Enhanced Metastatic Potential in a 3D Tissue Scaffold toward a

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Enhanced Metastatic Potential in a 3D Tissue Scaffold Toward a Comprehensive In Vitro Model for Breast Cancer Metastasis Gowri Balachander, Sai Balaji, Annapoorni Rangarajan, and Kaushik Chatterjee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b09064 • Publication Date (Web): 24 Nov 2015 Downloaded from http://pubs.acs.org on December 7, 2015

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ACS Applied Materials & Interfaces

Enhanced Metastatic Potential in a 3D Tissue Scaffold Toward a Comprehensive In Vitro Model for Breast Cancer Metastasis

Gowri Manohari Balachander1, Sai A. Balaji2, Annapoorni Rangarajan1,2$, Kaushik Chatterjee1,3*

1

Center for Biosystems Science and Engineering, Indian Institute of Science, Bangalore-560012, India

2

Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science,

Bangalore-560012, India 3

Department of Materials Engineering, Indian Institute of Science, Bangalore-560012, India

Authors to whom all correspondence should be addressed: *[email protected]; +91-80-22933408 $

[email protected]; +91-80-22933263

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ACS Paragon Plus Environment


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Abstract

Metastasis is clinically the most challenging and lethal aspect of breast cancer. While animal based xenograft models are expensive and time consuming, conventional two-dimensional (2D) cell culture systems fail to mimic in vivo signaling. In this study we have developed a three-dimensional (3D) scaffold system that better mimics the topography and mechanical properties of the breast tumor, thus recreating the tumor microenvironment in vitro to study breast cancer metastasis. Porous poly (εcaprolactone) (PCL) scaffolds of modulus 7±0.5 kPa, comparable to that of breast tumor tissue were fabricated, on which MDA-MB-231 cells proliferated forming tumoroids. A comparative gene expression analysis revealed that cells growing in the scaffolds expressed increased levels of genes implicated in the three major events of metastasis viz., initiation, progression and the site specific colonization compared to cells grown in conventional 2D tissue culture polystyrene (TCPS) dishes. The cells cultured in scaffolds showed increased invasiveness and sphere formation efficiency in vitro, and increased lung metastasis in vivo. A global gene expression analysis revealed a significant increase in the expression of genes involved in cell-cell & cell-matrix interactions and tissue remodeling, cancer inflammation, and the PI3K/Akt, Wnt, NF-kappaB and HIF1 signaling pathways– all of which are implicated in metastasis. Thus, culturing breast cancer cells in 3D scaffolds that mimic the in vivo tumor-like microenvironment enhances their metastatic potential. This system could serve as a comprehensive in vitro model to investigate the manifold mechanisms of breast cancer metastasis.

Keywords: cancer; breast cancer; metastasis; mechanotransduction; tissue scaffolds

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1. Introduction Breast cancer is the most prevalent form of cancer in women worldwide. About 90% of mortality in breast cancer is due to metastasis; the most common secondary sites are the lung, liver and bone.1 Metastasis is a multi-step process wherein the cancer cells migrate from the primary tumor into the vasculature, survive in the circulation, extravasate into the secondary site and establish a tumor.2 Each of the steps of metastasis involves a plethora of changes in gene expression and signaling pathways that assist the cells to form a secondary tumor. More often the epithelial cells gain mesenchymal properties which helps them in migration and invasion,3 activation of stress response pathways that help them survive in circulation,4 and secretion of soluble factors and extracellular matrix proteins that help in the colonization of the secondary site.5 Understanding the molecular mechanisms that orchestrate metastasis provides useful insights into developing therapeutics that prevent or treat metastasis. The culture and characterization of metastatic breast cancer cell lines on planar two-dimensional (2D) plastic dishes is the most commonly adopted system to study signaling events in breast cancer metastasis in vitro. While this approach is convenient and inexpensive, it suffers from serious limitations. On a 2D substrate, the cells become flat, lose their in vivo morphology, and a vast majority of cell-cell and cellmatrix interactions are reduced.6 As a consequence, the cellular signaling that occurs through these interactions is altered.7 Moreover, materials such as tissue culture polystyrene (TCPS) and glass are extremely stiff, of the order of GPa, compared to the mechanical properties of soft human tissues which are of the order of kPa.8 As cellular behavior is widely recognized to be influenced by the substrate stiffness,9 culturing cells on an extremely stiff substrate like TCPS could lead to altered signaling. On the other hand, although in vivo animal models involving spontaneous10 or experimental11 metastasis serve as good model systems for metastasis, they are very expensive and time consuming. To overcome these drawbacks, novel systems are being identified wherein the cells are grown in three dimensional (3D) scaffolds which could better mimic the tissue micro-environment and help recapitulate 3



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in vivo morphology and signaling.12,13 Studies have reported the characterization of breast cancer cells in biologically derived 3D gel matrices including collagen,14 reconstituted basement membrane (matrigel),15 and fibrin.16 Such matrices, though they promote cell attachment and proliferation, are not ideal systems to mimic the in vivo physiology of cancer metastasis due to the following reasons. The mismatch of elastic modulus with the tumor, and hence the varied mechanotransduction could lead to altered signaling, resulting in a different phenotype.17 A matrix of specific extracellular matrix (ECM) molecules like fibrin or collagen might not be able to mimic the extensive qualitative and quantitative changes in the ECM of the primary tumor in a temporal manner.18 This is also undesirable because the formation of a metastatic niche by the secretion of ECM is a primary requisite for successful colonization.19 The presence of excess amounts of ECM in matrigel prevents the cells from secreting their own native ECM molecules.20 Moreover, it has been shown that cancer cells in matrigel form spheriods that are 99±20 µm in diameter which is less than the maximum diffusion limit of oxygen, ≅ 150 µm,21 and, hence can never form a hypoxic core which is observed in most tumors.22 Hence matrices prepared from synthetic polymers that lack ECM based cell-binding sites, yet cytocompatible, are being exploited to study tumor signaling.22,23 Tumor models of oral squamous cell carcinoma have been developed using porous synthetic scaffolds in which cancer cells exhibited increased angiogenic potential as compared to cells cultured in conventional dishes, and are more representative of the cells in vivo.22 Given the potential advantages of porous synthetic scaffolds, we intended developing a comprehensive in vitro model for breast cancer metastasis using a similar approach. As an alternative to ECM-based matrices, the use of synthetic, biocompatible polymers that lack the cell-adhesive sites to facilitate de novo ECM synthesis by the cancer cells and offer greater process ability was considered. It was observed that culturing cells on fast degrading scaffolds could reduce their viability due to the accumulation of the acidic degradation products.24 Hence, poly (ε-caprolactone) (PCL), that degrades slowly, was used for this work. MDA-MB231, a transformed metastatic breast cancer cell line widely used to study breast cancer metastasis,25 was 4


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cultured in porous PCL scaffolds and scored for various gene expression and functional aspects of the different stages of metastasis namely initiation, progression and site-specific colonization. In vivo tumorigenicity and metastatic potential were assayed. Global gene expression changes were analyzed using gene expression microarrays.

2. Materials and methods 2.1. Preparation of porous PCL scaffolds Macro-porous scaffolds of PCL (average molecular weight Mn = 80,000 g/mol, Sigma) were prepared in 96 well plates by following reported procedures.13 Sodium chloride (Sigma) crystals were sieved to defined size range of 250-425 µm. 0.13 g of the salt was added to each well of a 96 well polypropylene plate (Sigma). 45 µL of 10% (wt/vol) PCL solution in chloroform was added, vacuum dried and leached in de-ionized water. 2.2. Structural and mechanical characterization of scaffolds The samples were dried and sputter coated with gold for analysis using a scanning electron microscope (SEM, ESEM Quanta 200, FEI)) for morphology, pore size and pore interconnectivity. X-ray microcomputed tomography (Xradia Versa XRM 500) was used to characterize the 3D architecture of the porous scaffolds. The X-ray voltage was set to 80 kV and the resolution was 3 µm. The distance between the sample and the detector was maintained at 25 mm. 1600 projections were obtained for each tomogram. The porosity of the scaffold was calculated by the liquid displacement method.26 The elastic modulus of the scaffolds was measured by dynamic compressive loading. The scaffolds were subjected to an isothermal frequency sweep using a DMA (TA Instruments, Q800). The frequency was ranged from 1 to 10 Hz with amplitude of 10 µm and a preload force of 0.01 N. The sample was 5



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compressed using a 15 mm compression clamp and the response to the isothermal strain sweep was measured at a constant furnace temperature of 37° C. 2.3. Cell culture MDA-MB-231 (ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma Aldrich) supplemented with 10% fetal bovine serum (FBS, Gibco, Invitrogen) and antibiotics streptomycin sulfate and benzyl penicillin at final concentrations of 100 µg/ml and 100 U/ml, respectively. The cells were cultured at 37oC in 5% carbon dioxide for up to 80% confluence prior to seeding on 3D porous PCL scaffolds or TCPS surfaces, which served as the conventional 2D controls. The scaffolds were exposed to ethylene oxide (Anaprolene) for 24 hours, degassed for 3 days and further exposed to UV 285 nm source for 30 min immediately prior to cell seeding. To wet these hydrophobic scaffold surfaces, the scaffolds were incubated in 0.2 mL complete culture medium for 1 day and 5 × 104 cells were seeded per scaffold (one well of the 96 well plate) in 0.2 mL medium. The medium was changed every two days for 7 days after which the media was refreshed daily. 2.4. Study of cellular morphology The cells were fixed with 3.7% paraformaldehyde for 15 min and permeabilized with 0.2% TritonX100 for 5 min. The cells were stained with 20 nM Phalloidin conjugated with Alexa Fluor 546 (Invitrogen) for 30 min at 37o C and 1 µg/ml of Hoecsht 33342 (Sigma Aldrich) for 5 min at 25° C. For immunofluorescence, the fixed and permeabilized samples were blocked with 0.2% fish skin gelatin (FSG) for 45 minutes and incubated with a primary antibody overnight at 4oC. Vimentin (Sigma), Nanog (Cell Signaling Technology (CST)), FAK (CST), Laminin (Sigma) and Fibronectin (Becton Dickinson) antibodies were diluted in a blocking buffer (0.2% FSG in PBS containing 0.02% Tween20). All the antibodies were used at a dilution of 1:200 except for Nanog, which was used at 1:100. FITC or Cy3 conjugated secondary antibodies corresponding to the origin of the primary antibody were added for 45 6


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min at 25o C. The nuclei were counter-stained with Hoecsht. The specificity of staining was ascertained by performing the same procedure in the absence of the primary antibody. The samples were imaged using an epi-fluorescence microscope (Olympus IX71). For immunocytochemistry, Z sections were taken using a laser scanning confocal microscope (Leica TCS SP5 II). The images were background corrected using their corresponding negative controls and contrasted to the same levels using Image J software and the maximum intensity projections are shown. For scanning electron microscopy, the cells were fixed with 2.5% gluteraldehyde for 12 hours at 4oC. The scaffolds were then dehydrated in a gradient of ethanol, 30, 50, 70, 90 and 100% each for a period of ten minutes. The samples were completely air-dried, sputter coated with gold and imaged. 2.5. Sphere formation in methylcellulose The sphere formation efficiency was assessed by retrieving the cells from twenty scaffolds and from 80% confluent TCPS dishes using 0.125% Trypsin-EDTA for 20 min and seeding 1× 105 cells (as single cells) in 2 ml of 1.5% methylcellulose (in DMEM F12 media supplemented with growth factors) as per reported procedures.27 The spheres were counted manually in twenty random 10x fields (Olympus IX71microscope). 2.6. RNA isolation and quantitative-reverse transcription PCR RNA was isolated from MDA-MB-231 cells grown in scaffolds for days 7 and 14 days using the RNeasy Mini kit (Qiagen). After lysis, the lysate was homogenized using the QIA shredder column following the manufacturer’s instructions. Cells grown in 60 mm TCPS dishes for up to 80% confluence were used as 2D controls and the RNA was isolated as above. A total of 0.5 to 2 µg of RNA was used for cDNA synthesis. The samples were first treated with 2 U/µl DNase to remove all traces of genomic DNA. cDNA synthesis was performed using High Capacity cDNA Reverse Transcription kit (Applied Biosystems) as per the manufacturer’s instructions. Quantitative Real time PCR (qRT-PCR) was carried 7



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out using DyNAmo Flash SYBR Green qPCR Kit (Thermo Scientific) with 10 ng of the cDNA as the template and ROX as a passive reference dye. Gene expression was normalized to HPRT and RPL35. Fold change was calculated using 2-∆∆ct. Gene expression across five individual biological experiments was analyzed. Paired t-test was performed, and p

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