ESCmicrostrands did not significantly alter glucose uptake and lactate production compared to that of noncocultured and empty microstrand controls (Figure 2c). Induction of Apoptosis/Necrosis and a Shift in Cell Cycle Profile in Triple Negative BCCs. Exposure of highly aggressive MDA-MB-231 BCCs to ESC-microstrands significantly increased both apoptosis and necrosis compared to the empty microstrand and noncocultured BCC controls based on results from an Annexin V-fluorescein isothiocyanate (FITC)

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RESULTS Creation and Optimization of a 3D ESC-Microstrand Model to Restrict Human Metastatic Triple Negative Breast Cancer. Alginate hydrogel microstrands were created by microfluidic synthesis (Figure 1a). Briefly, 1.5% alginate solution alone or with cells was pumped into a solution of 50 mM CaCl2 at a constant rate using a syringe pump in order to form alginate microstrands with a uniform diameter. The diameter of the syringe tip greatly impacted the diameter of the resultant alginate microstrands, and the diameter of the alginate microstrands did not change after 15 min of pumping (Figure 1b). Alginate microstrands containing mouse ESCs (ESC-microstrands), formed at a rate of 100 μL/min for 10 min, had an average diameter of 200 ± 23 μm (Figure 1c). Alginate microstrands with a 200 μm diameter were used instead of the smaller 100 μm D

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Figure 3. Coculture with ESC-microstrands induced triple negative BCC apoptosis and necrosis. (a) Significantly increased MDA-MB-231 BCC apoptosis following coculture compared to an empty microstrand and noncocultured control. (b) Significantly increased necrosis in triple negative BCC and decreased 3T3 fibroblast necrosis following coculture with ESC-microstrands compared to the empty microstrand and noncocultured controls. **** = p < 0.0001, ** = p < 0.01, and * = p < 0.05.

Figure 4. Coculture of triple negative BCCs with ESC-microstrands caused a shift from the highly proliferative G2/M phase to the G1 and S phases of the cell cycle. (a) Representative morphological examples (left to right: BF = brightfield, GFP = green fluorescent protein, and DRAQ5 nuclear stain) of MDA-MB-231 BCCs in the M phase of the cell cycle. (b) Representative morphological examples of cells in the SubG1 phase of the cell cycle. (c) Shift of MDA-MB-231 BCCs out of the G2/M phase of the cancer cell cycle and into the S and SubG1 phases after coculture with ESC-microstrands. (d) The increase and decrease in the number of BCCs in the SubG1 and G2/M phases of the cell cycle, respectively, were statistically significant compared to those of the empty microstrand and noncocultured controls. ** = p < 0.01.

decreased necrosis in the normal 3T3 fibroblasts; however, only the latter was significant compared to that of both controls. Coculture with ESC-microstrands induced the highest cell death, whether through apoptosis or necrosis, in MDA-MB-231 BCCs

assay (Figure 3). The upsurge in necrotic cells was more pronounced compared to apoptotic cells in the cocultured MDAMB-231 BCC population. Coculture with ESC-microstrands also induced apoptosis in nonaggressive MCF7 BCCs and E

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Figure 5. Coculture with ESC-microstrands reduced both mitochondrial respiration and glycolysis and reversed the Warburg effect in triple negative BCCs. (a) Reduced mitochondrial respiration of MDA-MB-231 BCCs following coculture with ESC-microstrands. (b) Decreased maximal mitochondrial respiration and reserve capacity OCR in cocultured MDA-MB-231 BCCs. (c) Reduced reliance on glycolysis in MDA-MB-231 BCCs following coculture with ESC-microstrands. (d) Reversed Warburg effect in cocultured MDA-MB-231 BCCs demonstrated as increased dependence on mitochondrial respiration, as opposed to glycolysis. **** = p < 0.0001, *** = p < 0.001, and ** = p < 0.01.

compared to that in MCF7 BCCs and 3T3 fibroblasts. DRAQ5 nuclear stain in combination with flow cytometry and green fluorescent protein (GFP)-expressing MDA-MB-231 cells was employed to further probe changes in cell death and proliferation following coculture with ESC-microstrands. Examples of cells in the G2/M (Figure 4a) and SubG1 (Figure 4b) phases of the cell cycle are provided. Compared to the noncocultured control (Figure 4c), cell cycle profiles indicated that coculture of triple negative MDA-MB-231 BCCs with ESC-microstrands causes a shift in the number of BCCs in the G2/M phase of the cell cycle to the SubG1 and S phases (Figure 4d). Specifically, data from multiple trials showed an increase in the number of cells in the SubG1 phase of the cell cycle and a decrease in the number of cells in the G2/M phase (Figure 4e), indicating that triple negative MDA-MB-231 BCCs cocultured with ESC-microstrands experienced augmented apoptosis/necrosis and diminished proliferation. The coefficient of variation (CV) values for the cell cycle profiles were within the target range (8.0% to 14.0%) for mammalian cells indicating that cell populations were homogeneous. Increased Reliance on Oxidative Phosphorylation over Glycolysis in Triple Negative BCCs. Highly aggressive cancer cells alter their energy metabolism in response to the tumor microenvironment during tumorigenesis and metastasis, and this metabolic change is frequently associated with drug resistance. Transformed energy metabolism is a hallmark of cancer malignancy and is characterized by a pronounced reliance on glycolysis over oxidative phosphorylation to meet the energy demands of the cell. A mitochondrial stress test demonstrated that cocultured MDA-MB-231 BCCs exhibited a marked decline in OCR, a reflection of oxidative phosphorylation (Figure 5a). The main differences were in the maximal and reserve capacity of

cocultured BCCs (Figure 5b). Since cocultured BCCs exhibited a significantly higher resting metabolism, these values were obtained by scaling to the second basal metabolic point of the noncocultured BCC control. To account for changes in cell number from cell death, all data points were normalized to total protein within each well. The reduction in OCR in cocultured BCCs was accompanied by a decrease in the ECAR, a reflection of glycolytic activity (Figure 5c). Finally, plotting the ratio of OCR to ECAR established that cocultured BCCs relied more heavily on oxidative phosphorylation compared to glycolysis even though both of these values declined in the cocultured BCC population (Figure 5d). Suppression of EMT in Triple Negative BCCs. Triple negative BCCs are capable of metastasizing and forming tumors at secondary sites within the body. Prerequisites for metastasis include the ability to invade surrounding tissue, intravasate blood vessels, and migrate throughout the body. These changes are governed by a process known as EMT, whereby cells lose attachments to surrounding cells and tissue and gain migratory abilities. We assessed the influence of ESC-microstrands on EMT in triple negative MDA-MB-231 BCCs by examining cell invasion, migration, and EMT marker expression. Results from a Matrigel invasion assay demonstrated that cocultured MDAMB-231 BCCs became less invasive than the noncocultured BCC control (Figure 6a). The mobility of noncocultured and cocultured BCCs was analyzed using 4 h time-lapse videos (Video S1), and individual cells were tracked using a Mastracker plugin. Total displacement calculations showed that cocultured MDA-MB-231 BCCs migrated approximately one-third as far as the noncocultured BCC control (Figure 6b). The total path length traveled was also significantly reduced following coculture with ESC-microstrands (Figure 6c). Finally, cocultured BCCs F

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Figure 6. Coculture with ESC-microstrands restricted the metastatic potential of triple negative BCCs. (a) Diminished cell invasion of MDA-MB-231 BCCs following coculture with ESC-microstrands. Decreased cell migration measured as (b) total displacement, (c) total path length, and (d) average velocity. (e) Confocal images of cocultured MDA-MB-231 BCCs compared to those of (f) the noncocultured BCC control costained with DAPI nuclear stain (blue) displayed augmented E-cadherin (green) and decreased vimentin (red) expression, indicating suppression of EMT. (g) Western blot analysis showed increased E-cadherin and decreased vimentin expression following coculture with ESC-microstrands. (h) Densitometry analysis indicated that the altered EMT marker expression following coculture was statistically significant compared to that of the noncocultured MDA-MB-231 BCCs. **** = p < 0.0001 and * = p < 0.05.

displayed an average velocity that was markedly less than noncocultured BCCs (Figure 6d). The relative protein expression levels of EMT markers, vimentin and E-cadherin, were probed using immunocytochemistry costaining with

confocal microscopy (Figure 6e and f) and Western blot analysis (Figure 6g and h). Cocultured MDA-MB-231 BCCs exhibited reduced levels of vimentin and increased E-cadherin intensity compared to those of the noncocultured BCC control (Figure G

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cancer cell behavior37 but not human triple negative BCCs (data not shown). We speculated that pluripotent soluble factor secretion of ESCs within alginate microbeads was limited by their large diameter (500−600 μm). Using alginate microstrands with a smaller diameter (≤200 μm) could overcome the diffusion limit for oxygen and nutrient transport. Moreover, the long tubular structure provides a higher surface area-to-volume ratio than other bulk hydrogel constructs, allowing high cellularity and free release of soluble factors secreted by ESCs. Hydrogel microstrands were studied for supporting ESC expansion and differentiation41−44 and reconstituting living tissues.45 Alginate hydrogel is an ideal candidate for mimicking the semisolid and fluidic characteristics of the embryonic microenvironment, which support ESC self-renewal and pluripotency.41 In addition, the alginate hydrogel system is inert and lacks biological recognition molecules (e.g., adhesive ligands and ECM proteins), rendering a 3D microenvironment for the study of cell−cell interactions. In our established cancer restriction model, cultivation of ESCs in alginate hydrogel microstrands offered a unique 3D embryoniclike microenvironment for study of the influence on triple negative BCC growth and metastatic potential. Cell proliferation assays and cell cycle analysis revealed reduced proliferative capacity of aggressive MDA-MB-231 BCCs following coculture with ESC-microstrands. MCF7 BCCs represent the less aggressive luminal subtype of human breast cancer, while metastatic MDA-MB-231 cells mainly comprise the aggressive basal subtype. Results from the proliferation assay indicated that reduced proliferation following exposure to ESCmicrostrands is unique to MDA-MB-231 BCCs. ESCs and MDA-MB-231 BCCs utilize similar signaling pathways to control proliferation, and these results suggest that hyperactivated signaling pathways specific to triple negative BCCs are being reversed by pluripotent signaling from ESC-microstrands. The significant decrease in MDA-MB-231 BCC proliferation following coculture with ESC-microstrands, coupled with the negligible impact of empty microstrands, showed that the alginate is not responsible for these observed changes. Since 3T3 fibroblasts have a mesenchymal phenotype, comparable to MDA-MB-231 BCCs, a similar effect was expected following coculture. Surprisingly, there was no noteworthy change in 3T3 fibroblast proliferation, and this may be due to their normal signaling pathway regulation compared to that of metastatic MDA-MB-231 BCCs. Cell cycle analysis data showed a shift of the cocultured MDA-MB-231 BCC population out of the G2/M phase signifying reduced proliferation. This change coincided with an increase in the number of cells within the S phase of the cell cycle, implying some form of S phase arrest within these cocultured BCCs. On the basis of cell cycle data, cocultured MDA-MB-231 BCCs developed a SubG1 peak indicative of the presence of apoptotic and/or necrotic cells. This was confirmed though further analysis of MDA-MB-231, MCF7, and 3T3 cells using an apoptosis assay, which showed that coculture with ESCmicrostrands had the greatest impact on apoptosis and necrosis within the metastatic MDA-MB-231 BCC population. The Warburg theory states that highly aggressive cancer cells exhibit increased reliance on glycolysis even in the presence of oxygen to meet their energy demands when compared to that of noncancerous cells, which rely primarily on the tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS). However, recent studies are emerging that imply oncogenic cellular metabolism is more complex than previously thought in that cancer cells are capable of converting to the TCA cycle and OXPHOS for ATP production when exposed to (1) an

6e−h), indicating a reversion of the EMT in triple negative cancer after coculture with ESC-microstrands. Application of the 3D ESC-Microstrand Model to Identify Soluble Factors Associated with Dysregulated Oncogenic Signaling in MDA-MB-231 BCCs. EGFR and canonical Wnt/β-catenin signaling are convergent in embryonic development and tumorigenesis. Relative mRNA expression levels of two soluble factors (NKD2 and TGF-α) associated with the EGFR and canonical Wnt/β-catenin signaling pathways were assessed using qRT-PCR following coculture with ESC-microstrands. These signaling pathways are precisely regulated in embryonic stem cells but hyperactivated in metastatic MDA-MB231 BCCs. NKD2 negatively regulates the canonical Wnt/βcatenin signaling pathway, and its expression is decreased in MDA-MB-231 BCCs. TGF-α is involved in a positive feedback loop in the EGFR signaling pathway and is up-regulated in MDAMB-231 BCCs. Coculture with ESC-microstrands caused a 2fold increase in NKD2 expression and decrease in TGF-α expression at mRNA level, respectively (Figure 7).

Figure 7. Coculture with ESC-microstrands altered the gene expression of two soluble factors associated with dysregulated EGFR and canonical Wnt/β-catenin signaling. Both signaling pathways are hyperactivated in MDA-MB-231 BCCs. Relative mRNA expression of NKD2, a negative regulator of the canonical Wnt/β-catenin signaling pathway, was increased following coculture with ESC-microstrands. TGF-α is involved in a positive feedback loop with the EGFR signaling pathway, and its relative mRNA expression was decreased following coculture with ESC-microstrands.

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DISCUSSION Breast cancer is the most prevalent type of cancer in the United States, and the National Cancer Institute predicts that the number of new diagnoses will increase by 50% by 2030.38 In order to successfully reverse this trend, it is critical to address current triple negative breast cancer treatment challenges, such as chemotherapeutic drug resistance and the lack of targeted therapies.39,40 This study provides a unique approach to addressing these issues through the establishment of a 3D ESC model to restrict and study triple negative breast cancer. Retaining undifferentiated ESCs in a 3D microenvironment allows for the recreation of in vivo embryonic conditions, promoting pluripotency and developmental soluble factor secretion and signaling networks. In order for this model to work properly, ESC survival and pluripotency must be maintained following encapsulation, and this was confirmed using live/dead staining and probing for pluripotent marker expression. Previous work from our group demonstrated that ESCs encapsulated in alginate microbeads could inhibit rat breast H

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The canonical Wnt/β-catenin and EGFR signaling pathways are hyperactivated in human breast cancer. NKD2 is hypermethylated and down-regulated in human breast cancer and is considered a tumor suppressor due to its negative regulation of the canonical Wnt/β-catenin signaling pathway.55 The increase in NKD2 mRNA expression following coculture with ESCmicrostrands denotes a reversal of its methylation status and may signify a renewed ability to direct canonical Wnt/β-catenin signaling pathway inhibition. Soluble TGF-α is a ligand for the EGFR signaling pathway, and its up-regulation in MDA-MB-231 BCCs contributes to hyperactivation of the EGFR signaling pathway. Coculture with ESC-microstrands led to a decrease in relative TGF-α mRNA expression, which may signify restored EGFR signaling pathway function. Both results indicate that the 3D ESC-microstrand model can be applied to identify soluble factors responsible for BCC inhibition. This study confirms that the ESC-derived microenvironment could restrict cancer cell proliferation and survival, which is consistent with reports based on ESC-conditioned matrix or media for inhibiting cancer cell growth.23−26 More importantly, we have shown for the first time that the ESC-derived microenvironment could not only reverse the Warburg effect in cancer cell metabolism, a hallmark of cancer that causes increased reliance on glycolysis over oxidative phosphorylation for rapid energy production, but also reverse EMT, a hallmark of cancer metastasis. In this work, the ability of this in vitro ESC model to reverse the metastatic potential of triple negative BCCs is more pronounced compared to the effects on proliferation, survival, and metabolism, indicating that this model could be successfully applied to the study of highly aggressive and resistant cancer cells. Future work will apply this model to pinpoint specific molecules within dysregulated triple negative breast cancer signaling pathways for targeted therapy. In addition to breast cancer, this cancer restriction model can be applied to other tumors for reprogramming to a less aggressive phenotype and therapeutic target discovery.

environment that is undergoing lactic acidosis, (2) specific anticancer drugs that may be contributing to cellular senescence, and/or (3) increased oxygen levels, such as what occurs at the periphery of a tumor.46−49 This behavior is well-documented in cancer stem cells, which are notorious for their extreme aggressiveness and ability to repopulate an entire tumor following treatment.50 This suggests that the decreases in both glycolytic rate and OCR in MDA-MB-231 BCCs following coculture with ESC-microstrands reflected the heterogeneous nature of the MDA-MB-231 BCC. It is unlikely that these changes were due to induced cellular senescence because these cocultured cancer cells did not have a senescent cell morphology (large and flat) and experienced increased apoptosis not conventionally observed in senescent cells. Furthermore, it is frequently senescent fibroblasts within the tumor microenvironment that contribute to cancer cell senescence through secretion of senescence-associated proteins.51 Recent studies suggest that the increased apoptosis following coculture with ESC-microstrands is directly attributable to inhibited OXPHOS and increased ROS production.52 These studies have triggered attempts to utilize drugs that specifically target mitochondrial respiration to kill cancer cells.48 For example, the type II diabetes drug, metformin, is being investigated as an anticancer drug as it has recently been shown to inhibit Complex I in OXPHOS.53 The increased cellular stress caused by ROS production was likely responsible for the depletion in reserve capacity OCR as it is widely accepted that cells undergoing extreme stress draw on their reserve capacity, which when depleted, results in protein damage and cell death.54 The cocultured cancer cells appeared to revert from abnormal cancer metabolism to a more typical metabolic profile in that they relied more heavily on OXPHOS than glycolysis for cellular metabolism. The fact that this did not coincide with measurable changes in ATP-linked OCR suggests that this effect was more influenced by decreased reliance on glycolysis, as opposed to, increased reliance on OXPHOS. Decreased glycolytic rate following coculture with ESC-microstrands did not coincide with decreased extracellular lactate production. This could be explained by considering that pyruvate is involved in other metabolic pathways which may contribute to its generation rate being equivalent to its removal rate even when the glycolytic rate is reduced.49 The fact that both OCR and glycolytic rate were suppressed in the restricted BCCs supports the notion that treatment with multiple drugs that individually target glycolysis and OXPHOS may produce superior results in the fight against highly metastatic cancer. Exposure of highly aggressive MDA-MB-231 BCCs to ESCmicrostrands induced a transformation from a metastatic mesenchymal to a benign epithelial phenotype. This was evident in that BCCs exhibited decreased ability to invade Matrigel, reduced migratory ability, and altered EMT marker expression. The greatest disparities between cocultured and noncocultured BCCs were observed in their migratory ability. The fact that both velocity and total displacement were decreased indicated that reduced migration in the cocultured BCCs was triggered by increased cell-to-cell contacts due to up-regulation of the epithelial marker E-cadherin and decreased expression of the mesenchymal marker vimentin, which is responsible for cellular motility through modulation of intermediate filament composition.31 This was further supported by immunocytochemistry and Western blot analysis for the EMT markers E-cadherin and vimentin. These results clearly demonstrated that ESC-microstrands restricted the metastatic potential of human triple negative BCCs through the reversal of EMT.

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CONCLUSION

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ASSOCIATED CONTENT

We have established an in vitro 3D ESC-based tumor suppressive microenvironment using alginate hydrogel microstrands. It restricts highly aggressive, metastatic BCC proliferation, metabolism, and survival. In particular, the ESC-microstrands markedly restrict the migration and invasion capacity of these highly aggressive cancer cells through the reversal of EMT. Our mechanistic studies indicate that pluripotent soluble factors secreted by ESCs could restore the regulation to specific signaling pathways that control EMT, and future studies will focus on elucidating the exact mechanism for restricting metastasis.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00237. Sequence of primers for qRT-PCR (PDF) Overall hindered migratory abilities in metastatic MDAMB-231 BCC migration after exposure to ESC-microstrands (AVI) Noncocultured BCCs as the control (AVI) I

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AUTHOR INFORMATION

Corresponding Author

*Phone: 518-956-7381. Fax: 518-956-8687. E-mail: YXie@ sunypoly.edu. ORCID

Yubing Xie: 0000-0003-1395-7365 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by National Science Foundation sponsored CBET 0846270 and DBI 0922830. We thank Dr. Nadine Hempel and Dr. Susan Sharfstein for metabolic analysis and Dr. Tom Begley and Dr. Lauren Endres for flow cytometry image analysis.

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