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Gelatin Methacrylate Hydrogels as Biomimetic Three-Dimensional Matrixes for Modeling Breast Cancer Invasion and Chemoresponse in Vitro Anuradha D. Arya,†,‡ Pavan M. Hallur,†,‡ Abhijith G. Karkisaval,§ Aditi Gudipati,† Satheesh Rajendiran,⊥ Vaibhav Dhavale,⊥ Balaji Ramachandran,⊥ Aravindakshan Jayaprakash,⊥ Namrata Gundiah,§ and Aditya Chaubey*,† †

Anti-Cancer Technologies Program, Mazumdar Shaw Center for Translational Research, Narayana Hrudayalaya Health City, Hosur Road, Bangalore 560 099, India § Department of Mechanical Engineering, Indian Institute of Science, Bangalore 560 012, India ⊥ In Vivo Pharmacology−Oncology, Syngene International Ltd., Plot Nos. 2 & 3, Bommasandra IV Phase, Jigani Link Road, Bangalore 560 099, India S Supporting Information *

ABSTRACT: Recent studies have shown that three-dimensional (3D) culture environments allow the study of cellular responses in a setting that more closely resembles the in vivo milieu. In this context, hydrogels have become popular scaffold options for the 3D cell culture. Because the mechanical and biochemical properties of culture matrixes influence crucial cell behavior, selecting a suitable matrix for replicating in vivo cellular phenotype in vitro is essential for understanding disease progression. Gelatin methacrylate (GelMA) hydrogels have been the focus of much attention because of their inherent bioactivity, favorable hydration and diffusion properties, and ease-of-tailoring of their physicochemical characteristics. Therefore, in this study we examined the efficacy of GelMA hydrogels as a suitable platform to model specific attributes of breast cancer. We observed increased invasiveness in vitro and increased tumorigenic ability in vivo in breast cancer cells cultured on GelMA hydrogels. Further, cells cultured on GelMA matrixes were more resistant to paclitaxel treatment, as shown by the results of cell-cycle analysis and gene expression. This study, therefore, validates GelMA hydrogels as inexpensive, cell-responsive 3D platforms for modeling key characteristics associated with breast cancer metastasis, in vitro. KEYWORDS: 3D culture, gelatin methacrylate, hydrogels, breast cancer, chemoresponse, invasiveness

1. INTRODUCTION Breast cancer is the most common malignancy in women worldwide. In most patients, the primary cause of death is distal metastasis.1 Understanding the biology of metastases is critical to improving treatment outcomes. However, central questions like the organ specificity of metastases and the role of the metastatic niche in the establishment of secondary tumors remain unanswered.2 A key challenge in these efforts has been the lack of easy-to-use tumor models that can realistically represent the metastatic condition.3 It is now understood that the stromal microenvironment functionally contributes to carcinogenic progression by influencing cell responses via mechanical, topographical, and biochemical cues.4,5 However, in vitro models based on two-dimensional (2D) culture systems have limited ability to simulate complex cellular behaviors associated with metastasis like adhesion, motility, invasion, dormancy, and the establishment of secondary tumors.6 © 2016 American Chemical Society

Resistance to chemotherapy is an additional complication of metastatic progression,7 with tremendous clinical impact. Elucidation of the basic mechanisms of chemoresistance and assaying of cellular response to drugs is mainly done using 2D monolayer cell cultures. However, extrapolation of the results obtained from such preclinical 2D models to malignant transformation has limited predictive power8 and could affect the success rate of assayed drugs in later phases of clinical trials.9 Therefore, complex in vitro culture models that can more realistically mimic in vivo cell behaviors are the need of the hour.10 In this context, three-dimensional (3D) culture systems are considered to be physiologically relevant for analysis of the Received: May 26, 2016 Accepted: August 5, 2016 Published: August 5, 2016 22005

DOI: 10.1021/acsami.6b06309 ACS Appl. Mater. Interfaces 2016, 8, 22005−22017

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ACS Applied Materials & Interfaces

Figure 1. GelMA-based hydrogel preparation. Critical steps of GelMA-based hydrogel preparation. Gelatin (1) and methacrylic anhydride [ratio 1:0.6 (w/w)] were reacted to add methacrylate pendant groups to gelatin (GelMA; 2). The methacrylated gelatin (GelMA polymer) was dissolved in PBS at 37 °C, mixed with the photoinitiator Irgacure-2959 (3), and cross-linked in the presence of 365 nm UV light (4). Gels thus polymerized within culture plates (5) were used for experiments.

gene function and cell phenotype, ex vivo.11 One of the earliest substrates used for culturing cells in 3D is matrigel, a solubilized extract derived from the Engelbroth−Holm−Swarm mouse sarcoma cells.12 However, being a biological product, its components including extracellular matrix (ECM) protein, growth factor, and endotoxin levels vary between lots. Moreover, it is pliable, and its mechanical properties cannot be modified to mimic that of native tissue.13 Therefore, in recent years, multidisciplinary approaches have focused on the development of alternatives suitable for modeling pathological conditions.14 Among the various scaffold-based matrix options available for 3D cell culture, hydrogels, which are macromolecular networks formed by hydrophilic polymers swollen in water or biological fluids, show the most promise mainly because of their similarity with a natural ECM.15 Hydrogels display favorable hydration properties and can typically be fabricated under cytocompatible conditions from natural or synthetic polymers. Hydrogels developed from poly(ethylene glycol) have been widely used for tissue engineering; however, they are generally biologically inert and need the incorporation of cell adhesion and degradation cues to render them cell-compatible.16 Hence, hydrogels made from natural polymers are desirable for specific applications. Gelatin is a natural polymer obtained by denaturing type 1 collagen from mammalian17−19 and nonmammalian20 sources. It retains natural cell-binding motifs and degradation sites. Compared to fish gelatin, which has inferior mechanical properties because of a lower denaturation temperature, gelatin from mammalian sources forms hydrogels more easily.20 Further, mammalian gelatin is rich in domains that bind to cell-surface receptors and to other ECM proteins, offering an excellent substrate for the attachment of adherent cells.21 Gelatin can be cross-linked by various methodsphysical, chemical and enzymaticto develop hydrogels suitable for use as cell-culture platforms. The choice of the cross-linking agent

largely depends on the end application. Aldehyde cross-linking agents are often cytotoxic,22 genipin, a natural cross-linker, requires long gelation times,23 and transglutaminase used for enzymatic cross-linking has been reported to be cytotoxic in long-term culture.24 Functionalization of gelatin with methacrylate, followed by photopolymerization, yields inexpensive and stable hydrogels.25 Photo-cross-linking is carried out using a water-soluble photoinitiator under UV light. Advantages of photo-cross-linking include a fast curing rate and better control of cross-linking, conferring the ability to control pore formation in hydrogels, which is critical for cell-culture applications. A common choice for a photoinitiator is 2-hydroxy-1-[4-(2hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959), which has high water solubility and low cytotoxicity.26 Several previous studies have demonstrated that gelatin methacrylate (GelMA) hydrogels developed using this method support the growth and proliferation of cancer cells cultured on them.27,28 Therefore, in recent years, GelMA hydrogels are increasingly used in biomedical applications.29−32 However, to determine the suitability of these hydrogels for specific applications, such as models for disease progression or as 3D drug assay systems, characterization of their physical properties and the molecular changes in cells following culture on such systems is essential. In this study, we investigate whether GelMA hydrogels are suitable in vitro 3D culture systems to model key characteristics of metastatic progression in breast cancer, in particular, invasiveness and chemoresponse.

2. MATERIALS AND METHODS 2.1. Fabrication of GelMA Hydrogels. GelMA was synthesized (Figure 1a) by the reaction of gelatin (type A, isolated from porcine skin; MP Biomedicals, USA) and methacrylic anhydride (SigmaAldrich, USA), following previously described protocols.25,27 After the dissolution of gelatin in phosphate-buffered saline (PBS; pH 7.4) at 50 °C, 20% (w/v) of methacrylic anhydride was added dropwise. The solution was vigorously stirred at room temperature for 22006


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ACS Applied Materials & Interfaces 1 h, diluted with double the quantity of PBS, and dialyzed for 3 days against distilled water at 37 °C. The solution was then freeze-dried in a lyophilizer (Alpha 1-2/LD plus, Martin Christ, Germany) to obtain methacrylamide-modified gelatin as a dry white powder. Previous studies have reported that this method results in GelMA with around 80% methacrylation27,33,34 of gelatin. A microporous 3D hydrogel of methacrylamide-modified gelatin was obtained by exposing varying percentages (5%, 7.5%, 10%, and 15%) of a GelMA solution in culture vessels (Eppendorf, USA) to 365 nm UV for 10 min in the presence of 0.5 mg/mL of the photoinitiator 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure2959, Sigma-Aldrich, USA). Hydrogels thus obtained were equilibrated with 100 μL of a culture medium for 24 h prior to seeding of the cells for experiments. 2.2. Analysis of the Mechanical Properties of GelMA Hydrogels. 2.2.1. Determination of the Compression Modulus and Viscoelastic Properties of GelMA Hydrogels. Monotonic compression and dynamic mechanical analysis (DMA) tests of GelMA hydrogels were performed with a Bose Electroforce 3200 (Bose Corp., USA) instrument using parallel compression plates. Unloaded gel heights and diameters were measured prior to the experiment using digital images (Nikon D7000) and ImageJ software.35 For monotonic compression studies, displacements were measured using a linear variable durable transducer and the corresponding forces with a load transducer (Bose Corp., ±22.5 N). GelMA hydrogels were preloaded to 7 g force, preconditioned at 10% strain for 30 cycles using sinusoidal loading at 0.05 Hz to obtain repeatable responses, and tested under quasi-static conditions using ramp loading at 0.01 mm/s until about a maximum of 45% strain value was reached.36,37 Customwritten programs in MATLAB (R2014b) were used to analyze the load−displacement data. The compression moduli of the gels were calculated from the linear part of the stress−strain curve at 1−5% incremental strain. In all, four batches of 10% (n = 16) and three batches of 15% GelMA gels (n = 12) were tested using monotonic compression. The 5% and 7.5% gels, being pliant, could not be similarly tested. To measure the viscoelastic properties of the hydrogels, we used dynamic mechanical testing. Samples from 10% (n = 8) and 15% (n = 8) GelMA groups were preloaded to 7 g and dynamically tested under small deformation (0−10% strain) compression loading using a cyclic sinusoidal load which varied from 0.05 to 130 Hz. The frequency of data acquisition was set to about 30 times the loading frequency. Custom-written codes in MATLAB were used to perform discrete Fourier transform infrared on the stress and strain data from each loading block corresponding to each frequency sweep.38 Stress and strain data from each loading block were next used to calculate the storage modulus (E′), loss modulus (E″), and phase angle (δ) for the hydrogels.39 2.2.2. Pore Size of GelMA Hydrogels. Samples of varying concentrations of GelMA hydrogels ranging from 5% to 15% were frozen at −80 °C and lyophilized. Cross sections of lyophilized samples of GelMA hydrogels were sputter-coated with 20-mÅ-thick platinum using a JEOL JFC 1600 fine coater for 90 s. 200× magnified images were recorded with a JEOL JSM-6480 LV scanning electron microscope operating at an acceleration voltage of 15 kV, under low vacuum. ImageJ software was used to calculate pore sizes of the hydrogels from the images.35 2.3. Decellularization of Primary Tumor Tissue. A primary breast tumor surgical biopsy obtained from a consenting patient was sliced into 0.5 cm pieces and decellularized by incubation in Tris-buffer containing 0.5% (w/v) sodium dodecyl sulfate (Sigma-Aldrich, USA) at 37 °C for 24 h, followed by treatment with DNase I (20 U/ml; Sigma-Aldrich, USA) and RNase A (0.2 mg/mL; Sigma-Aldrich, USA) containing 50 mmol/L MgCl2 (Merck, USA) for 24 h, under continuous shaking at 37 °C. Aseptic PBS was employed after all procedures to remove the residual substances. The decellularized tissue was fixed in 2.5% glutaraldehyde (Sigma-Aldrich, USA) for scanning electron microscopy (SEM) imaging. The cross sections of the freeze-dried samples were sputter-coated with 20-mÅ-thick platinum using a JEOL JFC 1600 fine coater for 90 s. The images

were recorded with a JEOL JSM-6480 LV scanning electron microscope operating at an acceleration voltage of 15 kV. 2.4. Cell Culture. The M D Anderson Metastatic Breast 231 (MDA MB 231) and 468 (MDA MB 468) cancer cell lines were generous gifts from Dr. Annapoorni Rangarajan, Indian Institute of Science, Bangalore, India, and Dr. Arkashubhra Ghosh, GROW Laboratories, Narayana Nethralaya, Bangalore, India, respectively. The basal medium used for all cell culture experiments was Dulbecco’s Modified Eagle’s medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen, USA), 1% nonessential amino acids (MP Biomedicals, USA) and antibiotics streptomycin sulfate and benzyl penicillin (Gibco, Invitrogen, USA) at final concentrations of 100 μL/mL and 100 U/mL, respectively. Cell cultures were maintained at 37 °C in an atmosphere of 5% CO2 prior to experiments. To generate 3D cultures on hydrogels, cells were seeded on top of GelMA hydrogels, at a concentration of 5 × 104 cells per well of a 24-well plate, for up to 7 days, with a partial medium change on alternative days. To generate 3D spheroids, MDA MB 231 cells were seeded under nonadherent conditions in ultralow-attachment 24-well plates (Corning, USA) at a concentration of 5 × 104 cells/well, for up to 5 days, with a partial medium change on alternative days. Bright-field microscopy was used to visualize cells. To obtain data on cell penetration into the hydrogels, green fluorescent protein (GFP)-tagged MDA MB 231 cells (Cell BioLabs, USA) were seeded on top of the hydrogels and cultured for 5 days. Optical slices were obtained from 3D cultures at intervals of 1 μm along the z axis using a Leica TCS SP5 confocal microscope. Z-stack images were compressed and reconstituted using Leica Application Suite Advanced Fluorescence software. Unless otherwise mentioned, all biological experiments were conducted using 10% GelMA hydrogels. Except for cell proliferation assays, all experiments were carried out for 5 days. Cells cultured on tissue culture polystyrene (TCPS) surfaces for up to 80% confluency served as the conventional 2D controls for all experiments against which cells cultured on GelMA hydrogels were evaluated. 2.5. Analysis of Cell Proliferation. AlamarBlue (Invitrogen, USA) was used to quantify metabolic activity in a proliferation assay. A total of 1 × 104 cells in 100 μL of culture media were cultured per well in both 2D TCPS dishes and 3D GelMA hydrogels of varying concentrations (5%, 10%, and 15%) in 96-well plates for 1−7 days. At specific time intervals, 4% (v/v) of AlamarBlue reagent was added to each well, incubated at 37 °C/5% CO2 for 4 h, and the absorbance was read at 570 and 590 nm. 2.6. RNA Isolation and Quantitative-Real Time Polymerase Chain Reaction (qRT PCR). RNA was isolated from cells using TRIZOL reagent following the manufacturer’s instructions. cDNA was synthesized from 1.0−2.0 μg of RNA using a high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA) as per the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was carried out using a Fast SYBR Green qPCR Kit (Roche) using cDNA as the template and as the passive reference dye. Gene expression was normalized to that of 18S rRNA, and the fold change was calculated using the 2−ΔΔCt method.40 The sequences of the primers used are listed in Table S1. 2.7. Cellular Migration and Invasion Assay. Cells cultured on 2D TCPS were harvested by trypsinization, and those cultured on 3D hydrogels were harvested by enzymatic degradation of the hydrogel using collagenase and suspended in a serum-free basal medium. For invasion assays, these cells were then plated (104 cells/chamber) onto invasion chambers (Sigma-Aldrich, 8.0 μm pore size) coated with 1 mg/mL matrigel (Sigma-Aldrich, USA). The upper chamber contained a serum-free basal medium. In the lower chamber, a medium with 10% FBS was used as a chemoattractant. After 24 h, the medium was removed and the chambers were washed twice with PBS; noninvading cells were removed from the upper surface of the membrane by gentle wiping with a cotton-tipped swab; invading cells on the lower surface of the membrane were fixed with 4% formaldehyde in PBS for 10 min, washed twice with PBS, permeabilized with methanol for 20 min, washed twice with PBS, stained with 0.4% crystal violet for 15 min, and washed twice with PBS. In each chamber, 10 fields were 22007


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Figure 2. Mechanical characterization of GelMA hydrogels. Stress−strain curves for (A) 10% GelMA hydrogels (n = 13) and (B) 15% GelMA hydrogels (n = 11). Compression modulus values (average ± standard deviation) are significantly higher for 15% GelMA samples compared to the 10% group (C). The error bar represents standard deviation. ** indicates p < 0.01. DMA for a representative sample from the 10% GelMA hydrogel group (D). photographed at a magnification of 10× and invading cells were counted in each field. The fold increase in invasion was calculated by normalizing the total number of invading cells from the hydrogel group to the total number of invading cells from the TCPS group. Migration assays were performed similarly but with uncoated inserts. 2.8. In Vivo Tumorigenicity Assay. All animal experiments were performed with approval from the Institutional Animal Ethics Committee (Protocol No. SYNGENE/IAEC/538/08-2015). 8-weekold female NOD-SCID mice used for in vivo animal experiments were housed under pathogen-free conditions. MDA MB 231 cells cultured on hydrogels were harvested by enzymatic degradation of the hydrogel and resuspended in 100 μL of a serum-free basal medium. Cells cultured on TCPS and treated similarly served as controls. A total of 1 × 106 cells were injected into each mouse via tail vein; three animals each were used per batch. Six weeks postinjection, animals were sacrificed and dissected to detect the presence of tumors. 2.9. Drug Sensitivity Assay. The metabolic activity of cells cultured under different conditions was analyzed with an MTT colorimetric assay. MDA MB 231 cells were seeded on the hydrogels and TCPS dishes at a density of 1 × 104 per well of a 96-well plate. Drug treatment was started after 72 h for the cells on the hydrogels and after 24 h for the cells cultured on TCPS dishes. Paclitaxel (SigmaAldrich, USA) concentrations ranging from 0.25 nM to 10 μM were used for assaying drug sensitivity. At predetermined times, 20 μL of a 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA) solution was added to each of the wells. After 2 h of incubation, the supernatant was carefully removed and 100 μL of a dimethyl sulfoxide (Sigma-Aldrich, USA) solution was added to dissolve the formazan crystals. After shaking for 10 min on a plate minishaker, 100 μL of solution from each well was transferred into the wells of a 96-well plate, and the absorbance was read at 570 nm with a

reference wavelength of 650 nm (Infinite F200 Pro, Tecan, USA). Four replicates were averaged for each concentration per experiment. IC50 values were derived for three time points (24, 48, and 72 h) using GraphPad Prism 5. 2.10. Cell-Cycle Analysis. Cells were retrieved from the GelMA hydrogels and TCPS dishes after 16 h of treatment with 3× and 10× the respective IC50 concentrations of paclitaxel, determined for 2D and 3D cultures in the MTT assay. The cells were fixed in 70% (v/v) cold ethanol and stored at −20 °C for 1 h, treated with RNase (10 μg/ mL), and stained with 40 μg/mL propidium iodide (PI; SigmaAldrich, USA) for 30 min in the dark. Cell-cycle distribution was determined by flow-cytometric analysis in a BD FACS Calibur instrument using the red fluorescence range of excited PI-stained nuclei as a measure of the DNA content. Linear displays of fluorescence emissions were used to compare cell-cycle phases and quantitate the cells with degraded sub-G1 DNA content characteristic of apoptotic cells. 2.11. Statistical Analysis. Unless otherwise indicated, data are the mean ± standard deviation of at least three individual biological experiments. All group differences were evaluated by a two-tailed unpaired Student’s t test, and p values of less than 0.05 were considered significant. Analysis of the data from flow-cytometry experiments was performed using software BD Cellquest Pro, version 6.0.

3. RESULTS 3.1. 3D Cell Culture Scaffolds Developed from GelMA Hydrogels Exhibit Mechanical and Morphological Properties Similar to Those of Breast Tissue. In this study, we aimed to fabricate biomimetic 3D culture matrixes from GelMA for modeling breast cancer metastatic progression. 22008


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Figure 3. Characterization of the physical properties of GelMA. SEM micrographs of (A) 10% GelMA hydrogels (bar = 50 μm) and (B) decellularized human breast tumor tissue (bar = 200 nm) showing gross morphological similarities. An increase in the polymer concentration resulted in hydrogels with smaller pore size; the results are average relative pore sizes of n = 3 gels each. The error bar represents standard deviation. *** indicates p < 0.001 (C). Confocal micrograph showing the penetration of GFP-tagged MDA MB 231 cells into 10% GelMA hydrogels (D).

mechanical experiments as described earlier. The results from a representative sample from the 10% GelMA hydrogel group are shown in Figure 2D. The specimen had a relatively constant value for E′ and E″ until ∼60 Hz, following which there are oscillations in the curves. tan δ, given by the ratio of E″ to E′, shows a similar behavior. These oscillations may be due to either microscopic local failures in the gels that were not directly visible or resonance. Storage and loss moduli from the 10% and 15% GelMA groups were calculated from the plateau region of the curves until ∼60 Hz. The storage modulus, E′, was higher than the loss modulus, E″, which reflects a more elastic behavior. The E′ values for 15% gels were 389.55% higher than those for 10% (6.03 ± 1.29 kPa); the corresponding E″ values were also 96.6% higher than 0.89 ± 0.53 kPa for the 10% hydrogels. SEM images of lyophilized 10% GelMA hydrogels (Figure 3A) indicated that qualitatively the gross morphology of GelMA hydrogels is similar to that of decellularized breast ECM (Figure 3B). Next, we used the SEM images to calculate the pore size of the hydrogels. Because the pore size of the hydrogels is influenced by the freezing process prior to lyophilization,41 analysis of the SEM images yields a relative (not absolute) pore size. We found that the average relative pore size decreased with an increase in the concentration of the hydrogel substrate (Figure 3C), with 10% GelMA hydrogels showing interconnected pores averaging 40 ± 4.5 μ size. SEM images of 5%, 7.5%, and 15% GelMA hydrogels are provided in the Figure S1A−C.

The protocol followed for the preparation of GelMA hydrogels is outlined in Figure 1. In order to ensure that the mechanical properties of our 3D culture matrixes are similar to that of the native breast tissue, we characterized the stiffness and elasticity of the GelMA hydrogels using compression and dynamic mechanical testing, respectively, and selected the matrix having stiffness and elasticity similar to that of native tissue, as reported in the literature, for biological experiments. Parts A and B of Figure 2 show stress−strain data corresponding to samples of GelMA hydrogels from three batches of 10% (n = 13) and 15% (n = 11) GelMA, respectively. These data demonstrate nonlinear stress−strain responses, characteristic of other hydrogels, and are fairly repeatable. The GelMA hydrogels did not catastrophically fail at strains of ∼40%. Higher toughness of these gels compared to glutaraldehyde cross-linked gelatin samples may be linked to the presence of methacrylate in the gels.38 To compare differences between the two hydrogel groups, we calculated compression moduli (Figure 2C). The 10% hydrogels had an average compression modulus of 4.81 ± 0.73 kPa compared to the significantly higher values of 19.25 ± 6.85 kPa for the 15% hydrogels (p < 0.01). Gels in the 15% group also did not fail during testing, nor did they show any signs of macroscopic damage. However, we note higher variance in the results from the 15% group compared to the 10% GelMA samples. To characterize the viscoelastic properties of gels having different concentrations of GelMA, we used dynamic 22009


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Figure 4. Phenotypic properties of breast cancer cell lines cultured on 2D TCPS and GelMA hydrogels. Bright-field microscopy images of MDA MB 231 cells in 2D culture at 10× magnification (A) and on GelMA hydrogels at (B) 10× magnification (bar = 100 μm) and (C) 20× magnification (bar = 50 μm). Quantitative analysis of mRNA levels of genes associated with stemness normalized to 18S rRNA expression revealed significantly increased levels of OCT4, NANOG, KLF4, and CXCR4 in 3D cultures compared to 2D cultures (D). MDA MB 231 cell proliferation on GelMA hydrogels versus TCPS assessed using an AlamarBlue assay. AlamarBlue reduction at 570 and 600 nm in MDA MB 231 cells (E). The error bar represents standard deviation. ** indicates p < 0.01, and *** indicates p < 0.001.

Figure 5. 3D culture enhances the invasiveness of breast cancer cells. Boyden chamber migration assay for MDA MB 231 cells cultured on TCPS (A) and on GelMA hydrogels (B). Quantification of the migrated cells (C). Boyden chamber invasion assay for MDA MB 231 cells cultured on TCPS (D) and on GelMA hydrogels (E). Quantification of the invaded cells (F). The error bar represents standard error of the mean. *** indicates p < 0.001.

Confocal images of GFP-tagged MDA MB 231 cells cultured on 10% GelMA hydrogels for 5 days indicated that cells penetrated to a depth of 0.5 mm below the surface of the hydrogel (Figure 3D), confirming that 10% GelMA hydrogels are sufficiently porous to allow cells and nutrients to infiltrate the hydrogel matrix. 3.2. GelMA Hydrogels Sustain 3D Spheroids of Breast Cancer Cells. We established 3D spheroids of breast cancer cells in vitro by culturing MDA MB 231 cells on GelMA hydrogels for up to 7 days under static culture conditions. 2D cultured cells displayed a flattened spindle-like morphology (Figure 4A). In 3D cultures, cells adhered to hydrogels within 24 h of culture and clusters of cells migrated toward each other, permitting the formation of loose aggregate spheroids (Figure 4B). Further, within 3 days of culture, cells in spheroids

developed prominent invadopodia and, by day 5, started to migrate out of the spheroids (Figure 4C). The ability of GelMA hydrogels to support spheroid cultures of cancer cells was also observed with the noninvasive cell line MDA MB 468 (Figure S2A−C); however, in this case, the spheroids formed were more compact. Because the formation of cell spheroids is associated with an increase in stemlike characteristics, we analyzed the expression of markers associated with stemness (OCT4, NANOG, KLF4, and CXCR4) in our breast cancer cell spheroids. Our data showed significant (p < 0.01) upregulation of all of the genes analyzed upon 3D GelMA culture (Figure 4D). Further, we compared the expression of these genes in cells cultured on 3D GelMA hydrogels to gene expression in spheroids established under nonadherent conditions (Figure S2D). It was observed 22010


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Figure 6. Breast cancer cells cultured on GelMA hydrogels exhibit a strong metastatic potential in vivo. MDA MB 231 cells cultured on TCPS and injected intravenously into NOD-SCID mice did not form tumor nodules in the either thoracic cavity (A) or lungs (B), whereas cells cultured on GelMA hydrogels show strong metastatic potential in vivo by forming multiple focal tumor nodules in the thoracic cavity (C) and lungs (D) when injected similarly. Quantitative analysis of mRNA of candidate genes associated with invasiveness in breast cancer (TGFβ1, MMP2, VEGF, COL1A1, ITGB1, and SPTAN1) normalized to the expression of 18s rRNA mRNA (E). The error bar represents standard deviation. * indicates p < 0.05, and ** indicates p < 0.01.

that 3D cultured cells displayed 5 times more migratory potential compared to 2D cultured cells in the migration assay (p < 0.001; Figure 5A−C). A similar migration assay carried out with MDA MB 468 cells showed a lesser, but significant (p < 0.05), difference in the ability of 3D cultured cells to migrate through Transwell inserts compared to 2D cultured cells, which may be linked to the noninvasive phenotype of these cells (Figure S3A−C). This was further corroborated by the results of the invasion assay carried out with MDA MB 231 cells (Figure 5D−F), which also showed a significant increase in the invasive potential (p < 0.001). Additionally, we compared the invasive potential of MDA MB 231 cells cultured on GelMA hydrogels to the invasive potential of similar cells cultured under nonadherent conditions (Figure S3D). The results indicate that breast cancer cell spheroids obtained on GelMA hydrogels have cells that are more invasive than cells from spheroids generated under ultralow-attachment conditions (p < 0.01). This was further supported by analysis of the expression of proinvasive genes in MDA MB 231 cells cultured under these two sets of experimental conditions. GelMA-cultured MDA MB 231 spheroids have significantly higher levels of genes associated with invasive characteristics than spheroids generated under nonadherent conditions (Figure S3E). In order to assess whether the increased invasiveness observed in 3D cultured cells, in vitro, translated to increased tumorigenicity in vivo, we injected MDA MB 231 cells cultured in 2D and on GelMA hydrogels intravenously into the tail veins of 8-week-old NOD-SCID mice. The lungs and thoracic cavity of mice injected with 2D cultured cells were tumor-free 6 weeks postinjection (Figure 6A,B), whereas all mice injected with 3D cultured cells showed the presence of tumor nodules in the lungs and thoracic cavity (Figure 6C,D) in the same time period.

that while nonadherent 3D spheroids had significantly higher levels of these genes compared to 2D cultures, spheroids generated on GelMA hydrogels showed the highest expression of these genes, signifying that GelMA hydrogels are better able to sustain stemlike characteristics in cultured cells. Next, we compared the proliferation of MDA MB 231 cells cultured on GelMA matrixes to that of 2D cultured cells using an AlamarBlue assay. Cells cultured on 2D TCPS reached peak proliferation on day 3, after which there was a steady decline in the multiplication rate, presumably because overconfluency led to cell death (Figure 4E). By day 5, a significant difference (p < 0.001) between the 2D and 3D cultures was apparent, with a decrease in AlamarBlue absorbance in the 3D cultures (Figure 4E) indicating that GelMA hydrogels are able to maintain cells longer than 2D cultures. A similar assay carried out with noninvasive MDA MB 468 cells showed a similar trend in proliferation (Figure S2E). Moreover, our data showed that cell proliferation correlates with the mechanical properties of hydrogels. Cells cultured on 10% GelMA hydrogels showed a higher proliferation rate compared to 15% GelMA in both cell lines (Figure S2F,G), suggesting that 10% GelMA hydrogels are better able to sustain long-term cultures of cells. Although cell proliferation was better in 5% hydrogels (Figure S2F,G) in both cell lines, its mechanical properties precluded its use for biological experiments. 3.3. 3D Cultured Breast Cancer Cells Display Increased Migratory Behavior and Differentially Express Invasiveness Markers. Local infiltration of tumor cells into adjacent tissue by migration is the first stage of metastasis. In order to assess whether our 3D culture scaffolds are able to enhance migratory behaviors in cells, we evaluated the ability of MDA MB 231 cells cultured on GelMA hydrogels to migrate through Transwell chambers without matrigel (in a migration assay) and with matrigel (in an invasion assay). We observed 22011


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Figure 7. Breast cancer cells cultured in 3D show decreased sensitivity to paclitaxel. MDA MB 231 cells incubated with various concentrations of paclitaxel were monitored using MTT assays after a 72 h incubation period (A). The error bar represents standard error of mean. *** indicates p < 0.001. Quantitative analysis of mRNA levels of chemoresistance genes normalized to the 18S rRNA expression revealed significantly increased levels of ABCB1, CAV1, COX2, TUBB3, and CD9 in GelMA hydrogels compared to 2D TCPS (B). The error bar represents standard deviation. ** indicates p < 0.01, and *** indicates p < 0.001.

To understand the molecular changes that were driving this increased invasiveness, we assessed the levels of mRNA of selected genes associated with invasiveness in the context of breast cancer (TGFβ1, MMP2, COL1A1, VEGF, ITGβ1, and SPTAN1) in MDA MB 231 cells that had been cultured in 2D and on GelMA hydrogels, using real-time qPCR. The transition from a 2D to a 3D culture format upregulated the expression of MMP2, VEGF, SPTAN1, and TGFβ1 significantly (p < 0.01; Figure 6E). COL1A1 and ITGβ1 were also upregulated to a lesser, but significant (p < 0.05), extent. The general trend of overexpression of these gene signatures indicates that culture in a 3D environment increases the invasiveness of breast cancer cells. 3.4. 3D Culture Mitigates Cell-Cycle Arrest, Decreass Chemosensitivity, and Increases the Expression of Genes Associated with Chemoresistance in Breast Cancer Cells. In our 3D model, we next assessed the response to paclitaxel, a microtubule inhibitor, by exposing breast cancer MDA MB 231 cells to the drug for 72 h and measured the cytotoxicity to assess cell survival. 2D cultured MDA MB 231 cells displayed sensitivity to paclitaxel at all time points assessed, with an IC50 value of 4.7 ± 0.5 nM (p < 0.001; Figure 7A). The corresponding response of similar cells cultured on 10% GelMA increased by a 3-fold order of magnitude, with an IC50 value of 1352 ± 46 nM (p < 0.001), demonstrating that 3D culture significantly altered the drug sensitivity of breast cancer cells (Figure 7A). A similar trend of decreased chemosensitivity upon the 3D culture was observed in MDA MB 468 cells (Figure S4A). To understand the gene expression changes that might be driving the 3D culture-induced decreased drug sensitivity, we analyzed the expression of putative chemoresistance markers TUBB3, CD9, COX2, CAV1, and ABCB1 (Figure 7B) in cells cultured in 2D and 3D, after drug treatment, by qPCR. All of the markers tested were significantly overexpressed (p < 0.001) in 3D cultures. Of these, TUBB3 is a marker specific for paclitaxel chemoresistance. This differential drug-induced cytotoxicity was further characterized by cell-cycle analysis. MDA MB 231 cells cultured on 10% GelMA hydrogels and treated with a lethal concentration of paclitaxel (3× of IC50) did not display G2M cell-cycle arrest (Figure 8A). Treatment of a 2D monolayer culture of MDA MB 231 cells results in an efficient cell-cycle arrest in G2-M (40%), whereas only 15% of 3D cultured cells displayed a similar arrest at G2-M (Figure 8B). A similar trend was observed with 10× IC50 concentrations of the drug

(Figure S5), confirming that the 3D culture influences the response of cells to chemotherapeutic drugs.

4. DISCUSSION Hydrogels are becoming increasingly popular matrix options for establishing 3D models of cancer because of their ease of preparation and cytocompatibility. Because the chemistry of the matrix is important in the regulation of the cellular phenotype, hydrogels made from biologically derived materials have attracted great interest as biomimetic substrates suitable for 3D culture.42 In this study, we used gelatin, which is essentially hydrolyzed collagen type 1, as the material of choice to establish 3D hydrogel cultures of breast cancer cells because of the physiological relevance of collagen in the development and maintenance of the breast microenvironment. Type 1 collagen is localized around the human mammary ducts.43,44 Further, recent analyses on primary breast tumor biopsies have identified morphological modifications in collagen deposition and arrangementTumor Associated Collagen Signatures, TACSwhich are linked to disease progression.45 Hence, collagen would be a preferred component of matrixes used to recapitulate the breast microenvironment in vitro. An adequate matrix pore size is important for the penetration of cells into gels, migration, proliferation, and exchange of oxygen, nutrients, and waste materials in and out of the 3D culture scaffolds.46 In this study, the porous structure of GelMA hydrogels provided the proper spatial scale for cell growth and proliferation, as indicated by the formation of 3D spheroids from breast cancer cells cultured on them. These spheroids are similar to those observed in standardized matrigel cultures of these cell lines.13 Moreover, it is becoming increasingly clear that the mechanical properties of tissues can indicate the onset of malignant conditions47,48 by influencing a wide range of fundamental cell behaviors, including the cell morphology,49 proliferation,50 motility,51 differentiation,52 and response to therapeutic agents.53 In particular, increased integrin-mediated signaling, a precursor for tumor progression, is correlated with an increase in the substrate stiffness.54 Hence, the creation of scaffolds with tunable properties to mimic the mechanical properties of the native tissue is essential to exploring the dynamic interactions between the cell and ECM under pathological conditions. In this study, we show that, by varying the percentage of GelMA used for preparing hydrogels, we can alter their mechanical properties to suit specific applications. Compression testing revealed that the stiffness of 10% GelMA 22012


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Figure 8. Cell-cycle analysis of MDA MB 231 cells cultured on 2D TCPS and GelMA hydrogels. Flow-cytometry analysis 18 h after MDA MB 231 cells were treated with a lethal concentration (3× of IC50) of paclitaxel revealed reduced G2/M arrest in cells cultured on GelMA hydrogels in comparison with cells cultured on 2D TCPS. Control indicates untreated cells; drug indicates cells treated with paclitaxel (A). Quantification of cellcycle analyses (B). The error bar represents standard deviation. * indicates p < 0.05. 2D-con: 2D cultured cells, not exposed to paclitaxel. 2D-drug: 2D cells treated with paclitaxel. GelMA-con: Cells cultured in 3D and not exposed to paclitaxel. GelMA-drug: Cells cultured on 3D and treated with paclitaxel.

hydrogels was within the range reported for breast tissue.55 Comparatively, the stiffness of the 2D control, TCPS (∼2.5 GPa),56 is 3−4 orders of magnitude higher. Further, the viscoelasticity of 10% GelMA hydrogels (0.89 ± 0.53 kPa) is also similar to that of breast tissue, as reported in the literature.57 Thus, we conclude that 10% GelMA hydrogels sufficiently recapitulate the mechanical properties of the breast microenvironment. Further, the results of proliferation studies demonstrate that GelMA hydrogels are able to sustain breast cancer cells longer compared to 2D cultures, with cells cultured on 10% GelMA

hydrogels showing a higher proliferation rate compared to 15% GelMA in both cell lines tested. This makes them robust systems for a long-term cell culture.58 In all, 10% GelMA hydrogels are suitable matrixes for mimicking the breast microenvironment in vitro. Both cell lines tested displayed an ability to form spheroids upon culture on GelMA hydrogels. Moreover, these cell spheroids concurrently showed an increased expression of markers associated with stemlike characteristics, signifying that these spheroids might be enriched in stemlike cell populations, 22013


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cancer cells cultured on 10% GelMA hydrogels provide a good model of early stages of breast cancer metastasis. A second characteristic that we evaluated in our 3D models was the response to chemotherapeutic drugs. We observed that 3D spheroids of breast cancer cells cultured on GelMA hydrogels show decreased sensitivity to taxane drugs like paclitaxel, as evidenced by the increased cell survival in a MTT assay. In breast cancer, taxane resistance is of particular concern because taxanes, alone or in combination with anthracyclines, are the first-line treatment regimen in early and advanced breast cancer.77 Deconstructing the molecular processes for the development of acquired taxane resistance is critical for innovative targeted therapies to control or eliminate resistant tumor cells. Taxanes bind microtubules and cause kinetic suppression of microtubule dynamics. The consequent arrest of the cell cycle at the mitotic phase has been considered to be the cause of taxane-induced cytotoxicity.78 However, resistant tumor cells evade this process either by acquiring stem-celllike characteristics and completely withdrawing from the cell cycle or by continuing to proliferate at a slow rate.79 We observed a reduction in the G2-M phase arrest following paclitaxel treatment in 3D cultured MDA MB 231 cells, indicating that 3D culture alleviates the response of these cells to taxanes. Elevated chemoresistance to anticancer drugs upon 3D culture has been reported in several cancer models.80,81 Recently, it was surmised that chemoresistance in 3D cultures might be driven by biochemical or mechanical cues from the microenvironment, resulting in the upregulation of downstream genes conferring drug resistance82 and the suppression of chemotherapy-induced apoptosis. This has given rise to the novel concept of cell adhesion mediated drug resistance (CAMDR).83,84 The integrin family of cellular adhesion molecules is a major class of receptors through which cells interact with the ECM. Integrins have been shown to participate in intracellular signal transduction pathways that may contribute to tumor cell growth and survival.67 Interactions between integrins and receptor tyrosine kinases in this signaling cascade are rendered possible by specific membrane proteins like CAV1.85 In our study, we observed overexpression of ITGβ1, signaling the activation of integrin-mediated signaling as well as upregulation of CAV1, thus supporting the possibility of CAM-DR. Moreover, the expression of class 3 β-tubulin (TUBB3), a predictive marker for the clinical outcome of taxane/ vinorelbine-based chemotherapy,86 was also elevated in 3D cultures. A similar trend in upregulation of all tested markers associated with chemoresistance was also observed in the noninvasive breast cancer cell line MDA MB 468 (Figure S4B). However, because we have only tested a limited number of genes, we cannot exclude other pathways, which may play a significant role in 3D culture-induced chemoresistance, as described in other solid tumor cells in vitro.87 The current drug-testing paradigm based on cells cultured in 2D monolayers unreliably estimates the sensitivity of cells to drugs, leading to an inaccurate representation of a drug’s effectiveness in in vivo conditions and resulting in a high failure-to-success rate of drugs during drug development.88 Hence, there is considerable interest in the drug-testing industry to transition cell culture from a 2D to a 3D format. A 3D assay system that more closely mimics the in vivo environment could optimize preclinical selection of the most effective drug candidates, thus increasing the predictive power of cell-based assays. Although 3D cell culture has not yet fully been incorporated as a routine drug development and testing

even in comparison to cells cultured under nonadherent conditions. A key process early in metastasis is the invasion of cancer cells into the surrounding matrix by forming invadopodia, which are cytoskeletal structures enriched in actin-associated proteins and having a MMP-mediated matrix degrading capacity.59−61 In our study, 3D cultured MDA MB 231 cells developed stellate projections of invadopodia within 3 days of culture and exhibited migration into the hydrogel matrix. Further, the formation of invadopodia was associated with an increased expression of MMP2 (Gelatinase A). Overexpression of MMP2 has been associated with tumor invasion, metastasis,62 and the risk of recurrence in certain subtypes of breast carcinoma.63 Similar increases in MMP production upon 3D culture have been previously reported in human lung cancer cells grown in an ex vivo 3D lung model.64 Overexpression of other genes associated with invasiveness was also observed in our 3D cultured breast cancer cells, suggesting that they may represent changes important for metastatic progression. For example, ITGβ1 plays a critical role in the maintenance of mammary tissue structure and function by mediating the interaction between cytoskeletal elements and the ECM.65 Its expression has been linked to therapeutic resistance in multiple cancer types66,67 and poor overall survival in patients with early-stage breast cancer.68 We observed an increased expression of ITGβ1 upon 3D culture of breast cancer cells, signaling the activation of cell-adhesion-mediated signaling pathways. Overexpression of COL1A1 was also observed upon culture of MDA MB 231 cells upon GelMA hydrogels. Increased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development69 and influences the diffusion of therapeutics into tumors.70 Our 3D cultured MDA MB 231 cells also showed an upregulation of TGFβ1 expression. Increased expression of TGFβ1 has been associated with malignant conversion and progression in breast, as well as gastric, endometrial, ovarian and cervical cancers, glioma, and melanoma.71,72 Importantly, mammary cell-specific expression of TGFβ ligands has been found to enhance breast-cancerassociated lung metastases in in vivo mouse models.73,74 Concurrent with the upregulation of TGFβ1 in our study, we observed abundant lung tumors earlier (at 6 weeks) in NODSCID mice injected with 3D cultured cells compared to mice injected with 2D cultured cells, which did not form tumors in the same duration. In most well-established models of breast cancer metastasis in mice, injection of MDA MB 231 cells via tail vein has been reported to give rise to lung tumors at 8 weeks.75,76 However, we had to end our study 6 weeks postinjection because the mice that were injected with 3D cultured cells became moribund in this duration. Our data therefore suggest that 3D culture of MDA MB 231 cells accelerates the kinetics of tumor formation in the lungs of NOD-SCID mice. This may be a result of the culture conditions and associated changes in the expression of key genes. Indeed, the expression of MMP2, CXCR4 and COX2, which have been reported to mediate breast cancer metastasis to lungs,75 was also found to be upregulated in our study. Moreover, 3D cultured cells also exhibited increased in vitro invasive capacity in a standard invasion assay. Thus, in our study, we observed correlations between the breast cancer cell culture conditions, in vitro and in vivo invasive capacity of cultured cells, and the expression of genes associated with invasiveness. Taken together, these results indicate that breast 22014


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tool, encouraging the validation of this tool for advanced cellbased in vitro screening strategies would go a long way in reducing the costs associated with drug development.

5. CONCLUSION In this study, a hydrogel cell culture matrix fabricated from gelatin methacrylate was investigated as a possible 3D model for studying the invasiveness and chemoresponse of breast cancer cells. The biochemical and mechanical properties of the hydrogels were tuned to provide an approximation of the native breast tissue microenvironment. Key characteristics of breast cancer cells cultured on these scaffolds were compared with 2D monolayer cultures. Breast cancer cells readily adhered, proliferated, and displayed increased invasiveness and increased tumorigenic ability in vivo when seeded on GelMA hydrogels, demonstrating their usefulness for creating complex 3D cultures for modeling advanced disease states like metastasis. Further, our data confirm decreased chemosensitivity of breast cancer cells to taxane drugs upon 3D culture, suggesting that GelMA hydrogels could be suitable matrixes to study tumor chemoresistance and identify pathways of breast cancer cell response to taxane drugs.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b06309. SEM images of GelMA hydrogels, bright-field images of MDA MB 468 cells, expression of genes associated with stemlike characteristics in cells, cell proliferation based on AlamarBlue Assay of MDA MB 468 cells, results from a migration assay carried out with MDA MB 468 cells, results from an invasion assay comparing MDA MB 231 cells, average relative fold expression of genes associated with invasive characteristics in cells, results from an MTT assay carried out on MDA MB 468 cells, average relative fold change of expression of markers associated with chemoresistance in MDA MB 468, results from flow cytometry analyses of MDA MB 231 cells, and sequences of RNA primers used for qRT-PCR (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ‡

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS A.C. acknowledges financial support from the Mazumdar Shaw Medical Foundation, Bangalore, India. A.J. acknowledges funding from Syngene International, Bangalore, India. N.G. acknowledges the Department of Biotechnology (Bioengineering and Biodesign) for project funding. The authors thank Priyanka Chevoor, GROW Laboratories, Narayana Nethralaya, Bangalore, India, for help with the flow-cytometry experiments.

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REFERENCES

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