Nanochip-Induced Epithelial-to-Mesenchymal Transition - American

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Biological and Medical Applications of Materials and Interfaces

Nanochip-induced Epithelial to Mesenchymal Transition: Impact of physical microenvironment on cancer metastasis. Udesh Dhawan, Ming-Wen Sue, Kuan Chun Lan, Waradee Buddhakosai, Pao Hui Huang, Yi Cheng Chen, Po-Chun Chen, and Wen-Liang Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19467 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

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ACS Applied Materials & Interfaces

Nanochip-induced Epithelial to Mesenchymal Transition: Impact of physical microenvironment on cancer metastasis. Udesh Dhawan1, Ming-Wen Sue2, Kuan Chun Lan2, Waradee Buddhakosai2, Pao Hui Huang2, Yi Cheng Chen3, Po-Chun Chen3*, Wen Liang Chen2* 1

Department of Materials Science and Engineering, National Chiao Tung University, 1001 University

Road, Hsinchu, Taiwan 300, ROC. 2

Department of Biological Science and Technology, National Chiao Tung University, 1001 University

Road, Hsinchu, Taiwan, 300, ROC. 3

Department of Materials and Mineral Resources Engineering, National Taipei University of

Technology, 1, Section 3, Zhongxiao E. Rd, Taipei, Taiwan, 10608, ROC. Corresponding author: Wen-Liang Chen, Ph.D., Professor Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 1001

University

Road,

Hsinchu,

Taiwan

300,

ROC

Phone: +886-3-5712121 ext. 59711 Email address: [email protected] Fax: +886-3-5729288 Po-Chun Chen, Ph.D., Professor, Department of Materials & Mineral Resources Engineering, National Taipei University of Technology, 1, Section 3, Zhongxiao E. Road, Taipei, Taiwan, 10608, ROC. Phone: +886 2 27712171 Email address: [email protected] Fax: +886 2 27317185 Keywords: Epithelial to Mesenchymal transition, Nanotopography, Artificial microenvironments, Tantalum oxide, Triple negative breast cancer, Nanodots 1

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ABSTRACT Epithelial to Mesenchymal transition (EMT) is a highly orchestrated process motivated by the nature of physical, chemical composition of Tumor Microenvironment (TME). The role of physical framework of the tumor microenvironment in guiding cells towards EMT is poorly understood. To investigate this, breast cancer MDA-MB-231 and MCF-7 cells were cultured on nanochips comprising of Tantalum oxide nanodots ranging in diameter from 10 to 200nm, fabricated through electrochemical approach and collectively referred to as artificial microenvironments. The 100 and 200nm nanochips induced cells to adopt an elongated or spindle shaped morphology. The key EMT genes, E-Cadherin, NCadherin and Vimentin, displayed the spatial control exhibited by the artificial microenvironments. The E-Cadherin gene expression was attenuated while of N-Cadherin and Vimentin was amplified by 100, 200nm nanochips indicating the induction of EMT. Transcription factors Snail, Twist were identified for modulating EMT genes in the cells on these artificial microenvironments. Localization of EMT proteins observed through Immunostaining indicted the loss of cell-cell junctions on 100, 200nm nanochips, confirming the EMT induction. Thus, by utilizing an in-vitro approach, we demonstrate how the physical framework of TME may possibly trigger or assist in inducing EMT in-vivo. Applications in the fields of drug discovery, biomedical engineering and cancer research are expected.

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INTRODUCTION Tumor microenvironment (TME) displays a highly complex scenario consisting of physical and chemical counter parts, working together to maintain its homogeneity 1. The physical portion of TME comprises of the Extracellular Matrix (ECM), blood vessels, and heterogeneous population of cells such as fibroblasts, immune cells, macrophages, lymphocytes2. The cellular interactions in the tumor microenvironment control the tumor growth, invasion, metastasis, resistance to Chemotherapy and ultimately the prognosis3. The TME is one of the key factors in promoting metastasis. Among the physical components, the organization as well as mechanical properties of the ECM have been seen to increase invasiveness4. Many studies have shown how matrix stiffness can affect the cell proliferation and migration, both of which are crucial for the cells to attain metastatic abilities5-6. The cellular cytoskeleton transfers physical as well as chemical signals from the ECM to the cell interior. Cellular surface receptors interact with the ECM to activate or to deactivate various signaling pathways. The ECM composition and architecture directs the cells towards metastasis7. However, the precise effect of differently sized components is unknown. A plethora of studies in the past have linked the metastasis to Epithelial to Mesenchymal transition (EMT) triggered by the tumor microenvironment8-9. Cancerous cells invade the tumor microenvironment by breaching the basement membrane in the TME. This is achieved by opting the EMT mechanism which involves loss of cell assembly, cell polarity, increased motility, reorganization of the cytoskeleton and increased invasiveness. Phenotypically, the cells acquire a spindle shape. Other factors such as Inflammation in the TME can also induce EMT. An interesting yet unexplored factor in this scenario is if the structure and size of ECM components, comprising the TME, pushes the cells to metastasis through EMT. While in-vivo, the physical composition of TME guides cellular behavior, invitro similar observations can be made on subjecting cells to diverse nanotopographies. This implies that Nanotopological cues can be analyzed to understand the in-vivo regulation of cellular behavior.

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Nano-topography can also regulate cellular behavior. Topographies like nano-dots, nanotube, nanoridge, nano-pore have been reported to affect the cell physiological behavior, including biocompatibility, cell growth, migration and cell adhesion10-12. Micro as well as Nano-topography can modulate the Cellular characteristics such as cell adhesion, morphology, cell attachment, migration, growth and differentiation by imitating the cellular microenvironment. The cells are able to convert Mechanical cues into Biochemical signals13. Tissue stiffness can affect the cell proliferation and migration14. Cellular surface receptors interact with the microenvironment to activate or to deactivate various signaling pathways. Studies have shown that 2D as well as 3D surfaces control the cellular morphology, proliferation and cytoskeleton organization. The tissue microenvironment consists of collagen and elastin fibers ranging from 10 to 300nm of diameter15. Therefore, it can be interpreted that cells respond to nano-surface in these dimensions. However, certain microenvironments, tumor microenvironment (TME), for instance is quite complicated and hard to understand due to the presence of cancerous as well as normal stroma in it. But, based on the previous studies, it may be possible to understand the role of specific TME components by engineering nanotopographies in its size range. The present study is based on the hypothesis that nanotopographies of different sizes can act as artificial microenvironments to the cancer cells, modulate the cellular behavior and serve as an alternate way to understand the in-vivo role of physical components of TME in inducing metastasis through EMT. To achieve this, a variety of nanochips, comprising of nanodot-shaped nanotopographies were engineered through a one/two-step electrolysis process. Breast cancer cell lines MDA-MB-231 and MCF-7 were seeded on different nanochips and modulation in cell morphology, behavior, regulation of EMT genes and transcription factors modulating the same were studied. The correlation of cellular characteristics to the nanochip size was also established. The primary goal of this study is to elucidate the role of physical composition of TME in triggering or modulating metastatic behavior of the cells. According to our results, these artificial microenvironments have the ability to induce metastatic

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abilities in the cells, and therefore, will find applications in the fields of drug development, cancer research and biomedical Engineering. EXPERIMENTAL SECTION Materials. Glutaraldehyde, Osmium tetroxide and Hexamethyldisilazane (HMDS) were purchased from Electron Microscopy Sciences (U.S.A). Paraformaldehyde was purchased from Alfa Aesar (Taiwan). Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute-1640 (RPMI) were purchased from GIBCO (Thermo Fisher Scientific Inc. U.S.A). Trypsin was purchased from Sigma-Aldrich (U.S.A). RNAzol RT solution was purchased from Molecular research center, Inc. (U.S.A). GScript First Strand Synthesis Kit was purchased from Genedirex (Taiwan). Taq DNA polymerase master mix red was purchased from Ampliqon (Denmark). Anti-Ecad, N-Cad, Vimentin, Snail, Twist (Rabbit anti Human) primary antibodies were purchased from Cell Signaling Technology (U.S.A). Donkey anti Rabbit Dy light 594 secondary antibodies were purchased from Thermo Fisher Scientific Inc., U.S.A. FITC-conjugated anti-rabbit secondary antibodies were purchased from Jackson Immunoresearch (U.S.A). Alexa Fluor 488 phalloidin and 568 phalloidin were purchased from Invitrogen. Bovine serum albumin (BSA) was purchased from GIBCO (Thermo Fisher Scientific Inc. U.S.A). Phosphate buffered saline (PBS) was purchased from Bio-tech (Taipei, Taiwan). Sulfuric acid (H2SO4), oxalic acid (H2C2O4), and phosphoric acid (H3PO4) were purchased from Sigma Chemicals (Perth, Western Australia), 6-inch silicon wafers, Aluminum ingots were purchased from Admat-Midas (Norristown, PA, USA). Other chemicals of analytical grade or higher were purchased from Sigma or Merck (USA). Fabrication of Artificial microenvironments. A 200-nm-thick tantalum nitride (TaN) thin film was sputtered onto a 6-in silicon wafer (Summit-Tech, West Hartford, CT, USA) followed by deposition of 400-nm-thick aluminum onto the top of a TaN layer using thermal coater. Highly uniform artificial microenvironments comprising of nanodot arrays were fabricated from 10nm to 200nm. Nanodot arrays of size smaller than 10nm could not be fabricated due to technical limitation. Anodization was carried 5



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out in 1.8 M sulfuric acid at 5 Volts, 90 min for the 10 nm nanodots and in 0.3 M oxalic acid at 25 Volts, 90 min for the 50 nm. 100 nm and 200 nm nanodots were fabricated by a two-step anodization method. In the first anodic oxidation step, anodization was carried out in 0.3 M oxalic acid at 40 Volts, 10 min, for 100 nm nanodots and in 5 % (w/v) H3PO4 at 100 Volts, 5 min, for 200 nm nanodots. The porous alumina was removed by immersion in 5% (w/v) H3PO4 for 70 min and 60 min for 100 nm and 200 nm nanodots, respectively. Second anodization step is repeated in the same way. Porous anodic alumina was formed during the anodic oxidation. The porous alumina was removed by immersing in 5 % (w/v) H3PO4 overnight. Characterization of artificial microenvironments. A thin layer of platinum (10nm) was sputtered onto the nanochips. The dimensions and homogeneity of nanodot arrays were measured and calculated from images taken by JEOL JSM-6500 TFE-SEM. For statistical analysis of size of nanodots, four different substrate fields were analyzed per wafer and three different wafers were analyzed per nanodot size. The morphology of nanodots was analyzed using Atomic force microscopy (AFM) and composition was analyzed using Energy-dispersive X-ray spectroscopy (EDX). Cell culture. MDA-MB-231 and MCF-7 cells were cultured in RPMI-1640 and DMEM respectively, supplemented with 10% FBS, 2 mM L-Gutamine and Earle’s BSS adjusted to contain 1.5g l-1 sodium bicarbonate, 0.1mM non-essential amino acids and 1.0 mM sodium pyruvate and incubated at 37ºC maintained at 5% CO2 concentration. To eliminate any possible contamination of nano/micro particles, cell culturing was performed in a class-10 clean room. Morphological observation with Scanning Electron Microscopy. MDA-MB-231 and MCF-7 cells were seeded at a density of 1x103 cells/cm2 on the artificial microenvironments for 0 (8 hours), 3, 5, and 7 days. After removing the culture medium, the wells were rinsed with Dulbecco’s Phosphate Buffered Saline (DPBS). The cells were fixed with 1.25% gluteraldehyde in PBS at room temperature for 15 minutes, followed by staining with 1% osmium tetraoxide for 30 minutes. Samples were then washed with PBS 3 times, 5 minutes each and finally immersed in 40% alcohol overnight. Dehydration 6


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was performed the next day using a series of ethanol concentrations (10 min incubation each in 50%, 60%, 70%, 80%, 90%, 95%, 100% ethanol). The samples were sputter-coated with Platinum and examined by Hitachi SU8010 Scanning electron microscope (SEM) at an accelerating voltage of 8 Kiloelectron volts (KeV). For statistical analysis of number of spindle shaped cells, cells were harvested after day 5 and cells with an elongated/spindle-shaped morphology were manually counted. Four different substrate fields were measured per sample and three separate samples were measured for each nanodot size Reverse transcription polymerase chain reaction (RT-PCR). The E-Cad, N-Cad, Snail, Slug, Twist mRNA levels in MBA_MB-231 and MCF-7 cells seeded on different nanodot arrays was determined by RT-PCR. Total RNA was extracted from the cells using RNAzol RT solution. An aliquot of cDNA was subjected to 35 cycles of PCR using a standard procedure of initiation at 65 °C for 5 mins, incubating at 37 °C for 2 mins, and inactivating at 70 °C for 15 mins. The amplified products were resolved in a 1 % agarose gel and visualized by Sybr safe staining. The forward and reverse primers of the genes and transcription factors used in this study are outlined in table 1.

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Gene

Forward

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Reverse

name Snail1

CTC TTC CTC TCC ATA CTT CAT CAA AGT CCT CCT G

CDH1

GTG G

CGA CAA AGG ACA GCC GGC GTA GAC CAA GAA TAT

CDH2

ATG

GCT TTC CAC TCT CTT ACT GTG CTT ACT GAA CTG A

VIM

TTG TC

AAG GGG ACC AAC GAG TGA CAT TCA GCA GGT TCT CT

CTT GG

GAPDH GCATCCTGGGCTACACTGA CCA CCA CCC TGT TGC TGT A Twist

GGA GTC CGC AGT CTT TCT GGA GGA CCT GGT ACG AG

AGA GG

Table 1: Forward and Reverse primers of EMT genes and transcription factors used in RT-PCR experiments. Immunostaining. Cells were harvested after 7 days and fixed using 4% paraformaldehyde in PBS for 15 min followed by 5 PBS washes. Cell membranes were permeabilised using 0.1% Triton X- 100 incubation for 15 min, followed by 5 washes in PBS. The membranes were then blocked overnight using 2% BSA in PBS followed by 5 PBS washes, the next day. The samples were then incubated overnight with 1:500 anti Actin (Mouse anti Human), 1:100 anti E-Cad (Rabbit anti Human), anti NCad, anti-vimentin, anti-Snail and anti-Twist (Rabbit anti Human) primary antibodies (diluted in 1% BSA solution) followed by 5 PBS washes, the next day. Finally, the samples were incubated with 1:1500 Donkey anti Rabbit Dy Light 594 or FITC-conjugated anti-rabbit or anti-mouse secondary 8


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antibodies (diluted in 1% BSA solution) for 1 hour at room temperature followed by 5 PBS washes. TO analyze the cell morphology, samples were incubated with Alexa Fluor 488 phalloidin, prepared in 2%BSA. Samples were incubated for 20 minutes followed by 5 PBS washes. Finally, all samples were mounted on glass slides and imaged using a Olympus microscope (Model No. 1X71S1F-3). All PBS washes were performed for 5 minutes each. Analysis of Mean fluorescence density on different artificial microenvironments. For relative comparison of the staining intensity on artificial microenvironments, ImageJ was used. To compare staining intensity of markers, the images was first analyzed with ImageJ to obtain the integrated fluorescence density. This value was then divided by the total number of cells in the image to obtain the staining intensity per cell. 3 images from 3 independent experiments were analyzed for each nanochip. Values were then analyzed to obtain the mean fluorescence intensity and plotted against the size of nanochip. Analysis of gene expression on different artificial microenvironments. To statistically analyze the modulation of different EMT genes on artificial microenvironments, the gene-band intensity was first analyzed with ImageJ. The band intensity was then normalized with respect to GAPDH and expressed as normalized gene expression against the size of different artificial microenvironment. At least 3 images from 3 independent experiments were analyzed for each EMT gene. The values were then expressed as mean and standard deviation. Statistics. At least 3 biological repeats and at least 3 technical repeats for each biological repeat were performed for each experiment on the nanochips. Data were expressed as mean and standard deviation. T-test was employed to determine data sets that differed significantly from one another, and significance was defined as a p-value < 0.05 or 0.01. Significant values were expressed with a * depicting a p value of < 0.05. Highly significant values were expressed with ** depicting a p value of

Nanochip-Induced Epithelial-to-Mesenchymal Transition - American

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