Evaluation of cancer cell deformability by microcavity array

Accepted Manuscript Evaluation of cancer cell deformability by microcavity array Tomoko Yoshino, Tsuyoshi Tanaka, Seita Nakamura, Ryo Negishi, Nozomi Shionoiri, Masahito Hosokawa, Tadashi Matsunaga PII:

S0003-2697(16)30440-7

DOI:

10.1016/j.ab.2016.12.026

Reference:

YABIO 12593

To appear in:

Analytical Biochemistry

Received Date: 12 September 2016 Revised Date:

14 December 2016

Accepted Date: 31 December 2016

Please cite this article as: T. Yoshino, T. Tanaka, S. Nakamura, R. Negishi, N. Shionoiri, M. Hosokawa, T. Matsunaga, Evaluation of cancer cell deformability by microcavity array, Analytical Biochemistry (2017), doi: 10.1016/j.ab.2016.12.026. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Evaluation of cancer cell deformability by microcavity array

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Tomoko Yoshino,a† Tsuyoshi Tanaka,a Seita Nakamura,a Ryo Negishi,a Nozomi

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Shionoiri,a Masahito Hosokawa,a and Tadashi Matsunagaa

a. Division of Biotechnology and Life science, Institute of Engineering, Tokyo

184-8588, Japan.

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University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo,

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† Corresponding author.

Phone: +81-42-388-7021 Fax: +81-42-385-7713

e-mail: [email protected]

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Abstract

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A cell entrapment device consisting of a microcavity array was used to analyze the deformability of MCF-10 human breast epithelial and MCF-7 human breast cancer cell lines by confocal laser scanning microscopy. Entrapment of up to 8 × 103 cells was

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achieved within 3 min. Protrusions were formed at the bottom surface of the array with

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a pore size of 3 µm. Protrusion length increased at higher filtration pressures and could be used to distinguish between MCF-7 and MCF-10 cells. These results indicate that our system is useful for high-throughput deformability analysis of cancer cells, which

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can provide insight into the mechanisms underlying tumor cell malignancy.

Keywords: Cell deformability, Breast cancer

ACCEPTED MANUSCRIPT Introduction

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Metastasis is a defining feature of cancer that involves the spread of tumor cells from a

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primary site to secondary tissues or organs. Malignant tumor cells exhibit increased

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deformability, making them highly invasive and metastatic [1-3]. Therefore, in addition to

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detecting cancer-specific markers, evaluating the stiffness/deformability of tumor cells could

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provide information on the mechanistic basis of metastasis.

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The deformability of cells can be estimated by evaluating their elastic and viscous properties

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using approaches such as micropipette aspiration, atomic force microscopy, and optical

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tweezers, among others [4-6]. Recently, microfluidics has been used to increase throughput in

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cell deformability analyses. Micropipette aspiration, which induces the formation of a

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hemispherical cell projection by applying negative pressure, has been integrated into

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microfluidic devices for this purpose. Microchannels are used to estimate cell stiffness by

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measuring migration time, buoyant mass, and the entry velocity of single cells into a

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microchannel

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cell-trapping/aspiration chambers has been proposed that would allow high-throughput

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measurement of cortical tension and Young’s modulus in single cells to enable an estimation

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of cell deformability [9]. Cancer cell metastasis can also be studied by genetic approaches

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including single-cell transcriptome analyses; however, these are limited by the difficulty in

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isolating target cells from microfluidic devices.

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We previously developed a microfluidic device consisting of a microcavity array for

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recovering cancer cells from whole blood based on differences in cell size and deformability

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[10-12]. Microcavity sizes and shapes were designed to capture cancer cells through

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application of negative pressure. This system enables efficient recovery of circulating tumor

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cells. Furthermore, cancer cells can be rapidly recovered from the microcavity array by

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hydrogel encapsulation for subsequent examination [13] by whole genome amplification and

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A microfluidic

pipette

array device

equipped

with

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[7,

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ACCEPTED MANUSCRIPT genetic analysis at the single-cell level.

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In this study, we used a cell entrapment device consisting of a microcavity array was used to

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analyze the deformability of MCF-10 human breast epithelial and MCF-7 human breast

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cancer cells. Cell morphology was evaluated at various filtration pressures by confocal laser

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scanning microscopy. We found that the normal and cancer cell lines could be distinguished

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based on the degree of cell membrane extension induced by filtration pressure. The utility of

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the microcavity array for assessing nucleus deformability was also investigated.

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33 Material and methods

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Cell culture

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MCF-7 human breast cancer cells were cultured in Eagle’s minimal essential medium (MP

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Bio, Santa Ana, CA, USA) supplemented with 1 mM sodium pyruvate, 10 µg/ml insulin, 1.5

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mg/ml

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penicillin-streptomycin. MCF-10 non-malignant human breast epithelial cells were cultured

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in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) supplemented with

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20 ng/ml epidermal growth factor, 0.5 µg/ml hydrocortisone, 10 µg/ml insulin, 5% (v/v) calf

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serum, and 1% (v/v) penicillin-streptomycin. Cultures were maintained at 37°C and 5% CO2

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for approximately 1 week. Prior to each experiment, confluent cell cultures were washed with

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phosphate-buffered saline (PBS, pH 7.4) followed by trypsinization and resuspension in PBS.

10%

(v/v)

fetal

bovine

serum,

and

1%

(v/v)

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bicarbonate,

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sodium

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Fabrication of a microcavity array

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A nickel microcavity array was fabricated by electroformation as previously described [14].

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The fabricated microcavity array had 10,000 (100 × 100) pores with sizes of 3, 8, 11, 15, and

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20 µm separated by a distance of 25 µm (Fig. 1A). The microfluidic device for measuring

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cell deformability was constructed by laminating the layers of the nickel microcavity array,

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poly(dimethylsiloxane) (PDMS) structure equipped with a microchannel on the underside.

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Each layer was assembled with spacer tapes. The microchannel at the bottom of the device

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was connected to a peristaltic pump that drew the sample solution through the device (Fig.

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1B).

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Figure 1. Cell entrapment device equipped with a microcavity array. (A) Scanning electron

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micrograph of the microcavity array (pore sizes: 3 µm). Scale bar: 20 µm. (B) Schematic

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image of the cell entrapment device.

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ACCEPTED MANUSCRIPT Measurement of ∆P across the microcavity array

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To measure the differential pressure across the microcavity array, another PDMS structure

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with microchannels was attached to the top of the cell entrapment device shown in Figure 1B.

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DTXPlus disposable blood pressure transducers (BD Biosciences, Franklin Lakes, NJ, USA)

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were connected to the up- and downstream points of the device, and a 5-ml syringe was

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connected further upstream of the channel. PBS or cell suspension was introduced into the

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device via the syringe and was drawn through by applying negative pressure at a flow rate of

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200 µl/min using a peristaltic pump connected to the microchannel at the bottom of the

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device. The difference in pressure between two points (∆P) was calculated with PowerLab

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(ADInstruments, Colorado Springs, CO, USA) and analyzed using Lab Chart & Scope

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software (ADInstruments).

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Confocal laser scanning microscopy

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Cultured cells were labeled with 5 µM CellTracker Green or CellTracker Orange for 30 min,

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then collected by centrifugation at 400 × g for 4 min, washed three times with PBS, counted,

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and re-suspended in PBS at a concentration of 1.6 × 104 cells/ml. Stained cell suspensions (2–

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8 × 103 cells/250–500 µl) were introduced into the microfluidic device and negative pressure

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was applied to the samples using a peristaltic pump connected to the vacuum line. A 400-µl

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volume of PBS was added to cells trapped on the microcavity array at a flow rate of 200 µl

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/min. Cell Tracker Green-stained MCF-7 or MCF-10 cells (8 × 103) or a mixture of the two

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cell types (stained with Cell Tracker Green and Orange, respectively) were also introduced

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onto the microcavity array. Trapped cells were observed with a confocal laser scanning

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microscope (FV1000-D IX81; Olympus, Tokyo, Japan), and 3D images of the trapped cells

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were acquired by imaging adjacent Z-axis optical sections separated by 0.2 µm with

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excitation wavelengths of 488 and 543 nm for Cell Tracker Green and Orange, respectively.

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ACCEPTED MANUSCRIPT To visualize nuclei and the F-actin cytoskeleton, cells were stained with ethidium homodimer

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(excitation wavelength: 528 nm; emission wavelength: 617 nm) (Invitrogen, Carlsbad, CA,

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USA) and Alexa Fluor 488-conjugated phalloidin, respectively. Image conversion into 3D

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and processing were accomplished using Volocity software (PerkinElmer), which was also

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used to measure cell membrane extension length of individual cells trapped on the

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microcavity array.

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Fluorescence microscopic observation of F-actin

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Confluent MCF-7 and MCF-10 cell monolayers were fixed in formalin for 10 min. After

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rinsing with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min and

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washed again with PBS and blocked with 1% bovine serum albumin in PBS for 20−30 min.

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F-actin was labeled with Alexa Fluor 488-conjugated phalloidin for 20 min. Cell nuclei were

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stained with 10 µg/ml Hoechst 33342 with excitation and emission wavelengths of 350 and

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461 nm, respectively) for 20 min. Images of stained cells were acquired using IN Cell

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Analyzer 2000 with 340–380/440–480 nm filters for Alexa Fluor 488, and 460–500/510–560

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nm filters for Hoechst.

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Results and discussion

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Estimation of differential filtration pressure (∆P) and cell deformability at various cell

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concentrations

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Microcavity flow rates (200–5000 µl/min) and pore sizes (3–20 µm) were first evaluated to

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establish the optimal conditions for cell recovery. A flow rate of < 200 µl/min and pore size <

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8 µm resulted in complete recovery of MCF-7 cells. The number of cells that were applied

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was less than the number of pores in the microcavity (1 × 104); we therefore used a maximum

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of 8 × 103 cells (80% coverage) in this study.

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ACCEPTED MANUSCRIPT The ∆P values of the 3 µm-microcavity array with different numbers of MCF-7 cells trapped

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on the surface are shown in Table 1. ∆P increased as a function of cell number due to the

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capping of microcavities by the cells. The morphology of MCF-7 cells trapped on the

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microcavity array was observed by confocal laser scanning microscopy. Representative

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three-dimensional (3D) images of cells stained with CellTracker Green are shown in Figure

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2A and B. The cells formed protrusions at the bottom surface that were presumed to be

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caused by the negative pressure applied from the underside of the array. We also performed

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the experiment using a microcavity array with larger pores (8 µm) and found that there was

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no deformation of MCF-7 cells even when 8 × 103 cells were introduced into the system (data

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not shown). We therefore used the 3 µm-microcavity array in subsequent experiments.

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Table 1. ∆P of a 3 µm-pore microcavity array as a function of MCF-7 cell number

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Number of introduced cells

Filtration pressure (kPa)

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0.8 ± 0.1

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4 × 103

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6 × 103

2.1 ± 0.6

8 × 103

3.7 ± 0.3

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1.1 ± 0.2

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Figure 2. Confocal laser scanning micrographs of MCF-7 cells trapped on the microcavity

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array. (A) Cell array and (B) single cell. Cells were stained with CellTracker Green. (C)

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Schematic illustration of a single cell trapped in a microcavity.

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The extension length of cellular protrusions (Fig. 2C)—defined as the length from the lower

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surface of the microcavity array to the top of the protruding cell—increased at higher ∆P in a

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linear fashion (Fig. 3A, B). These results indicate that cell deformability can be estimated by

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the extension length of individual cells when known numbers of cells are trapped on the

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array.

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Figure 3. Relationship between ∆P and extension length of MCF-7 cells trapped on the

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microcavity array. (A) XZ-slice images and (B) extension length of cells at each ∆P. Scale

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bar: 10 µm.

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Comparison of MCF-7 and MCF-10 cell deformability

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MCF-7 cells are more deformable than non-tumorigenic MCF-10 cells [15]. To assess the

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potential for distinguishing between normal and malignant cells using our cell deformability

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assay, we analyzed the extension length of MCF-10 and MCF-7 cells (8 × 103 each) trapped

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on microcavity arrays and stained with CellTracker Green by confocal microscopy (Fig. 4A,

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B). The extension length of MCF-7 cells was 2-fold greater than that of MCF-10 cells (Table

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2), suggesting that the two cell lines were distinguishable by cell deformability as estimated

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by extension length. The higher standard deviation (SD) among MCF-7 cells indicated

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greater variability in their deformability as compared to MCF-10 cells.

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Figure 4. XZ-slice images of (A, C) MCF-7 and (B, D) MCF-10 cells trapped either

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separately (A, B) or in a mixture (C, D) on the microcavity array. Scale bar: 10 µm.

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To confirm the utility of the microcavity array for evaluating cell deformability, a mixture of

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MCF-7 and MCF-10 cells (8 × 103) stained with Cell Tracker Green and Cell Tracker Orange,

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respectively, was applied to the array and analyzed as described above (Fig. 4C, D). The two

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cell lines were also distinguishable under these conditions based solely on extension length,

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which again showed greater variability in MCF-7 (Table 2).

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Table 2. Extension lengths of MCF-7 and MCF-10 cells trapped on the microcavity array Cell line

Extension length (µm)

MCF-7

3.0 ± 0.6

MCF-10

1.5 ± 0.3

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2.5 ± 0.8 (MCF-7) MCF-7 & MCF-10

1.5 ± 0.3 (MCF-10)

Filtration pressure: 3.7 kPa

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Applied cell numbers: 8000 cells Value: Mean ± S. D.

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We found that up to 8 × 103 cells were trapped within 3 min using the microcavity array. It is

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possible to enhance the performance by increasing the number of microcavities up to 105 [16].

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Although 3D imaging by confocal microscopy required approximately 3 min for 102 cells, a

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microscope with a microlens array that generates a multi-beam laser would drastically reduce

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the imaging time by eliminating the scanning step. Thus, cell entrapment using the

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microcavity array is a powerful tool for rapid evaluation of cancer cell deformability.

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Deformability of the cell nucleus

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Cell stiffness depends on the integrity of cytoskeletal actin. Filamentous (F-)actin is a major

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component of the cytoskeleton that stabilizes the plasma membrane. The F-actin network was

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clearly observed in MCF-10 but not MCF-7 cells (Fig. S1), as previously reported [15]. We

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speculated that the difference in cell deformability between the two cell lines was due to

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differences in F-actin expression, which also influences nuclear stiffness [17]. We therefore

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examined the deformability of cell nuclei by confocal laser scanning microscopy (Fig. 5) and

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introduced onto the microcavity array with a 3-µm pore size. These results indicate that the

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deformability of the nucleus can serve as another means of distinguishing between normal

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and cancer cells.

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It is well recognized that change in the structure and organization of cytoskeleton causes

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larger deformability in various cancers, such as bladder cells [18], prostate cells [19], ovarian

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cells [20] and thyroid cells [21]. These changes are generally related to partial loss of actin

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filament or disorganization of microtubules, resulting in the lower density of cellular scaffold.

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Furthermore, recently, HER2 overexpression causes deformation of the cell membrane [22].

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Thus, the determination of cell stiffness will enable a more effective detection and

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identification of cancer cells.

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Figure 5. (A) Confocal laser scanning micrographs of MCF-7 cells trapped on the

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microcavity array. Nuclei were stained with Hoechst 33342 (red) and F-actin was labeled

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with Alexa 488-conjugated antibody (green). (B) Schematic illustration of a single cell

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trapped on a microcavity. Scale bar: 2.5 µm.

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Conclusions

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In this study, a cell entrapment device consisting of a microcavity array that was originally

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developed for cancer cell recovery and subsequent genetic analysis at the single-cell level

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was used to analyze cell deformability. The extension length of cell protrusions could be used

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to estimate the deformability of the plasma membrane and nucleus and thereby distinguish

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between normal and cancer cells. Our system provides a tool for high-throughput analysis of

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cancer cell deformability that can improve our understanding of the mechanisms underlying

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tumor cell malignancy.

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