Isolation of Phenotypically Distinct Cancer Cells Using Nanoparticle

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Isolation of Phenotypically-Distinct Cancer Cells Using Nanoparticle-Mediated Sorting Brenda Green, Leyla Kermanshah, Mahmoud Labib, Sharif Ahmed, Pamuditha Silva, Laili Mahmoudian, I-Hsin Chang, Reza M Mohamadi, Jonathan V. Rocheleau, and Shana O. Kelley ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 26 May 2017 Downloaded from http://pubs.acs.org on May 27, 2017

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ACS Applied Materials & Interfaces

Isolation of Phenotypically-Distinct Cancer Cells Using Nanoparticle-Mediated Sorting Brenda J. Green†€, Leyla Kermanshah†€, Mahmoud Labib‡, Sharif U. Ahmed‡, Pamuditha N. Silva†, Laili Mahmoudian‡, I-Hsin Chang‡, Reza M. Mohamadi‡, Jonathan V. Rocheleau† and Shana O. Kelley†‡§* †

Institute of Biomaterials and Biomedical Engineering, University of Toronto, 144 College Street, Toronto, M5S 3M2, Canada, ‡Department of Pharmaceutical Sciences, 144 College Street, University of Toronto, Toronto M5S 3M2, Canada, §Department of Biochemistry, Faculty of Medicine, University of Toronto, Toronto M5S 1A8, Canada

KEYWORDS: breast cancer, magnetic nanoparticles, microfluidics, phenotype, epithelial to mesenchymal transition ABSTRACT: Isolating subpopulations of heterogeneous cancer cells is an important capability for the meaningful characterization of circulating tumor cells at different stages of tumor progression and during the epithelial to mesenchymal transition (EMT). Here, we present a microfluidic device that can separate phenotypically-distinct subpopulations of cancer cells. Magnetic nanoparticles coated with antibodies against the epithelial cell adhesion molecule (EpCAM) are used to separate breast cancer cells in the microfluidic platform. Cells are sorted into different zones based on the levels of EpCAM expression, which enables the detection of cells that are losing epithelial character and becoming more mesenchymal. The phenotypic properties of the isolated cells with low and high EpCAM are then assessed using matrix-coated surfaces for collagen uptake analysis, and an NAD(PH) assay that assesses metabolic activity. We show that low-EpCAM expressing cells have higher collagen uptake and higher folateinduced NAD(P)H responses compared to high-EpCAM expressing cells. Additionally, we tested SKBR3 cancer cells undergoing chemically induced hypoxia. The induced cells have reduced expression of EpCAM, and we find that these cells have higher collagen uptake and NAD(P)H metabolism relative to non-induced cells. This work demonstrates that nanoparticle-mediated binning facilitates the isolation of functionallydistinct cell subpopulations and allows surface marker expression to be associated with invasiveness, including collagen uptake and metabolic activity.

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INTRODUCTION Circulating tumor cells (CTCs) have heterogeneous phenotypes, which may arise from epigenetic changes, environmental cues or differentiation.1 Aggressive tumors release thousands of cancer cells into the circulation each day; however, less than 3% of these disseminated cancer cells metastasize.2,3 Studies have suggested that subpopulations of circulating tumor cells (CTCs) have a more aggressive phenotype and a greater capacity to seed metastasic tumors.4, 5 Cancer cells may acquire metastatic potential by undergoing the epithelial to mesenchymal transition (EMT).2, 6, 7 EMT is a process where epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory properties enabling them to move through adjacent tissues and enter the blood circulation. Metastases may introduce metabolic changes in the cell, and increase the capacity to withstand oxidative stress caused by hostile environments.8

Molecular diversity among cancer cells has been recognized as a major driving force for the evolution of the disease, and can occur outside of the primary tumor.9 Cytometry techniques such as PCR-activated cell sorting, flow cytometry and deformability assays can be applied to identify cell subtypes; however, they do not provide a measure of invasiveness.10-12 Currently, the characterization of CTCs can involve immunostaining, flow cytometry, quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), whole genome amplification, RNA-sequencing or xenograft studies.9, 13-18 Live-cell functional assays are a relatively unexplored area for CTCs, but could advance our understanding of cellular phenotypes correlated with invasiveness. Existing functional assays that have been


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Figure 1. Phenotypic profiling of cancer cell subpopulations. (a) Schematic showing the separation of cancer cells into 4 zones of a microfluidic device that processes a sample in the presence of an external magnetic field. Cells are incubated with magnetic nanoparticles (MNPs) labelled with EpCAM. Cells that have high levels of EpCAM and subsequently high number of MNPs are captured in zone 1 and 2, whereas cells with low levels of EpCAM, and low number of MNPs, are captured in zone 3 and 4. The linear velocity in the device decreases in a stepwise manner in each zone, to increase the probability of cell capture in the apex of the X-structures. Viable cells can be released from each zone. (b) Phenotypic analysis of isolated tumor cells. Viable cells are assessed using a fluorescent collagen uptake assay and a metabolic NAD(P)H assay. Low-EpCAM cells have increased collagen uptake, and increased NAD(P)H response relative to high-EpCAM cells. Scale bars are 5µm.

applied to CTCs include detection of specific proteins secreted during the in vitro culture of CTCs, collagen adhesion assays to detect invasive cancer cells, and in vivo transplantation of patient-derived CTCs into immunodeficient mice.15,

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approaches are limited by the low yield of CTCs from patients, but have the ability to detect metastases-initiating cells. Microfluidic cell sorting technologies have the potential to enhance characterization of heterogeneous cell populations, and examples include aptamer-mediated separation23,


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basement-membrane coated chips24, lateral displacement microarrays25, and single-cell chemotaxis chips.26 These methods enable the separation of cells based on their surface marker expression level, adhesion capacity, transportability and migration potential, respectively. In our lab, we have used a combination of microfluidic devices and magnetic nanoparticles to not only capture CTCs, but also divide them into subpopulations according to levels of protein surface expression. 27,28 This technology has been used to detect heterogeneous populations of cancer cells from cancer patients27, 29, and to profile CTCs from tumor-bearing animal models.30 Here, we show that this technology is capable of isolating subpopulations of cells that exhibit different biochemical and functional phenotypes. We analyze cellular subpopulations isolated using a microfluidic device with functional assays that monitor collagen uptake and NAD(P)H metabolism of cancer cells (Figure 1). SKBR3 breast cancer cells are sorted based on EpCAM expression levels, released from the zones of the microfluidic device, and then subjected to functional assays. We demonstrate phenotypic differences in an EMT model, and show that low-EpCAM expressing cells have properties that correlate with invasive cell behaviour. Altogether, separating subpopulations of cells on the basis of surface marker expression levels yields groups of cells with distinct functional phenotypes.


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RESULTS AND DISCUSSION Invasive cancer cells are thought to be a rare subpopulation of the bulk group of circulating tumor cells, but it remains unknown how best to identify them.3,

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plasticity requires both epithelial and mesenchymal properties, and can contribute to the invasion/metastatic cascade.32 The separation of cancer cells into distinct zones of a microfluidic device using nanoparticles enhances the ability to identify cells with varying levels of EMT markers. A Hypoxia-Driven Model of EMT. In order to generate cells that could be analyzed to visualize varied phenotypic properties, a SKBR3 cell model of EMT (SKBR3-EMT) was created in order to explore the phenotypic differences of cells undergoing EMT in the microfluidic device. The model was created based on a previously-described method that relies on the chemical induction of hypoxia in cell culture.33,

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progression, cancer cells grow rapidly in an avascular environment; therefore, oxygen becomes scarce in the inner layers of the cells. A substantial body of evidence indicates that the hypoxic tumor microenvironment plays a pivotal role in the induction of EMT and consequently the emergence of CTCs.33-36 Cancer cells can adapt to hypoxic conditions by regulation of a transcription factor called hypoxia-inducible factor 1-alpha (HIF-1α). In the presence of oxygen, HIF-1α is constantly synthesized and rapidly degraded through a multistep process catalyzed by prolyl hydroxylase enzymes (PHDs) and the Von Hippel–Lindau (VHL) tumor suppressor proteins, while in the absence of oxygen, HIF-1α accumulates in the cell. The accumulation of HIF-1α results in the regulation and transcription of genes involved


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in EMT and metastases, including HIF-1, VEGF, vimentin, MMP2, MMP9, µPAR, PAI-1, c-Met, TWIST and CCR7.33-36 SKBR3 cells were selected to model EMT, as they are a non-aggressive cell line with high levels of EpCAM, enabling us to monitor changes in epithelial status. Several methods have been reported for induction of hypoxia in cell cultures, including incubation with cobalt chloride (CoCl2).33,

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Cobalt chloride mimics the hypoxic

microenvironment of tumor cells by interfering with the degradation of HIF-1α by inhibition of VHL and therefore stabilizing HIF-1α. The successful induction of EMT was confirmed by monitoring mRNA and protein levels using qPCR and flow cytometry, respectively. Gene expression data demonstrated that epithelial genes (EpCAM, cytokeratin 7, cytokeratin 8) were downregulated and mesenchymal genes (Snail1, Slug and Vimentin) were upregulated after 24, 48 and 72 hours of treatment with CoCl2 (Figure S2a). On the protein level, epithelial markers (EpCAM, E-Cadherin, and cytokeratin) were downregulated while the mesenchymal marker N-Cadherin was upregulated after 72 hour treatment with CoCl2 (Figure S2b). These results confirm the induction of EMT in SKBR3 cells (SKBR3-EMT), and these cells were then included in the studies described below focused on investigating phenotypic changes. Nanoparticle-Mediated Separation of Cell Subpopulations. Devices that can sort heterogeneous cancer cells based on phenotypic differences will advance our understanding of the metastatic cascade.26 In our previous work, we demonstrated the principle

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Figure 2. Microfluidic profiling of breast cancer cells. Cells are labelled with anti-EpCAM magnetic nanoparticles and captured in the microfluidic device. (a) Cell sorting profile of MCF-7 and MDA-MB-231 cells. (b) Cell sorting profile of SKBR3 and SKBR3-EMT cells. SKBR3-EMT cells are treated with CoCl2 for 72 hours. (c) Flow cytometric analysis of EpCAM levels in MDA-MB-231, SKBR3, SKBR3-EMT and MCF-7 cells. (d) Cell sorting profiles of low numbers of MCF-7 and MDA-MB-231 cells spiked in whole blood. Cells are captured and then immunostained with cytokeratin-APC, DAPI and CD45-FITC. Cancer + + cells are identified as CK /DAPI /CD45 . Experiments are repeated in triplicate. Standard errors of the mean are shown. Statistics are performed with one-way ANOVA followed by the Tukey multiple comparisons (p

Isolation of Phenotypically Distinct Cancer Cells Using Nanoparticle

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