micromachines
Article
Investigation of the Mechanical Properties of the
Human Osteosarcoma Cell at Different Cell
Cycle Stages †
Guocheng Zhang 1 , Na Fan 1 , Xiaoying Lv 3 , Yiyao Liu 3 , Jian Guo 1,4 , Longxiang Yang 1 ,
Bei Peng 1,2, * and Hai Jiang 1, *
1
2
3
4
*
†
School of Mechatronics Engineering, University of Electronic Science and Technology of China,
Chengdu 611731, Sichuan, China; [email protected] (G.Z.); [email protected] (N.F.);
[email protected] (J.G.); [email protected] (L.Y.)
Center for Robotics, University of Electronic Science and Technology of China, Chengdu 611731, Sichuan, China
School of Life Science and Technology, University of Electronic Science and Technology of China,
Chengdu 611731, Sichuan, China; [email protected] (X.L.); [email protected] (Y.L.)
School of Materials Science and Engineering, Xiangtan University, Xiangtan 411105, Hunan, China
Correspondence: [email protected] (B.P.); [email protected] (H.J.);
Tel.: +86-28-61831723 (B.P.); +86-28-61830242 (H.J.)
This paper is an extended version of our paper published in the 2016 International Conference of
Microfluidics, Nanofluidics and Lab-on-a-Chip (2016 ICMFLOC) was held in Dalian, China, 10–12 June 2016.
Academic Editors: Yongxin Song, Junsheng Wang and Dongqing Li
Received: 3 February 2017; Accepted: 10 March 2017; Published: 15 March 2017
Abstract: The mechanical properties of a single cell play substantial roles in cell mitosis, differentiation,
and carcinogenesis. According to the difference of elastic modulus between the benign cell and
the tumor cell, it has been shown that the mechanical properties of cells, as special biomarkers,
may contribute greatly to disease diagnosis and drug screening. However, the mechanical properties
of cells at different cell cycle stages are still not clear, which may mislead us when we use them as
biomarkers. In this paper, the target regions of the human osteosarcoma cell were precisely scanned
without causing any cell damage by using an atomic force microscopy (AFM) for the first time.
Then, the elasticity properties of the human osteosarcoma cells were investigated quantitatively
at various regions and cell cycle stages. The 32 × 32 resolution map of the elasticity showed that
the elastic modulus of the cells at the interphase was larger than that at the telophase of mitosis.
Moreover, the elastic modulus of the cell in the peripheral region was larger than that in the nuclear
region of the cell. This work provides an accurate approach to measure the elasticity properties of
cells at different stages of the cell cycle for further application in the disease diagnosis.
Keywords: mechanical property; elastic modulus; atomic force microscopy; human osteosarcoma cells;
cell cycle
1. Introduction
The mechanical properties of the cell change with the alteration of the cell function; these properties
play important roles in adhesion, migration, mitosis and differentiation [1,2]. For instance, the tumorigenesis
and oncogenic progression may lead to mechanical and structural variations. Therefore, the mechanical
properties of cells have been employed as the biomarkers in the cancer diagnosis [3,4].
Numerous methods have been developed to study the mechanical properties of cells, including
micropipette aspiration [5], utilization of optical tweezers [6], magnetometric analysis [7] and atomic
force microscopy (AFM)-based methods [8]. Jiang et al. [9] measured the regional cell stiffness with
optical magnetic twisting cytometry and investigated the relationship between the cytoskeleton and the
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mechanical properties of the primary human aortic smooth muscle cells. Rebelo et al. [10] studied the
viscoelastic properties of cells from different kidney cancer phenotypes by using AFM and conducted
the elastic modulus map. They analyzed the elastic modulus distribution of cells and found that the
non-tumorigenic cells were less deformable but more viscous than the cancerous cells. Cross et al. [11]
investigated the nanomechanical responses of metastatic cancer cells and benign mesothelial cells by
using AFM and the results showed that the metastatic tumor cells were more than 80% softer than the
benign cells. Costa et al. [12] reported that the periphery of bovine pulmonary artery endothelial cells
was two or three times as stiff as that of the cell body.
However, variation in the mechanical properties of cells could also occur at the different cell
cycle stages, which might mislead us when we use them as biomarkers. The mechanical properties of
the eukaryotic cells are dominated by their cytoskeleton, which may experience huge changes and
remodelling during the cell cycle [13–15]. Usually, the actin filaments dominate the mechanic properties
of the cell. At the interphase, the cell includes an extensive actin network. However, once the cell
enters the mitosis stage, the network will be deconstructed and rearranged. For most cells, the shape
of the cell may undergo a process from spindle to round with furrows during the cell cycle until the
cell divides into two daughter cells [16–18]. The aforementioned studies neglected the variations of the
state and shape of the cell during the cell cycle which has an obvious effect on the mechanical properties
of cells. Therefore, the investigation of accurate and practical measurement of mechanical properties
at the different cell cycle stages is immensely significant. For example, Stewart et al. [19] introduced
an approach to measure the mechanical properties of globular cells during the mitosis by using the AFM.
They found that the mechanical stress at the interphase was higher than that at the anaphase.
In this paper, the elasticity properties of the human osteosarcoma (U-2 OS) cells at the different
cell cycle stages and the relationship between the elastic modulus and the indentation depth were
investigated quantitatively by AFM in the force–volume mode. In addition, the elastic modulus of the
nuclear and peripheral regions of the U-2 OS cells were surveyed at the interphase and the telophase
of mitosis. To our knowledge, this is the first time that the elastic properties at the different regions of
the cell and the different cell cycle stages have been compared. This work also provides an accurate
approach to measure the elasticity properties of cells at the different cell cycle stages. Such findings
can be applied in the disease diagnosis and provide a strong reference.
2. Methods and Materials
2.1. Cell Culture
U-2 OS cells (ATCC® ) were cultured in McCoy’s 5A Medium and supplemented with 10% fetal
bovine serum (v/v, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) at 37 ◦ C under 5% CO2
atmosphere. The cells were seeded onto poly-L-lysine-coated 35-mm Petri dishes. The test was
conducted when the cell density increased to approximately 30%. Before testing, the cells were
washed with phosphate-buffered saline (PBS) three times to remove the unattached cells and organic
medium. They were finally re-suspended in 1 mL PBS buffer for living cell imaging and elasticity
mapping experiments.
2.2. Actin and Nucleic Acid Staining
Actin and nucleic acid staining were used for identifying the morphology of the cell
nucleus and F-actin, and the shapes at different cell cycle stages. DNA and actin were
stained with 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI) and phalloidin (Sigma-Aldrich,
St. Louis, MO, USA), respectively. Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 15 min
at 4 ◦ C and permeabilized with 0.1% Triton X-100, 1% Bovine Serum Albumin (BSA) (Sigma-Aldrich).
Next, cells were incubated with Phalloidin and DAPI for 1 h at 4 ◦ C in the dark. Confocal images were
collected on an Olympus Fluoview FV1000 microscope (Olympus, Tokyo, Japan).
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2.3. Cell Imaging by Atomic Force Microscopy (AFM)
The morphologies of cells were scanned by an AFM (Agilent 5500, Agilent Technologies,
Santa Clara, CA, USA) with a maximum x-y scan range of 90 µm and a z range of 7 µm in contact mode.
The curvature radius of the silicon nitride tips (MSCT–AUHW, Veeco, Town of Oyster Bay, NY, USA)
R is approximately 10 nm and the spring constant k of the rectangle cantilever is calibrated to
be ~0.02 N/m by the thermal noise method [20]. Before the experiments, the silicon nitride tips
were exposed to ultraviolet light for 0.5 h to eliminate the organic contaminates so as to reduce the
measurement error. The topographic images and deflection images of cells were recorded during the
tips canning process, respectively.
2.4. AFM Force Spectroscopy Analysis
From the force spectroscopy obtained by AFM, the elastic modulus of cells can be obtained by the
Hertz–Sneddon model [21]. The elastic modulus of the cell E was calculated by the following equation
because the pyramidal Si3 N4 tip was used in the experiment:
F=
2 E
tan ∂δ2
π 1 − ν2
(1)
where F is the normal force, v is Poisson’s ratio (about 0.5 used in cell [22]), ∂ is the half-open
angle of the pyramidal Si3 N4 tip, and δ is the probe indentation depth of the tip. The indentation
depth δ can be obtained from δ = ∆z − ∆d (∆z = z − z0 is the piezo-actuator displacement and
∆d = d − d0 is the cantilever deflection, where d and z represent the cantilever deflection and
piezo-displacement respectively). (z0 , d0 ) represents the contact point. In this study, the elasticity
mapping experiments were conducted by using 10 individual cells and five pairs of daughter cells in the
force–volume mode with a resolution of 32 × 32 (32 × 32 measuring points for force–distance curves).
This specification allowed us to obtain simultaneously the force–distance curves in every area of the
single cell. All measurements were taken at a scan rate of 1 Hz.
3. Results and Discussion
3.1. Analysis of Elastic Modulus under Different Indentation Depths
To obtain the accurate results of the elastic modulus in the different regions of cells, the force
(indentation depth) must be appropriate, otherwise, the AFM probe will have a big chance to contact
the nucleus or the substrate, resulting in measurement errors. Figure 1a shows three representative
force–distance curves obtained from the substrate, the nuclear and the peripheral regions of an
osteosarcoma cell, respectively. In this study, the peripheral and nuclear regions are defined by the
height (H) of the cell area (peripheral region: H < 1 µm; nuclear region: H > 1 µm). To ensure the
accuracy of measurement, the force–distance curves were fitted under a series of indentation depths
by the Hertz–Sneddon model. Figure 1b demonstrates the relationship between the elastic modulus
and the indentation depth which is extracted from the force–indentation curves shown in Figure 1a.
As reported previously, the variation of the elastic modulus with indentation depth is mainly ascribed
to the distribution of the cytoskeleton of the cell [23,24]. In the nuclear region (red line) as shown in
Figure 1b, the elastic modulus E starts with 12 kPa at the indentation depth of 0.1 µm, then drops
quickly to less than 4 kPa at the indentation depth of 0.5 µm. After that, the elastic modulus remains
at the level of ~1.9 kPa over the range from 0.8 µm to 1.7 µm. The elastic modulus increases slightly
when the indentation depth was larger than 1.7 µm because the AFM and the nucleus are in contact.
Conversely, in the the peripheral region (green line) as shown in Figure 1b, the elastic modulus
remained at the level of ~6 kPa over a small range around 0.5 µm, and then increases dramatically.
The reason for this is the contact between the AFM probe and the substrate. Therefore, in the following
tests, the loading force was set up at 0.5 nN (the indentation depth was around 500 nm) to eliminate
the effect of the substrate or the nucleus.
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in the2017,
following
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nm)
to eliminate the effect of the substrate or the nucleus.
3 of 9
in the following tests, the loading force was set up at 0.5 nN (the indentation depth was around 500
nm) to eliminate the effect of the substrate or the nucleus.
Figure Figure
1. (a)1.Three
representative
curves
obtained
from
the substrate,
the
nuclear
(a) Three
representative force–distance
force–distance curves
obtained
from the
substrate,
the nuclear
and
peripheralregions
regions of
of an
an osteosarcoma
osteosarcoma cell,
(b) (b)
the the
relationship
between
the elastic
and peripheral
cell,respectively;
respectively;
relationship
between
the elastic
modulus
andindentation
the indentation
depth
at
thenuclear
nuclear and
and peripheral regions,
respectively.
modulus
and1.the
depth
at
the
regions,
respectively.
Figure
(a) Three representative
force–distance
curvesperipheral
obtained from
the substrate,
the nuclear and
peripheral regions of an osteosarcoma cell, respectively; (b) the relationship between the elastic
modulus
theat
indentation
depthCell
at the
nuclear
and peripheral regions, respectively.
Images of and
Cells
the Different
Cycle
Stages
3.2. AFM Images of Cells at the Different Cell Cycle Stages
3.2. AFM
Figure 2a,b shows a single osteosarcoma cell at the interphase when plenty of DNA and proteins
Figure
2a,bImages
shows
a the
single
osteosarcoma
cellStages
at the interphase
whenlasts
plenty
DNA
proteins
3.2. synthesized
AFM
of Cells
at
thespreads
Different
Cell Cycle
are
and
cell
completely.
Typically,
the interphase
for atofleast
90%and
of the
are synthesized
the
cell
completely.
the interphase
lasts
forregion,
at
least
entireFigure
timeand
of
the
cell
cycle.
Once
the flat lamellipodia
is observed
at the
peripheral
the90%
cell of the
2a,b
shows
a spreads
single
osteosarcoma
cellTypically,
at the interphase
when
plenty
of DNA
and
proteins
adheres
tightly
on
the
substrate.
Figure
2c,d
shows
the
cell
at
the
telophase
of
mitosis
after
a
mother
entire time
of
the
cell
cycle.
Once
the
flat
lamellipodia
is
observed
at
the
peripheral
region,
are synthesized and the cell spreads completely. Typically, the interphase lasts for at least 90% of thethe cell
cell
hastime
divided
daughter
cells.
flat lamellipodia
observed
at the
region.
of the
theinto
cell two
cycle.
Once
the
flat
lamellipodia
observed
at
the peripheral
region,after
the
cell
adheresentire
tightly
on
substrate.
Figure
2c,dNo
shows
theiscell
at is
the
telophase
of peripheral
mitosis
a mother
Two
cross-sectional
height
profiles
(Figure
2e)
are obtained
from
the positions
of the marked
lines in
adheres
tightly
on
the
substrate.
Figure
2c,d
shows
the
cell
at
the
telophase
of
mitosis
after
a
mother
cell has divided into two daughter cells. No flat lamellipodia is observed at the peripheral region.
Figure
As shown
in Figure
2e, cells.
the black
curve
indicates that
the nucleus
is in
the center
of the
cell has2a,c.
divided
into two
daughter
No flat
lamellipodia
is observed
at the
peripheral
region.
Two cross-sectional
height
profiles
(Figure
2e)
are
obtained
from
the apositions
of the (red
marked
lines in
cell
the interphase.
it proceeds
to are
the obtained
telophasefrom
of mitosis,
cleavage
furrow
curve)
Twoduring
cross-sectional
heightWhen
profiles
(Figure 2e)
the positions
of the
marked lines
in
Figure which
2a,c. Asanshown
in Figure
the black
curve
indicates
that the [25].
nucleus
is inmembrane
the center of the
important
sign
of 2e,
the
mitosis
is clearly
The
Figure is
2a,c. As
shown in
Figure
2e,final
the stage
black of
curve
indicates
that visible
the nucleus is
in cell
the center of thein
cell during
the
interphase.
When
it
proceeds
to
the
telophase
of
mitosis,
a
cleavage
furrow
(red
the
vicinity
of
the
equatorial
plate
concaves
inward,
and
the
cell
is
split
into
two
daughter
To curve)
cell during the interphase. When it proceeds to the telophase of mitosis, a cleavage furrow (redcells.
curve)
this,
another
osteosarcoma
cell
was
stained
at
the
telophase
of
mitosis
as
shown
in
Figure
3.
which prove
is
an
important
sign
of
the
final
stage
of
mitosis
is
clearly
visible
[25].
The
cell
membrane
which is an important sign of the final stage of mitosis is clearly visible [25]. The cell membrane inIt
is
the
immunofluorescence
image
highly
corresponded
to the
AFM
image.
theobvious
vicinity
of
the
equatorial
plate
inward,
and
the cell
split
twointo
daughter
cells. To cells.
in the vicinity
ofthat
the
equatorial
plateconcaves
concaves
inward,
and
theis cell
isinto
split
two daughter
prove
anotherosteosarcoma
osteosarcoma cell
at the
telophase
of mitosis
as shown
Figurein
3. It
To prove
this,this,
another
cellwas
wasstained
stained
at the
telophase
of mitosis
as in
shown
Figure 3.
is
obvious
that
the
immunofluorescence
image
highly
corresponded
to
the
AFM
image.
It is obvious that the immunofluorescence image highly corresponded to the AFM image.
Figure 2. Atomic force microscopy (AFM) images of the osteosarcoma cell. (a) Topographic image
of the cell at the interphase; (b) deflection image of the cell at the interphase; (c) topographic image
of the cell at the telophase of mitosis; (d) deflection image of the cell at the telophase of mitosis;
(e) two cross-sectional profiles of the cell shown in (a) and (c) (the positions of the cross-sections were
marked by black and red lines, respectively).
Micromachines 2016, 8, 89
Figure 2. Atomic force microscopy (AFM) images of the osteosarcoma cell. (a) Topographic image of
Figure
Atomic
force microscopy
(AFM)
images
of the
cell. (a)
Topographicimage
imageofof
the cell2.at
the interphase;
(b) deflection
image
of the
cellosteosarcoma
at the interphase;
(c) topographic
the
deflection
image of image
the cellofatthe
the
interphase;
(c) topographic
thecell
cellatatthe
theinterphase;
telophase of(b)
mitosis;
(d) deflection
cell
at the telophase
of mitosis;image
(e) twoof
the
cell at the telophase
of the
mitosis;
(d) deflection
ofpositions
the cell at
of mitosis;
(e) two
cross-sectional
profiles of
cell shown
in (a) andimage
(c) (the
ofthe
thetelophase
cross-sections
were marked
Micromachines 2017, 8, 89
cross-sectional
profiles
the cell shown in (a) and (c) (the positions of the cross-sections were marked
by black and red
lines,of
respectively).
by black and red lines, respectively).
3 of 9
5 of 10
Figure
3. Immunofluorescence
image
two
daughter
cells
the
telophaseofofmitosis.
mitosis.The
TheF-actin
F-actinand
Figure
3. Immunofluorescence
image
of of
two
daughter
cells
atat
the
telophase
0
and
the
nucleus
were
stained
with
Phalloidin
(red)
and
4’,6-diamidino-2-phenylindole
theFigure
nucleus
were stained with Phalloidin
(red)daughter
and 4 ,6-diamidino-2-phenylindole
dihydrochloride
3. Immunofluorescence
image of two
cells at the telophase of mitosis.
The F-actin
dihydrochloride
(DAPI)
respectively.
(DAPI)
(blue),
respectively.
and
the nucleus
were(blue),
stained
with Phalloidin (red) and 4’,6-diamidino-2-phenylindole
dihydrochloride (DAPI) (blue), respectively.
3.3. Elastic Modulus Map of the Cell at the Interphase
3.3. Elastic Modulus Map of the Cell at the Interphase
3.3. Elastic
Modulus
Map
the Cell
at the Interphase
Figure
4a shows
anofelastic
modulus
map with 32 × 32 measuring points of a single osteosarcoma
Figure 4a shows an elastic modulus map with 32 × 32 measuring points of a single osteosarcoma
cell Figure
at the interphase
(Figure
2a).
It
reveals
the
distinction
of the elastic
modulus
in both
the entire
4a shows(Figure
an elastic
modulus
map
with
32 × 32 measuring
points
of a single
cellregion
at the of
interphase
2a).
It reveals
the
distinction
of region)
the elastic
modulus
inosteosarcoma
both
entire
the
cell
(including
the
nuclear
region
and
peripheral
and
the
substrate
by
thethe
color
cell at
the
interphase
(Figure
2a).
It reveals
theand
distinction
of the
elasticand
modulus
in both by
the
entire
region
of
the
cell
(including
the
nuclear
region
peripheral
region)
the
substrate
the
color
change. The nuclear region was softer than the peripheral region. Figure 4b shows the histogram of
region of the cell (including the nuclear region and peripheral region) and the substrate by the color
change.
The nuclear
was
softer than
the peripheral
region.4100
Figure
4b shows curves
the histogram
the detailed
elasticregion
modulus
distribution
obtained
by calculating
force–distance
chosen of
change. The nuclear region was softer than the peripheral region. Figure 4b shows the histogram of
the from
detailed
elasticmodulus
modulusmaps
distribution
obtained
by calculating
4100 force–distance
chosen
the elastic
of 10 individual
cells.
The elastic modulus
of the cell in curves
the nuclear
the detailed elastic modulus distribution obtained by calculating 4100 force–distance curves chosen
from
the
elastic
modulus
maps
of
10
individual
cells.
The
elastic
modulus
of
the
cell
in
the
nuclear
region at the interphase ranged from 0.50 kPa to 7.5 kPa with an average value of 2.8 ± 1.5 kPa. Its
from the elastic modulus maps of 10 individual cells. The elastic modulus of the cell in the nuclear
elastic
modulus
was less
than that
in 0.50
the peripheral
which
from
1.0 kPa
to 7.5
kPakPa.
region
at the
interphase
ranged
from
kPa to 7.5region,
kPa with
anranged
average
value
of 2.8
± 1.5
region at the interphase ranged from 0.50 kPa to 7.5 kPa with an average value of 2.8 ± 1.5 kPa. Its
with anmodulus
average value
of 4.4
± 1.6that
kPa.
Its elastic
was less
than
in the peripheral region, which ranged from 1.0 kPa to 7.5 kPa
elastic modulus was less than that in the peripheral region, which ranged from 1.0 kPa to 7.5 kPa
with
anan
average
value
with
average
valueofof4.4
4.4±± 1.6
1.6 kPa.
kPa.
Figure 4. (a) The elastic modulus map of a single cell at the interphase with the resolution of
32 × 32 data points; (b) the histogram of the elastic modulus distribution in the whole region (gray),
in the nuclear region (green), and in the peripheral region (red); (c) large lamellipodia area surrounded
by black dashed ellipse in the peripheral region.
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Figure 4. (a) The elastic modulus map of a single cell at the interphase with the resolution of 32 × 32
data points; (b) the histogram of the elastic modulus distribution in the whole region (gray), in the
nuclear
region
(green), and in the peripheral region (red); (c) large lamellipodia area surrounded by6 of 10
Micromachines 2017,
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black dashed ellipse in the peripheral region.
As mentioned above, the mechanical properties of the cell depend on the actin filaments of the
the
cytoskeleton. The actin filaments include long stress fibers and short actin filaments, some of which
are
body [26,27].
[26,27].
are located
located beneath
beneath the cell membrane and the others may extend all over the entire cell body
In this experiment, the indentation depth was much greater than the thickness of the cell membrane.
membrane.
Therefore,
that
thethe
difference
of elastic
modulus
in distinct
regions of
the cellofwas
Therefore,ititexplained
explained
that
difference
of elastic
modulus
in distinct
regions
thegenerated
cell was
due
to the distribution
actin filaments
in the
cell body.
this, To
the validate
actin filaments
andactin
the
generated
due to the of
distribution
of actin
filaments
in To
thevalidate
cell body.
this, the
nucleic
acidand
of an
stained with phalloidin
DAPI,
respectively.
redDAPI,
color
filaments
theosteosarcoma
nucleic acidcell
of were
an osteosarcoma
cell were and
stained
with
phalloidinThe
and
demonstrates
the distribution
of the actin filaments
in the cell,
as shown
in Figure 5.
is cell,
found
the
respectively. The
red color demonstrates
the distribution
of the
actin filaments
in It
the
asthat
shown
in Figure
5. It is
that
the actin filaments
are highly
concentrated
in the
peripheral
region.
In
actin
filaments
arefound
highly
concentrated
in the peripheral
region.
In addition,
large
lamellipodia
areas
addition,
large lamellipodia
areas in the
peripheral
regionhelps
are observed
in Figure
4c, which
helps
to
in
the peripheral
region are observed
in Figure
4c, which
to promote
cell spreading
and
drive
promote cell
spreading
and drive
retrograde flow.
formation
pseudopodia
alsoofresulted
from
retrograde
flow.
The formation
of pseudopodia
alsoThe
resulted
fromof
the
recombination
the skeleton,
the recombination
of the skeleton,
especially
the actin
filaments which
wereand
stiffer
than the
especially
the actin filaments
which were
stiffer than
the intermediate
filaments
microtubules
intermediate
filaments
and[28,29].
microtubules appearing in the cell body [28,29].
appearing
in the
cell body
Figure 5.
5. Immunofluorescence
Immunofluorescence image
image of
of aa single
single cell
cell at
at the
the interphase.
interphase. The
The F-actin
F-actin and
and the
the nucleus
nucleus
Figure
were
stained
with
Phalloidin
(red)
and
DAPI
(blue),
respectively.
were stained with Phalloidin (red) and DAPI (blue), respectively.
3.4. Elastic
Elastic Modulus
Modulus Map
Map of
of the
the Cell
Cell at
at the
the Telophase
Telophase of
of Mitosis
Mitosis
3.4.
Figure 6a
6a illustrates
illustrates the
the elastic
elastic modulus
modulus map
map of
of two
two daughter
daughter cells
cells at
at the
the telophase
telophase of
of mitosis
mitosis
Figure
(see
Figure
2c).
No
significant
difference
of
the
elastic
modulus
is
measured
between
the
two
(see Figure 2c). No significant difference of the elastic modulus is measured between the two daughter
daughter
cells
in
both
the
nuclear
region
and
the
peripheral
region.
Figure
6b
is
the
histogram
of
the
cells in both the nuclear region and the peripheral region. Figure 6b is the histogram of the elastic
elastic modulus
distribution
obtained
by calculating
1970 force–distance
from
five
modulus
distribution
obtained
by calculating
1970 force–distance
curves curves
chosen chosen
from five
elastic
elastic
modulus
maps
(five
pairs
of
daughter
cells),
respectively.
The
elastic
modulus
of
the
daughter
modulus maps (five pairs of daughter cells), respectively. The elastic modulus of the daughter cell in
cell nuclear
in the nuclear
region
at the telophase
of mitosis
ranges
0.55.5
kPa
to with
5.5 kPa,
with an value
average
the
region at
the telophase
of mitosis
ranges from
0.5from
kPa to
kPa,
an average
of
value
of
2.0
±
0.68
kPa.
Consistent
with
the
cells
at
the
interphase,
the
elastic
modulus
is
stiffer
in
the
2.0 ± 0.68 kPa. Consistent with the cells at the interphase, the elastic modulus is stiffer in the peripheral
peripheral
region
varying
kPa
to 7.0
kPa withvalue
an average
of 3.5
1.3 kPa. the
In addition,
region
varying
from
0.5 kPafrom
to 7.00.5
kPa
with
an average
of 3.5 ±value
1.3 kPa.
In ±addition,
cleavage
the
cleavage
furrow
is
found
to
contribute
significantly
to
the
stiffness
due
to
the
high
concentration
furrow is found to contribute significantly to the stiffness due to the high concentration of the F-actin
of the
the furrow
F-actin region
in the furrow
in
[9,30]. region [9,30].
Micromachines 2017, 8, 89
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3 of 9
Figure
Figure 6.
6. (a)
(a) The
The elastic
elastic modulus
modulus map
map of
of aa single
single cell
cell at
at the
the telophase
telophase of
of mitosis
mitosis with
with aa resolution
resolution of
of
32
×
32;
(b)
the
histogram
of
the
elastic
modulus
distribution
of
one
daughter
cell
in
the
whole
32 × 32; (b) the histogram of the elastic modulus distribution of one daughter cell in the whole region
region
(gray),
(gray), the
the nuclear
nuclear region
region (green),
(green),and
andthe
theperipheral
peripheralregion
region(red).
(red).
3.5.
3.5. Comparison of the Elastic Modulus
Modulus between
between Two
Two Stages
Stages
A
of the
the average
average elastic
elasticmodulus
modulusbetween
betweenthe
thetwo
twostages
stages
shown
Figure
7.
A comparison of
is is
shown
in in
Figure
7. A
A
significant
distinction
theaverage
averageelastic
elasticmodulus
moduluswas
wasobtained
obtainedbetween
between the
the two
two stages in the
significant
distinction
ofofthe
the
nuclear
respectively.
TheThe
average
elastic
modulus
at theat
interphase
was higher
nuclear and
andperipheral
peripheralregions,
regions,
respectively.
average
elastic
modulus
the interphase
was
than
that
at the
of mitosis
in bothin
the
nuclear
region region
and theand
peripheral
region.region.
The major
higher
than
thattelophase
at the telophase
of mitosis
both
the nuclear
the peripheral
The
reason
is that is
thethat
cellthe
hascell
anhas
unstable
structure
at the telophase
of mitosis
due to due
the decreasing
of the
major reason
an unstable
structure
at the telophase
of mitosis
to the decreasing
adhesion
between
the cell the
andcell
theand
extracellular
matrix [16],
which
highbear
amounts
of stress.
of the adhesion
between
the extracellular
matrix
[16],cannot
whichbear
cannot
high amounts
Moreover,
in this experiment,
the spreading
area of thearea
cell at
(Figure 2a)(Figure
was measured
of stress. Moreover,
in this experiment,
the spreading
of the
the interphase
cell at the interphase
2a) was
2 , whereas the2spreading area of the single daughter cell at the telophase of mitosis
to
be about to
1500
measured
beµm
about
1500 μm , whereas the spreading area of the single daughter cell at the
2 . It is obvious that the2 actin stress fibers are more abundant on the larger
(Figure
2b)of
was
about(Figure
800 µm2b)
telophase
mitosis
was about 800 μm . It is obvious that the actin stress fibers are more
spreading
area,
cause the
larger
elastic
abundant on
thewhich
largermay
spreading
area,
which
maymodulus.
cause the larger elastic modulus.
Micromachines 2017, 8, 89
Micromachines 2016, 8, 89
8 of 10
3 of 9
Figure 7.
7. Comparison
Comparisonof
ofthe
theelastic
elasticmodulus
modulusof
ofcells
cellsatatthe
theinterphase
interphaseand
andthe
thetelophase
telophaseofofmitosis.
mitosis.
Figure
Dataare
arepresented
presented as
as mean
mean ±±standard
standarderror
errorofofthe
themean
mean(**
(**denoted
denotedpp<<0.01).
0.01).
Data
4.
4.Conclusions
Conclusions
In
paper,we
wequantitatively
quantitativelyevaluated
evaluated
elastic
modulus
of OS
U-2cells
OS in
cells
the nuclear
In this paper,
thethe
elastic
modulus
of U-2
thein
nuclear
region
region
and
the
peripheral
region
at
the
interphase
and
the
telophase
of
mitosis,
respectively.
The
and the peripheral region at the interphase and the telophase of mitosis, respectively. The results
results
indicated
the stiffness
of the peripheral
region
the
cell was significantly
than
indicated
that thethat
stiffness
of the peripheral
region of the
cell of
was
significantly
higher than higher
that of other
that
of other
of thethe
cell
at either or
thethe
interphase
telophase
of mitosis.
However,
the
regions
of theregions
cell at either
interphase
telophaseor
of the
mitosis.
However,
the elastic
modulus
at
elastic
modulus
at
the
interphase
was
greater
than
that
at
the
telophase
of
mitosis
in
the
nuclear
the interphase was greater than that at the telophase of mitosis in the nuclear region and the peripheral
region
the peripheral
region, respectively.
Thisthe
investigation
that the property
variationcan
of the
region,and
respectively.
This investigation
reveals that
variation ofreveals
the mechanical
not
mechanical
property
can
not
only
occur
in
a
cancer
cell,
but
also
in
a
healthy
cell
which
is
at
the
only occur in a cancer cell, but also in a healthy cell which is at the division phase. Our findings in
division
phase.
Our findings
in this study
canaccurate
contribute
to providing
accurate
cancermodulus
diagnoses
this study
can contribute
to providing
more
cancer
diagnosesmore
by using
the elastic
as
by
using the elastic modulus as a biomarker.
a biomarker.
Acknowledgments: The
supports provided
provided by
by the
the National
National
Acknowledgements:
The authors
authors would like to acknowledge the partial supports
NaturalScience
ScienceFoundation
Foundationof
ofChina
China(Nos.
(No. 51575090,
11272083
No. 11502049),
of International
Natural
51575090, No.
11272083
and and
11502049),
Project ofProject
International
Science
Science and Technology Cooperation and Exchange of Sichuan (No. 2017HH0072), the Fundamental Research
and Technology Cooperation and Exchange of Sichuan (No. 2017HH0072), the Fundamental Research Funds for
Funds for the Central Universities (No. ZYGX2014Z004, No. ZYGX2016KYQD118 and No. ZYGX2015J084),
the
Central
Universities
(Nos.
ZYGX2014Z004,
ZYGX2016KYQD118
and
ZYGX2015J084),
the China
the China
Postdoctoral
Science
Foundation
Grant (No.
2016M590873) and the
National
Youth Top-Notch
Talent
Postdoctoral
Science
Foundation
Grant
(No.
2016M590873)
and
the
National
Youth
Top-Notch
Talent
Support
Support Program.
Program.
Author Contributions: Guocheng Zhang carried out the experiments and drafted the manuscript. Hai Jiang
revised the
manuscript. Guocheng
Bei Peng, Na
Fan, carried
Jian Guo
and
Jiang participated
in thethe
design
of the experiment.
Authors
Contributions:
Zhang
out
theHai
experiments
and drafted
manuscript.
Hai Jiang
Xiaoying Lv cultured and seeded the cells. Yiyao Liu and Longxiang Yang contributed to the valuable discussion.
revised the manuscript. Bei Peng, Na Fan, Jian Guo and Hai Jiang participated in the design of the experiment.
All authors read and approved the final manuscript.
Xiaoying Lv cultured and seeded the cells. Yiyao Liu and Longxiang Yang contributed to the valuable
Conflicts ofAll
Interest:
authors
declare no
of interest.
discussion.
authorsThe
read
and approved
theconflict
final manuscript.
Conflicts
of Interest: The authors declare no conflict of interest.
References
1.
Bao, G.; Suresh, S. Cell and molecular mechanics of biological materials. Nat. Mater. 2003, 2, 2715–2797.
References
[CrossRef] [PubMed]
1.2. Bao,
Cell and
molecular
mechanics
of biological
materials.
Nat. Mater.
2003, 2, 2715–2797.
Zhu,G.;
C.;Suresh,
Bao, G.;S.Wang,
N. Cell
mechanics:
mechanical
response,
cell adhesion,
and molecular
deformation.
2. Annu.
Zhu, C.;
Bao,
G.;
Wang,
N.
Cell
mechanics:
mechanical
response,
cell
adhesion,
and molecular
Rev. Biomed. Eng. 2000, 2, 189–226. [CrossRef] [PubMed]
deformation.
Annu. Rev.
Biomed.
Eng. S.
2000,
2, 189–226.
3.
Good,
D.W.; Stewart,
G.D.;
Hammer,
Elasticity
as a biomarker for prostate cancer: A systematic review.
3.
Good,
D.W.;
Stewart,
G.D.;
Hammer,
S.
Elasticity
BJU Int. 2014, 113, 523–534. [CrossRef] [PubMed] as a biomarker for prostate cancer: A systematic review.
Int. 2014,
113, 523–534.
4. BJU
Lekka,
M. Atomic
force microscopy: A tip for diagnosing cancer. Nat. Nanotechnol. 2012, 7, 691–692.
4.
Lekka,
M.
Atomic
force
[CrossRef] [PubMed] microscopy: A tip for diagnosing cancer. Nat. Nanotechnol. 2012, 7, 691–692.
5.5. Ward,
Li,Li,
W.;
Zimmer,
S.; S.;
Davis,
T. T.
Viscoelastic
Ward, K.;
K.A.;
W.I.;
Zimmer,
Davis,
Viscoelasticproperties
propertiesofoftransformed
transformedcells:
cells:Role
Roleinintumor
tumorcell
cell
progression
and
metastasis
formation.
Biorheology
1990,
28,
301–313.
progression and metastasis formation. Biorheology 1990, 28, 301–313.
6.
Guck, J.; Schinkinger, S.; Lincoln, B.; Wottawah, F.; Ebert, S.; Romeyke, M.; Lenz, D.; Erickson, H.M.;
Ananthakrishnan, R.; Mitchell, D. Optical Deformability as an inherent cell marker for testing malignant
transformation and metastatic competence. Biophys. J. 2005, 88, 3689–3698.
7.
Goldmann, W.H.; Galneder, R.; Ludwig, M.; Xu, W.; Adamson, E.D.; Wang, N.; Ezzell, R.M. Differences in
Micromachines 2017, 8, 89
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
9 of 10
Guck, J.; Schinkinger, S.; Lincoln, B.; Wottawah, F.; Ebert, S.; Romeyke, M.; Lenz, D.; Erickson, H.M.;
Ananthakrishnan, R.; Mitchell, D. Optical Deformability as an inherent cell marker for testing malignant
transformation and metastatic competence. Biophys. J. 2005, 88, 3689–3698. [CrossRef] [PubMed]
Goldmann, W.H.; Galneder, R.; Ludwig, M.; Xu, W.; Adamson, E.D.; Wang, N.; Ezzell, R.M. Differences in
elasticity of vinculin-deficient F9 cells measured by magnetometry and atomic force microscopy. Exp. Cell Res.
1998, 239, 235–242. [CrossRef] [PubMed]
Kuznetsova, T.G.; Starodubtseva, M.N.; Yegorenkov, N.I.; Chizhik, S.A.; Zhdanov, R.I. Atomic force
microscopy probing of cell elasticity. Micron 2007, 38, 824–833. [CrossRef] [PubMed]
Jiang, Q.; Chen, P.; Li, J. Spatial distribution of cytoskeletal mechanical properties in vascular smooth muscle cells.
J. Biomed. Sci. Eng. 2015, 8, 350. [CrossRef]
Rebelo, L.M.; Desousa, J.S.; Mendes, F.J.; Radmacher, M. Comparison of the viscoelastic properties of cells
from different kidney cancer phenotypes measured with atomic force microscopy. Nanotechnology 2013, 24,
1–11. [CrossRef] [PubMed]
Cross, S.E.; Jin, Y.S.; Tondre, J.; Wong, R.; Rao, J.; Gimzewski, J.K. AFM-based analysis of human metastatic
cancer cells. Nanotechnology 2008, 19, 384003. [CrossRef] [PubMed]
Costa, K.D.; Sim, A.J.; Yin, F.C. Non-Hertzian approach to analyzing mechanical properties of endothelial
cells probed by atomic force microscopy. J. Biomech. Eng. 2006, 128, 176–184. [CrossRef] [PubMed]
Sosa, M.S.; Bragado, P.; Aguirreghiso, J.A. Mechanisms of disseminated cancer cell dormancy: An awakening field.
Nat. Rev. Cancer 2014, 14, 611–622. [CrossRef] [PubMed]
Mavrakis, M.; Azougros, Y.; Tsai, F.C.; Bertin, A.; Alvarado, J.; lv, F.; Kress, A.; Brasselet, S.; Lecuit, T.
Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles. Nat. Cell Biol.
2014, 16, 322–334. [CrossRef] [PubMed]
Lancaster, O.M.; Baum, B. Shaping up to divide: Coordinating actin and microtubule cytoskeletal remodelling
during mitosis. Semin. Cell Dev. Biol. 2014, 34, 109–115. [CrossRef] [PubMed]
Heng, Y.W.; Koh, C.G. Actin cytoskeleton dynamics and the cell division cycle. Int. J. Biochem. Cell Biol. 2010,
42, 1622–1633. [CrossRef] [PubMed]
Matzke, R.; Jacobson, K.; Radmacher, M. Direct, high-resolution measurement of furrow stiffening during
division of adherent cells. Nat. Cell Biol. 2001, 3, 607–610. [CrossRef] [PubMed]
Mathieu, P.S.; Loboa, E.G. Cytoskeletal and focal adhesion influences on mesenchymal stem cell shape,
mechanical properties, and differentiation down osteogenic, adipogenic, and chondrogenic pathways.
Tissue Eng. Part B. Rev. 2012, 18, 436–444. [CrossRef] [PubMed]
Stewart, M.P.; Toyoda, Y.; Hyman, A.A. Tracking mechanics and volume of globular cells with atomic force
microscopy using a constant-height clamp. Nat. Protoc. 2012, 7, 143–154. [CrossRef] [PubMed]
Hutter, J.L.; Bechhoefer, J. Calibration of atomic-force microscope tips. Rev. Sci. Instrum. 1993, 64, 1868–1873.
[CrossRef]
Sneddon, I.N. The relation between load and penetration in the axisymmetric boussinesq problem for
a punch of arbitrary profile. Int. J. Eng. Sci. 1965, 3, 47–57. [CrossRef]
Alcaraz, J.; Buscemi, L.; Grabulosa, M.; Trepat, X.; Fabry, B.; Farre, R.; Navajas, D. Microrheology of human
lung epithelial cells measured by atomic force microscopy. Biophys. J. 2003, 84, 2071–2079. [CrossRef]
Lekka, M. Discrimination between normal and cancerous cells using AFM. Bionanoscience 2016, 6, 1–16.
[CrossRef] [PubMed]
Suresh, S. Biomechanics and biophysics of cancer cells. Acta Biomater. 2007, 3, 413–438. [CrossRef] [PubMed]
Clark, A.G.; Paluch, E. Mechanics and regulation of cell shape during the cell cycle. Results. Probl. Cell Differ.
2011, 53, 31–73. [PubMed]
Pogoda, K.; Jaczewska, J.; Wiltowska-Zuber, J.; Klymenko, O.; Zuber, K.; Fornal, M.; Lekka, M. Depth-sensing
analysis of cytoskeleton organization based on AFM data. Biophys. Struct. Mech. 2012, 41, 79–87. [CrossRef]
[PubMed]
Haupt, B.J.; Pelling, A.E.; Horton, M.A. Integrated confocal and scanning probe microscopy for biomedical
research. Sci. World J. 2006, 6, 1609–1618. [CrossRef] [PubMed]
Stewart, M.P.; Helenius, J.; Toyoda, Y.; Ramanathan, S.P.; Muller, D.J.; Hyman, A.A. Hydrostatic pressure
and the actomyosin cortex drive mitotic cell rounding. Nature 2011, 469, 226–230. [CrossRef] [PubMed]
Micromachines 2017, 8, 89
29.
30.
10 of 10
Pollard, T.D.; Borisy, G.G. Cellular motility driven by assembly and disassembly of actin filaments. Cell 2003,
112, 453–465. [CrossRef]
Sanger, J.M.; Sanger, J.W. Assembly of cytoskeletal proteins into cleavage furrows of tissue culture cells.
Microsc. Res. Tech. 2000, 49, 190–201. [CrossRef]
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