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Microfluidic chip bio-sensor for detection of cancer cells

Authors:
Hesam Babahosseini at National Institutes of Health
Hesam Babahosseini
Vaishnavi Srinivasaraghavan at Virginia Tech (Virginia Polytechnic Institute and State University)
Vaishnavi Srinivasaraghavan
Masoud Agah at Virginia Tech (Virginia Polytechnic Institute and State University)
Masoud Agah
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Abstract and Figures

Mechanical properties of single cells are associated with their disease status. Thus, cell biomechanics can serve as a reliable biomarker to distinguish cancerous cells from normal ones. Previously, it has been shown that the average deformability of cancerous cells is significantly larger than that of normal cells. In this paper, to compare the deformability of benign and tumor cells, we designed a microfluidic device with a narrow constriction straight channel and two reservoirs. We expect that softer cells will travel faster through the channel than stiffer cells. Hence, we can correlate the measured transit times for the cells to their stiffness. The results show that the average transit time of non-malignant breast cells (MCF 10A) through the channel is 1.9-fold larger than malignant breast cells (MDA-MB-231) and the average transit time of benign early stage mouse ovarian cancer cells is 1.8-fold larger than aggressive late stage ones.
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Figure 1. Optical image of the fabricated microfluidic chip with a zoomed i
n
view of the constriction channel.
Microfluidic Chip Bio-Sensor for Detection of
Cancer Cells
Hesam Babahosseini*, Vaishnavi Srinivasaraghavan*, and Masoud Agah
VT MEMS Lab, The Bradley Department of Electrical and Computer Engineering
Virginia Tech, Blacksburg, VA, USA
Email: {hbabahosseini, vaishnas, agah}@vt.edu
Abstract—Mechanical properties of single cells are associated
with their disease status. Thus, cell biomechanics can serve as a
reliable biomarker to distinguish cancerous cells from normal
ones. Previously, it has been shown that the average
deformability of cancerous cells is significantly larger than that
of normal cells. In this paper, to compare the deformability of
benign and tumor cells, we designed a microfluidic device with a
narrow constriction straight channel and two reservoirs. We
expect that softer cells will travel faster through the channel
than stiffer cells. Hence, we can correlate the measured transit
times for the cells to their stiffness. The results show that the
average transit time of non-malignant breast cells (MCF 10A)
through the channel is 1.9-fold larger than malignant breast
cells (MDA-MB-231) and the average transit time of benign
early stage mouse ovarian cancer cells is 1.8-fold larger than
aggressive late stage ones.
I. INTRODUCTION
Progression of cancer is dependent on alteration of the cell
cytoskeleton [1, 2]. Cancer cells become softer and more
compliant to facilitate motility [3]. In fact, aggressive cancer
cells have the ability to deform and squeeze through tissue
matrix to access the circulatory system and subsequently move
through small leaks in blood vessel walls to establish new
tumors via the metastasis process [4, 5].
Metastasis is the predominant cause of death in cancer. To
effectively treat cancer, the cancer cells must be detected at
the earliest possible stage before it metastasizes to other parts
of the body. Hence, it is important to find a biomarker which
can allow us to distinguish cancerous cells with high
sensitivity so that it can be diagnosed and treated early.
Prior study on the deformability of adherent breast cells by
our group using atomic force microscopy (AFM) determined
that the average Young’s modulus of MDA-MB-231, the
metastatic breast cancer cells, is 0.51±0.35 kPa and
significantly lower (p<0.0001) than that of MCF 10A, the
non-tumorigenic breast epithelial cells, which is 1.13±0.84
kPa [6]. Besides that, our group recently reported that the
cancer progression of mouse ovarian surface epithelial
(MOSE) cells from early/benign to late/aggressive stage is
associated with a decrease in the average Young’s modulus
from 1.09±0.68 kPa to 0.54±0.28 kPa [7]. The populations of
elasticity measurements fitted by log-normal distributions
revealed that cancerous cells had a wider distribution
compared to the cells from the earlier stages of cancer [6, 7].
Microfluidic technology has emerged as a faster and easier
alternative to AFM technique because it can study the overall
cell mechanical properties and can also provide an
environment to mimic in vivo processes such as migration of
cells through capillaries during metastasis [8].
In this paper, we propose a capillary-like poly
(dimethylsiloxane) (PDMS) microchannel microfluidic
channel, in which cells are pulled through a narrow
constriction channel using negative pressure.
II. DESIGN AND FABRICATION
The microfluidic device was made from PDMS and is
shown in Fig. 1. The device consists of two major parts; (i) the
narrow (6µm-wide) and shallow (12µm-deep) constriction
channel which is straight and 300µm-long and (ii) the delivery
channel which is 200µm-wide and ~60µm-deep. The delivery
channel was designed to establish a continuous free flow of
un-deformed single cells suspended in culture medium. The
principle of trapping of the cells at the entrance of the
constriction channel is based on the hydrodynamic resistance
inside the microfluidic channel and follows the principle
previously reported by [9]. The constriction channel was
designed with a rectangular cross section (6µmx12µm) to
enable deformation of the cells when they are pulled through
the channel.
This work was primarily supported by the National Science Foundatio
n
under Award Number ECCS-IDR 0925945.
*Both authors contributed equally to this research.
Figure 3. Image showing a cell travelling through the constriction channel.
The process flow for the fabrication of the microfluidic
device is shown in Fig. 2. The fabrication was done using a
two-step etch process to obtain the shallow constriction
channel and the deep main channel. Briefly, photoresist
(Shipley 1827) was spun coated on a silicon wafer and
patterned using photolithography. A shallow etch was done
using deep reactive ion etching (DRIE) to etch to a depth of
12µm. The wafer was then cleaned and the photoresist was
stripped before a second round of photolithography using a
thicker photoresist (AZ9260). To fabricate the delivery
channel, the wafer was etched for 20 min in the DRIE to
obtain an overall depth of ~60µm. The photoresist was
stripped and the wafer was cleaned before depositing a layer
of polytetrafluoroethylene (PTFE) to enable easy peel-off of
PDMS. PDMS pre-polymer was mixed with the curing agent
in 1:10 ratio and poured over the silicon master. The wafer
was then placed in a degasser to remove air bubbles in the
PDMS before curing at 120ºC for 40 min. The devices were
allowed to cool and then peeled off and diced. Inlet and outlet
ports were punched into the devices and they were bonded to
glass slides using oxygen plasma.
III. EXPERIMENT METHODS
A. Prepration of cell samples
Non-invasive MCF10A and highly invasive MDA-MB-
231 breast cells were used in this work. The cells were
purchased from the American Type Culture Collections
(ATCC). MDA-MB-231 cells were maintained in F12:DMEM
(50:50) culture medium which contained 10% fetal bovine
serum (FBS), 4 mM glutamine and penicillin-streptomycin
(100 Units/ml). MCF10A cells were grown in Hams
F12:DMEM (50:50), 2.5 mM L-glutamine, 20 ng/ml
epidermal growth factor (EGF), 0.1 μg/ml cholera toxin (CT),
10 μg/ml insulin, 500 ng/ml hydrocortisone and 5% horse
serum. Cells were grown at 37°C in humidified 5% CO2-95%
air atmosphere.
Mouse Ovarian Surface Epithelial (MOSE) was used as a
second cell type to confirm the results. MOSE are an
alternative model for lethal human ovarian cancer. The MOSE
cell lines have been developed and grown in vitro from a
benign/healthy state to a malignant/cancerous one as described
in [10]. According to their passage number, they are
categorized into early/benign (passage No. 15-25) and
late/aggressive (passage No.155-171). MOSE Cells were
maintained in plastic T-75 cm2 culture flasks in High Glucose
Dulbecco’s Modified Eagle’s Medium (DMEM-HG) culture
medium containing 40 mL/L (4%) Fetal Bovine Serum (FBS),
3.7g/L of Sodium Bicarbonate (HCO3), 10 mL/L of Insulin-
Transferin-Selenium (ITS), and 10 mL/L (1%) of Penicillin-
Streptomycin solution.
The cells are suspended in culture medium at 1x105
cells/ml concentration for the tests.
B. Exterimental Setup
A poly(dimethylsiloxane) (PDMS) microchannel of similar
dimensions as that of blood capillaries was fabricated to
mimic the microvascular environment. The microchip has a
mechanism to deliver, trap, and pass the cells continuously
through a constriction channel while cell transit time is
measured by processing the video images obtained. Flow is
established in the delivery channel by a difference in the level
of solution in the reservoirs at the inlet and outlet of the
channel. Single cells are trapped at the entrance and passed
continuously through the constriction channel of the
microfluidic device (Fig. 3). Once a cell is captured into the
trap and is traveling through the constriction channel, another
cell does not come in. A syringe pump was used to apply
negative pressure in the constriction channel to pull the cells
through. The flow rate was set at 50 µl/min for the breast cells
to ensure that the transit time was within the range of
discernible times using the high speed videos obtained.
Imaging was done using an inverted Zeiss microscope and
Zen lite 2011 software was used to capture high speed videos.
Figure 2. Process flow of the fabrication process.
IV. RESULTS
The measured transit times for breast cells are shown in
Table 1. Transit time for a cell is the total time taken the cell
to squeeze into and flow through the constriction channel.
These two periods can be called entry and travel times. Entry
time is the time taken for the cell to deform and enter
completely into the microchannel and travel time is the time it
takes to pass through the microchannel.
As shown in Table 1, the average, standard deviation, and
peak values of transit times for MCF10A cells are larger than
that for MDA-MB-231 cells. The transit time reduces from
0.203±0.226 s (n=120) for MCF-10A to 0.115±0.0747 s
(n=100) for cancerous MDA-MB-231 breast cells. The
histograms of measured transit times are shown in Fig. 4 and
Fig. 5. Similar to the elasticity measurements, the transit time
measurements are not normally distributed and are best
described by log-normal distributions. The peak values can be
extracted by fitting the Gaussian function to the distribution
profile of the populations. The peak transit times of MCF10A
and MDA-MB-231 are located at 0.07 s and 0.04 s,
respectively. The histogram of MDA-MB-231 is sharper and
shifted towards lower transit time in comparison to MFC10A.
Based on the t-test statistical significance analysis, the average
transit time of cancerous and normal cells are significantly
different (p<0.0002) (Shown in Fig. 6 in log scale).
As the cell sizes are roughly the same for both cell types,
the above result indicates that MCF-10A cells are less
deformable than MDA-MB-231 cells. This is consistent with
our results found previously using AFM in [6]. Hence,
suspended cancerous MDA-MB-231 cells are softer than
suspended benign MCF-10A cells. Moreover, the results also
show a direct correlation between stiffness of adherent and
suspended cells.
The measurements were made in multiple experiments to
ensure repeatability. The measured results from a sample size
as small as 15-20 cells were consistent with the range
presented for each cell type. The measurements from 15-20
cells were obtained within 1-2 minutes on average. Therefore,
an important advantage of this proposed microfluidic
technique is the ability to achieve a higher throughput than
AFM technique which albeit its precision on a single cell
level, requires a skilled operator and is very low throughput
(<1 cell/2 min).
To examine the versatility of the microfluidic technique,
similar experiments were conducted on early and late MOSE
cells. The dimensions of the constriction channel were the
same as the devices used with breast cells. The experiments
were conducted with a flow rate of 20 µl/min. The results
show that the average transit time of early stage MOSE cells
through the channel is 0.104±0.142 s (n=345) and decreases to
0.058±0.062 s (n=195) for late stage MOSE cells. The
difference in the average transit time of early and late MOSE
cells is statistically significant (P<0.0002). The distributions of
the measured transit times were described by a log-normal
distribution scheme and there was a noticeable shrink and
shifting pattern to smaller values in the distribution of cells
from early to late stage of MOSE cells.
TABLE I. ELASTICITY AND TRANSIT TIME CHARACTERISTICS OF
NORMAL AND CANCEROUS BREAST CELLS.
Cell Type Elasticity (kPa) Transit time (sec.)
mean±std. peak mean±std. peak
MDA-MB-231 0.51±0.35 0.2 0.115±0.0747 0.04
MCF10A 1.13±0.84 0.43 0.203±0.226 0.07
Figure 4. Image showing histogram of transit time through the
constriction channel for MCF10A cells.
Figure 5. Image showing histogram of transit time through the
constriction channel for MDA-MB-231 cells.
Figure 6. The transit time measurements of malignant MDA-MB-231
and non-malignant MCF10A breast cells have a significant difference
(p
<0.0002
)
.
V. CONCLUSION
A microfluidic chip with a narrow constriction channel
was designed and the transit time of suspended cells through
the channel was correlated with the Young’s modulus
measurements of adherent cells from atomic force
microscopy. The transit times of aggressive/late stage cells of
both breast and ovarian cancer were lesser than the
benign/early stage cells. This work demonstrates that the
presented microfluidic platform can be a high-throughput
alternative for AFM while providing information regarding
(relative) cell stiffness, the homogeneity of the cell sample,
and the health status of the cells.
ACKNOWLEDGMENT
The authors would like to thank Dr. Eva M. Schmelz at the
Department of Human Nutrition, Foods and Exercise of
Virginia Tech for providing us the ovary cells.
REFERENCES
[1] M. Nikkhah, J. Strobl, E. Schmelz, P. Roberts, H. Zhou, and M. Agah,
"MCF10A and MDA-MB-231 human breast basal epithelial cell co-
culture in silicon micro-arrays," Biomaterials, vol. 32, pp. 7625-7632,
2011.
[2] M. Nikkhah, J. Strobl, and M. Agah, "Attachment and Response of
Fibroblast and Human Breast Cancer Cells to Isotropic Silicon
Microstructures of Different Geometries," Biomedical Microdevices,
vol. 11, pp. 429-441, 2009.
[3] S. Suresh, "Biomechanics and biophysics of cancer cells," Acta
Biomater., vol. 3, pp. 413-438, 2007.
[4] J. B. Wyckoff, J. G. Jones, J. S. Condeelis, and J. E. Segall, "A critical
step in metastasis: in vivo analysis of intravasation at the primary
tumor," Cancer Res., vol. 60, pp. 2504-2511, 2000.
[5] G.Y.H. Lee and C.T. Lim, "Biomechanics approaches to studying
human diseases," Trends Biotechnol., vol. 25, pp. 111-118, 2007.
[6] M. Nikkhah, J. S. Strobl, R. De Vita, and M. Agah, "The cytoskeletal
organization of breast carcinoma and fibroblast cells inside three
dimensional isotropic silicon microstructures," Biomater., vol. 31, pp.
4552-4561, 2010.
[7] A. N. Ketene, E. M. Schmelz, P. C. Roberts, and M. Agah, "The effects
of cancer progression on the viscoelasticity of ovarian cell cytoskeleton
structures " Nanomed: Nanotechnology, Biol and Med., vol. 8, pp. 93-
102, 2012.
[8] H. W. Hou, Q. S. Li, G. Y. H. Lee, A. P. Kumar, C. N. Ong, and C. T.
Lim, "Deformability study of breast cancer cells using microfluidics,"
Biomed Microdevices, vol. 11, pp. 557-564, 2009.
[9] W. Kim and A. Han, "A micro-aspirator chip using vacuum expanded
microchannels for high-throughput mechanical characterization of
biological cells," in Int. Conf. on Miniaturized Systems for Chemistry
and Life Sciences (MicroTAS 2010), Groningen, The Netherlands,
October 2010, pp. 253-255.
[10] P. C. Roberts, E. Motillo, A. C. Baxa, H. H. Q. Heng, N. Doyon-Reale,
L. Gregoire, W. D. Lancaster, R. Rabah, and E. M. Schmelz,
"Sequential Molecular and Cellular Events during Neoplastic
progression: A Mouse Syngeneic Ovarian Cancer Model," Neoplasia,
vol. 7, pp. 944-956, 2005.
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  • Nov 2015
  • LAB CHIP
Cancer progression and physiological changes within the cells are accompanied by alterations in the biophysical properties. Therefore, the cell biophysical properties can serve as promising markers for cancer detection and physiological activities. To aid in the investigation of the biophysical markers of cells, a microfluidic chip has been developed which consists of a constriction channel and embedded microelectrodes. Single-cell impedance magnitudes at four frequencies and entry and travel times are measured simultaneously during their transit through the constriction channel. This microchip provides a high-throughput, label-free, automated assay to identify biophysical signatures of malignant cells and monitor the therapeutic efficacy of drugs. Here, we monitored the dynamic cellular biophysical properties in response to sphingosine kinase inhibitors (SphKIs), and compared the effectiveness of drug delivery using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with SphKIs versus conventional delivery. Cells treated with SphKIs showed significantly higher impedance magnitudes at all four frequencies. The bioelectrical parameters extracted using a model also revealed that the highly aggressive breast cells treated with SphKIs shifted electrically towards that of a less malignant phenotype; SphKI-treated cells exhibited an increase in cell-channel interface resistance and a significant decrease in specific membrane capacitance. Furthermore, SphKI-treated cells became slightly more deformable as measured by a decrease in their channel entry and travel times. We observed no significant difference in the bioelectrical changes produced by SphKI delivered conventionally or with NPs. However, NPs-packaged delivery of SphKI decreased the cell deformability. In summary, this study showed that while the bioelectrical properties of the cells were dominantly affected by SphKIs, the biomechanical properties were mainly changed by the NPs.
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Using Nanotechnology and Microfluidics in Search of Cell Biomechanical Cues for Cancer Progression
Article
Full-text available
"...the use of dynamic loading paradigms to evaluate cell biomechanical responses may provide powerful biomarkers for single cancer cell detection and personalized medicine applications."
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Deformability study of breast cancer cells using microfluidics
Article
Full-text available
  • Jun 2009
  • BIOMED MICRODEVICES
Cell deformability is an important biomarker which can be used to distinguish between healthy and diseased cells. In this study, microfluidics is used to probe the biorheological behaviour of breast cancer cells in an attempt to develop a method to distinguish between non-malignant and malignant cells. A microfabricated fluidic channel design consisting of a straight channel and two reservoirs was used to study the biorheological behaviour of benign breast epithelial cells (MCF-10A) and non-metastatic tumor breast cells (MCF-7). Quantitative parameters such as entry time (time taken for the cell to squeeze into the microchannel) and transit velocity (speed of the cell flowing through the microchannel) were defined and measured from these studies. Our results demonstrated that a simple microfluidic device can be used to distinguish the difference in stiffness between benign and cancerous breast cells. This work lays the foundation for the development of potential microfluidic devices which can subsequently be used in the detection of cancer cells.
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Sequential Molecular and Cellular Events during Neoplastic Progression: A Mouse Syngeneic Ovarian Cancer Model
Article
Full-text available
  • Nov 2005
Studies performed to identify early events of ovarian cancer and to establish molecular markers to support of early detection and the development of chemopreventive regimens have been hindered by a lack of adequate cell models. Taking advantage of the spontaneous transformation of mouse ovarian surface epithelial (MOSE) cells in culture, we isolated and characterized distinct transitional stages of ovarian cancer as the cells progressed from a premalignant nontumorigenic phenotype to a highly aggressive malignant phenotype. Transitional stages were concurrent with progressive increases in proliferation, anchorage-independent growth capacity, in vivo tumor formation, and aneuploidy. During neoplastic progression, our ovarian cancer model underwent distinct remodeling of the actin cytoskeleton and focal adhesion complexes, concomitant with downregulation and/or aberrant subcellular localization of two tumor-suppressor proteins E-cadherin and connexin-43. In addition, we demonstrate that epigenetic silencing of E-cadherin through promoter methylation is associated with neoplastic progression of our ovarian cancer model. These results establish critical interactions between cellular cytoskeletal remodeling and epigenetic silencing events in the progression of ovarian cancer. Thus, our MOSE model provides an excellent tool to identify both cellular and molecular changes in the early and late stages of ovarian cancer, to evaluate their regulation, and to determine their significance in an immunocompetent in vivo environment.
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A MICRO-ASPIRATOR CHIP USING VACUUM EXPANDED MICROCHANNELS FOR HIGH-THROUGHPUT MECHANICAL CHARACTERIZATION OF BIOLOGICAL CELLS
Article
Mechanical properties of cells can serve as biomarkers to identify cells depending on their disease states, and thus have the potential to be used in disease diagnostic applications. However, existing techniques are not capable of quanti-fying these properties in statistically significant quantities. To address this, we propose a high-throughput micro-aspirator chip, which can deliver, trap, and deform multiple cells simultaneously at single-cell resolution without skill-dependent micromanipulation. The mechanical property of HeLa cells was measured using the micro-aspirator chip and no dependency of HeLa cell Young's modulus on the diameter of the cells was found. INTRODUCTION Mechanical properties of living cells have been recently shown to be directly related to the cell types and their condi-tions. Of particular interest is the fact that cancer cells, especially metastatic cancer cells, have vastly different stiffness compared to benign cells. Micropipette aspiration and atomic force microscopy (AFM) are the most commonly used techniques for measuring mechanical properties of single cells [1, 2]. Though powerful and versatile, both traditional glass micropipette aspiration technique and AFM have two drawbacks. First, micromanipulation of glass micropipettes and AFM tips require expertise and extensive operator skills. Second, the serial manipulation process severely limits the throughput. To address these limitations, we have developed a high-throughput micro-aspirator chip that can deliver, trap, and deform multiple cells simultaneously with single-cell resolution without skill-dependent micromanipulation. The micro-aspirator chip is composed of arrays of 40 cell traps paired with one aspiration channel each through which negative pressure can be applied to aspirate the trapped cell. The principle of cell trapping is based on flow resis-tance inside the microfluidic channel (Figure 1) [3]. Once the first trap is filled with a cell, the next cell coming in passes by the trap and is captured in the next trap. After all traps are filled with cells, negative pressure can be applied to the 40 aspiration channels simultaneously using hydrostatic pressure. One of the key design concept of this device is that the aspiration channel is positioned at the center of the cell both in vertical and horizontal direction to obtain a good seal be-tween the cell and the aspiration channel, just like a traditional micropipette (Figure 2). Devices have been reported pre-viously to make raised aspiration channels for planar patch-clamping devices [4, 5], but not for cell stiffness analyses. Figure 1: Schematic diagram showing the cell trapping and cell aspiration principle. (1, 2) When the trap is empty, flow resistance along the straight channel is lower than that of the loop channel, and the main flow stream goes through the cell trap. (3) Once a cell is trapped, the main flow stream goes through the loop channel, carrying the second cell to the next trap. (4) Negative pressure is applied to aspirate the trapped cell.
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Biomechanics and biophysics of cancer cells
Article
  • Jul 2007
  • ACTA MATER
The past decade has seen substantial growth in research into how changes in the biomechanical and biophysical properties of cells and subcellular structures influence, and are influenced by, the onset and progression of human diseases. This paper presents an overview of the rapidly expanding, nascent field of research that deals with the biomechanics and biophysics of cancer cells. The review begins with some key observations on the biology of cancer cells and on the role of actin microfilaments, intermediate filaments and microtubule biopolymer cytoskeletal components in influencing cell mechanics, locomotion, differentiation and neoplastic transformation. In order to set the scene for mechanistic discussions of the connections among alterations to subcellular structures, attendant changes in cell deformability, cytoadherence, migration, invasion and tumor metastasis, a survey is presented of the various quantitative mechanical and physical assays to extract the elastic and viscoelastic deformability of cancer cells. Results available in the literature on cell mechanics for different types of cancer are then reviewed. Representative case studies are presented next to illustrate how chemically induced cytoskeletal changes, biomechanical responses and signals from the intracellular regions act in concert with the chemomechanical environment of the extracellular matrix and the molecular tumorigenic signaling pathways to effect malignant transformations. Results are presented to illustrate how changes to cytoskeletal architecture induced by cancer drugs and chemotherapy regimens can significantly influence cell mechanics and disease state. It is reasoned through experimental evidence that greater understanding of the mechanics of cancer cell deformability and its interactions with the extracellular physical, chemical and biological environments offers enormous potential for significant new developments in disease diagnostics, prophylactics, therapeutics and drug efficacy assays.
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The Effects of Cancer Progression on the Viscoelasticity of Ovarian Cell Cytoskeleton Structures
Article
  • Jun 2011
Unlabelled: Alterations in the biomechanical properties and cytoskeletal organization of cancer cells in addition to genetic changes have been correlated with their aggressive phenotype. In this study, we investigated changes in the viscoelasticity of mouse ovarian surface epithelial (MOSE) cells, a mouse model for progressive ovarian cancer. We demonstrate that the elasticity of late-stage MOSE cells (0.549 ± 0.281 kPa) were significantly less than that of their early-stage counterparts (1.097 ± 0.632 kPa). Apparent cell viscosity also decreased significantly from early (144.7 ± 102.4 Pa-s) to late stage (50.74 ± 29.72 Pa-s). This indicates that ovarian cells are stiffer and more viscous when they are benign. The increase in cell deformability directly correlates with the progression of a transformed phenotype from a nontumorigenic, benign cell to a tumorigenic, malignant one. The decrease in the level of actin in the cytoskeleton and its organization is directly associated with the changes in cell biomechanical property. From the clinical editor: The authors have investigated changes in the viscoelasticity of mouse ovarian surface epithelial (MOSE) cells and demonstrated that ovarian cells are stiffer and more viscous when they are benign.
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The cytoskeletal organization of breast carcinoma and fibroblast cells inside three dimensional (3-D) isotropic silicon microstructures
Article
  • Mar 2010
Studying the cytoskeletal organization as cells interact in their local microenvironment is interest of biological science, tissue engineering and cancer diagnosis applications. Herein, we describe the behavior of cell lines obtained from metastatic breast tumor pleural effusions (MDA-MB-231), normal fibrocystic mammary epithelium (MCF10A), and HS68 normal fibroblasts inside three dimensional (3-D) isotropic silicon microstructures fabricated by a single-mask, single-isotropic-etch process. We report differences in adhesion, mechanism of force balance within the cytoskeleton, and deformability among these cell types inside the 3-D microenvironment. HS68 fibroblasts typically stretched and formed vinculin-rich focal adhesions at anchor sites inside the etched cavities. In contrast, MCF10A and MDA-MB-231 cells adopted the curved surfaces of isotropic microstructures and exhibited more diffuse vinculin cytoplasmic staining in addition to vinculin localized in focal adhesions. The measurement of cells elasticity using atomic force microscopy (AFM) indentation revealed that HS68 cells are significantly stiffer (p < 0.0001) than MCF10A and MDA-MB-231 cells. Upon microtubule disruption with nocodazole, fibroblasts no longer stretched, but adhesion of MCF10A and MDA-MB-231 within the etched features remained unaltered. Our findings are consistent with tensegrity theory. The 3-D microstructures have the potential to probe cytoskeletal-based differences between healthy and diseased cells that can provide biomarkers for diagnostics purposes.
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Attachment and response of human fibroblast and breast cancer cells to three dimensional silicon microstructures of different geometries
Article
  • Apr 2009
  • BIOMED MICRODEVICES
The paper reports the development of three dimensional (3-D) silicon microstructures and the utilization of these microenvironments for discriminating between normal fibroblast (HS68) and breast cancer cells (MDA-MB-231). These devices consist of arrays of microchambers connected with channels and were fabricated using a single-mask, single-isotropic-etch process. The behavior and response of normal fibroblast and breast cancer cells, two key cell types in human breast tumor microenvironments, were explored in terms of adhesion and growth in these artificial 3-D microenvironments having curved sidewalls. Breast cancer cells formed stable adhesions with the curved sidewalls however fibroblasts stretched and elongated their cytoskeleton and actin filaments inside the microchambers. Statistical analysis revealed that fibroblast cells grew on both flat silicon surfaces and inside the microchambers regardless of microchamber depth. However, the localization of breast cancer cells in these same substrates was dependent on the microchamber depth. After 72 h in culture, the ratio of the number of breast cancer cells on flat surfaces compared to breast cancer cells inside the microchambers was significantly decreased within the deeper microchambers; for microchambers having depths 88 mum less than 5% of the breast cancer cells grew on the flat surfaces. This behavior was sustained for 120 h, the longest time point examined. The results suggest that certain 3-D silicon microstructures have potential application as a tool to detect breast cancer cells and also as a platform for separating normal fibroblasts from breast cancer cells for cancer diagnosis applications.
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A critical step in metastasis: In vivo analysis of intravasation at the primary tumor
Article
  • Jun 2000
Detailed evaluation of all steps in tumor cell metastasis is critical for evaluating the cell mechanisms controlling metastasis. Using green fluorescent protein transfectants of metastatic (MTLn3) and nonmetastatic (MTC) cell lines derived from the rat mammary adenocarcinoma 13762 NF, we have measured tumor cell density in the blood, individual tumor cells in the lungs, and lung metastases. Correlation of blood burden with lung metastases indicates that entry into the circulation is a critical step for metastasis. To examine cell behavior during intravasation, we have used green fluorescent protein technology to view these cells in time lapse images within a single optical section using a confocal microscope. In vivo imaging of the primary tumors of MTLn3 and MTC cells indicates that both metastatic and nonmetastatic cells are motile and show protrusive activity. However, metastatic cells show greater orientation toward blood vessels and larger numbers of host cells within the primary tumor, whereas nonmetastatic cells fragment when interacting with vessels. These results demonstrate that a major difference in intravasation between metastatic and nonmetastatic cells is detected in the primary tumor and illustrate the value of a direct visualization of cell properties in vivo for dissection of the metastatic process.
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Biomechanics approaches to studying human diseases
Article
  • Apr 2007
  • TRENDS BIOTECHNOL
Nanobiomechanics has recently been identified as an emerging field that can potentially make significant contributions in the study of human diseases. Research into biomechanics at the cellular and molecular levels of some human diseases has not only led to a better elucidation of the mechanisms behind disease progression, because diseased cells differ physically from healthy ones, but has also provided important knowledge in the fight against these diseases. This article highlights some of the cell and molecular biomechanics research carried out on human diseases such as malaria, sickle cell anemia and cancer and aims to provide further important insights into the pathophysiology of such diseases. It is hoped that this can lead to new methods of early detection, diagnosis and treatment.
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