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Measuring Microenvironment Mechanical Stress of Rat Liver
During Diethylnitrosamine Induced Hepatocarcinogenesis by
Atomic Force Microscope
ZHAO GANG, QIN QI, CUI JING, AND CHUNYOUWANG*
Pancreatic Surgical Center, Union Hospital, Wuhan City, Hubei Province 430022, People’s Republic of China
KEY WORDS liver cirrhosis; hepatic cell carcinoma; carcinogenesis; stiffness; AFM
ABSTRACT We developed a highly sensitive method to detect liver tissue stiffness with atomic
force microscopy (AFM), and investigated the physical features of hepatocarcinogenesis. Wistar
rats received weekly intraperitoneal injections of diethylnitrosamine (DEN) or saline (control) fol-
lowed by a 2-week wash-out period. Liver samples were harvested at 10, 14, or 18 weeks for patho-
logical examination and stress detection. Previously normal liver tissues developed fibrosis and
carcinoma after DEN administration. Although the elastic modulus (E) values of the normal (sa-
line; 0.18 6 0.04 MPa), fibrotic (8 weeks DEN; 0.25 6 0.06 MPa) and cirrhotic (12 weeks DEN; 0.39
6 0.06 MPa) tissues were significantly different, there was no significant difference between the E
values of the cirrhotic and the hepatic cell carcinoma (16 weeks DEN; 0.42 6 0.07 MPa) tissues.
Thus, tissue stiffness quantitatively increases during hepatocarcinogenesis, and AFM can be used
to sensitively and precisely detect liver stiffness at the microscopic level. Microsc. Res. Tech.
72:672–678, 2009. VVC 2009 Wiley-Liss, Inc.
INTRODUCTION
As in many carcinomas, the carcinogenesis of hepatic
cellular carcinoma (HCC) is a multi-step process
involving various genetic alterations that ultimately
lead to malignant hepatocyte transformation. The
main established risk factors for HCC are chronic viral
hepatitis B and C infection and aflatoxin B1 (McGlynn
and London, 2005). In most (70–90%) HCC cases, there
is underlying liver cirrhosis. The risk of HCC differs
among hepatitis C patients, depending on the degree of
liver fibrosis and the time of hepatic C virus (HCV)
acquisition. The risk of HCC for cirrhotic HCV patients
is 5.8%, which is obviously higher than HCV patients
with lower levels of fibrosis (0.5–2.6%, Kiyosawa,
2002). Thus, HCC is a complex neoplasm that often
occurs in the preneoplastic cirrhotic liver, and survival
as well as treatment options depend on both HCC and
cirrhosis variables. However, the exact role of cirrhosis
in HCC carcinogenesis is seldom addressed.
Cirrhosis is associated with the massive deposition
of matrix in the extracellular microenvironment and
increased tissue stiffness, which can also be observed
in HCC. Extracellular matrix (ECM) orientation medi-
ates tension-dependent cell migration to orchestrate
developmental processes such as gastrulation, and ma-
trix rigidity influences cell growth, viability, differen-
tiation, and motility (Tzvetkova-Chevolleau et al.,
2008). Tumor rigidity likely reflects an elevation in in-
terstitial tissue pressure and solid stress due to a per-
turbed vasculature and tumor expansion. An increase
in the elastic modulus (E) value of transformed cells is
mediated by an altered cytoarchitecture, and matrix
stiffening linked to fibrosis. Tumor rigidity may influ-
ence the treatment efficacy and enhance tumor metas-
tasis. Paszek et al. (2005) showed that mammary dif-
ferentiation and tissue homeostasis were most favored
by a soft tissue microenvironment; the breast desmo-
plasia and tissue stiffening that accompany mammary
tumor development might therefore actively promote
the malignant behavior of the gland.
The mechanical characteristic of macroscopic tissue
is typically defined as stiffness or rigidity; however,
when we focus on the interactions between cell and its
microenvironment from a microscopic perspective, this
physical property should be described as stress. Atomic
force microscopy (AFM) can be used to uncover the
nanomechanical properties of small regions of materi-
als, such as single molecules or individual cells (Parot
et al., 2007). To fully understand the processes underly-
ing the interaction of cells with their environments, a
combination of AFM experiments is essential (Ludwig
et al., 2008).
We hypothesized that the increased ECM stiffness of
the cirrhotic liver may mediate hepatocarcinogenesis.
We used diethylnitrosamine (DEN) to simulate the car-
cinogenesis of a rat liver from fibrosis to HCC, and
developed a new protocol for stiffness measurement
with AFM. Furthermore, we quantified the elastic
properties of normal, fibrotic, cirrhotic, and carcinoma
liver tissues throughout carcinogenesis.
Z. G. and Q. Q. contributed equally to this work.
*Correspondence to: Chunyou Wang, Pancreatic Surgical Center, Union Hospi-
tal, Jiefang Avenue 1277, Wuhan city, Hubei Province 430022, People’s Republic
of China. E-mail: chunyouwang52@163.com
Received 15 November 2008; accepted in revised form 27 February 2009
Contract grant sponsor: The National Natural Science Foundation of China;
Contract grant number: 30600594
DOI 10.1002/jemt.20716
Published online 7 April 2009 in Wiley InterScience (www.interscience.
wiley.com).
VVC 2009 WILEY-LISS, INC.
MICROSCOPY RESEARCH AND TECHNIQUE 72:672–678 (2009)
MATERIALS ANDMETHODS
Experimental Design
All animal care and experimentation conformed to
the Guide for the Care and Use of Laboratory Animals
from the National Academy of Sciences. Sixty Wistar
rats weighing 180–200 g were divided into control, fi-
brosis, cirrhosis, and HCC groups. To simulate hepato-
carcinogenesis from fibrosis to carcinoma, rats within
the fibrosis, cirrhosis, and HCC groups received weekly
intraperitoneal (i.p.) injections of DEN (Sigma-Aldrich,
St. Louis, MO) at 50 mg/kg for 8, 12, and 16 weeks,
respectively, and were sacrificed at 2 weeks after the
last injection (to allow recovery from acute necrosis).
Rats in the control group were injected with saline and
sacrificed at three time points corresponding to the
three treated groups. At the time of sacrifice, animals
were anesthetized with an i.p. injection of thiopental.
Liver samples were harvested for pathological exami-
nation and stiffness measurement with AFM.
Histology and Hematoxylin and Eosin Staining
Tissue slices were rinsed in ice cold PBS and fixed in
10% buffered formalin for 1 h. Slices were dehydrated
in a series of increasing concentrations of ethanol to
100% and then xylene. Slices were embedded in paraf-
fin and sectioned at a thickness of 4 lm on a micro-
tome. Sections were mounted on slides, rehydrated,
stained in hematoxylin and eosin successively, and
then covered with a glass coverslip. Images were
acquired by an optical microscope.
Atomic Force Microscopy
An AFM (Picoscan, 5500, Agilent, CA) equipped
with a standard stage suite for fluid cells was used to
measure the material properties of the liver. The can-
tilever probe used for all indentations had a square-
based pyramidal silicon-nitride tip with a 208 half-
opening angle on a V-shaped 200-lm-long silicon
nitride cantilever with a nominal spring constant (k)
of 0.56 N/m (Agilent). Cantilevers were calibrated by
measuring the thermally induced motion of the
unloaded cantilever.
Liver Sample Preparation
Focal necrosis develops during hepatocarcinogenesis,
particularly in the center of tumor nodules. Liver sam-
ples were harvested from freshly sacrificed rats at four
typical homogeneous solid sites without focal necrosis
by visual inspection under stereomicroscope. One liver
section (� 2 mm thickness) was sliced out at each site
by a vibratome (752 M Vibroslice, Campden Instru-
ments, Loughborough, UK). The specimens, usually
harvested from several rats at once, were stored in cold
PBS supplemented with a protease inhibitor cocktail
(Complete, Boehringer-Mannheim, Mannheim,
Germany). Polystyrene Petri dishes (35 mm diameter,
Corning) were used as fluid cells.Liver sections were
glued to the dishes with histoacryl tissue glue
(B. Braun Surgical, Melsungen, Germany) and the
samples were covered with PBS.
AFM Indentation
Force curves were collected by monitoring the canti-
lever deflection while ramping the piezo scanner in z,
with the xy scanning disabled, resulting in a plot of
force versus sample position (Burnham and Colton,
1989). To achieve a constant and well-defined maxi-
mum applied load, the maximum tip deflection value
was set to 1,000 nm, which indicates the desired
increase in deflection beyond that of the undeflected
cantilever probe (Stolz et al., 2004). Lattice indenta-
tions arranged in 9 3 9 arrays were performed on each
section. A spacing of 10 lm was chosen so that each
indention would not interfere with the preceding and
the following indentations.
Data Analysis of the Force Curves
The slope of a force curve (Fig. 2) describes the elas-
tic properties of a sample in a qualitative way. To calcu-
late E from the force curves, we employed Sneddon’s
modification of the Hertzian model (Costa and Yin,
1999; Domke and Radmacher, 1998; Weisenhorn et al.,
1993) for the elastic indentation of a flat, soft sample
by a stiff cone. The model gives the flowing relation
between the applied loading force F and the indenta-
tion depth d:
F ¼ ð2=pÞ½E=ð1� m2Þd2 tan a� ð1Þ
where E is the Young’s modulus, a is the half-opening
angle of the AFM tip, and m is the Poisson’s ratio of the
sample (assumed to be 0.5). We assumed that a smaller
Poisson’s ratio would increase the estimation of E. Our
primary goal, however, was to demonstrate a relative
difference in the E values, rather than an absolute
value.
On a stiff sample, the cantilever deflection d(z) will be
equal to the piezo movement z, whereas on a soft sam-
ple the deflection is decreased because of elastic inden-
tation d(z) 5 z 2 d. Hooke’s law connects the deflection
of the cantilever and the applied loading force via the
known spring constant k of the cantilever:
F ¼ kdðzÞ ¼ kðz� dÞ ð2Þ
We combined Eqs. (1) and (2) to obtain E as a func-
tion of the measured quantities z and d. Fitting this
function to the force-curve data not only yields E but
also the position Z0. Z0 indicates the position where the
cantilever initially contacts the sample, which corre-
sponds to the real sample height at the point where the
force curve is recorded.
During the measurements, the AFM tip may indent
into ruptured cells, the hepatic sinus space, or the vas-
cular lumen, leading to abnormal or nonexistent
curves. The abnormal force curves exhibiting a positive
precontact slope or too little pre- or postcontact data
were eliminated from the analysis. In addition, for
some factors, the measurement interfered with noise
signals, resulting in waves with smooth curves; such
curves were also excluded from analysis.
Due to the liver tissue viscosity, hydrodynamic drag
can be seen as a separation of the off-surface parts of
the approaching and retracting force curves, which
adds a constant external force to the loading force of
the cantilever. The higher the scan velocity, the more
the approach and retract curves are separated
(Vinckier and Semenzaa, 1998). By indenting slowly
Microscopy Research and Technique
673MICROENVIRONMENT MECHANICAL STRESS BY AFM
enough, the viscous contributions are small and force
measurements are dominated by elastic behavior. The
force curve was obtained in the contact mode at a tip
velocity (0.5 lm/s) previously determined to be slow
enough to minimize the amount of hysteresis, yet fast
enough to maximize the number of force curves that
can be captured in a given experiment. We used 50–
75% of the approaching curve to calculate the indenta-
tion, because use of the retracting curve leads to an
incorrect measurement of indentation.
Statistical Analysis
The mean elastic modulus for each sample was calcu-
lated from the force curve, which was fit using the cri-
teria above. Data from each group were expressed as
the mean 6 SD by frequency analysis. The Levene ho-
mogeneity test indicated unequal variances (P < 0.05).
Therefore, the means were analyzed by one-way analy-
sis of variance with a Brown Forsythe test to account
for unequal variances, followed by Bonferroni posthoc
tests using SPSS (SPSS, Chicago, IL) to evaluate dif-
ferences between regions. A P-value < 0.05 was consid-
ered statistically significant.
RESULTS
Histological Changes of Rat Livers During
DEN-Induced Hepatic Carcinogenesis
None of the control group rats died during the
experiment, and five were sacrificed at each time point.
Seven of the rats in the other three groups died during
the experiment, and 14, 13, and 11 of the DEN-admin-
istered rats were sacrificed at 10, 12, and 18 weeks,
respectively.
The liver tissues of the control group rats contained
intact lobules, and neither inflammation nor fibrosis
was found in the tissue sections (Fig. 1A).
The liver samples of rats that received DEN for 8
weeks (fibrotic group) were enlarged with a smooth
surface. Under the microscope, there was apparent
degeneration and necrosis of hepatocytes located at the
portal area and limiting plate, and many inflammatory
cells had infiltrated into the portal space and lobules.
Fibrous tissues surrounding the portal space had pro-
liferated and formed fibrous septa in the lobules,
accompanied by a normal or mussy lobular structure;
however, no liver cirrhosis was seen (Fig. 1B).
The liver samples harvested after DEN was adminis-
tered for 12 weeks (cirrhotic group) were malformed,
stiffened, and shrunk. The normal lobular architecture
was disturbed and pseudolobules had formed. Hepato-
cytes in the pseudolobules were arranged turbulently,
with absent or deviated central veins. Numerous small
bile ducts could be seen in the fibrous tissue, accompa-
nied by lymphocyte infiltration (Fig. 1C).
After DEN was administered for 16 weeks (HCC
group), the rat livers were enlarged. There were sev-
eral gray nodes in the liver, with diameters ranging
from 1–2 cm. Hemorrhage and necrosis could be seen
on the surface. Microscopy revealed that most of the
cancer nodules were surrounded by sclerotic tissue
(Fig. 1D).
Fig. 1. Pathological examination of livers harvested from the con-
trol group and the DEN-induced carcinogenesis groups. Images show
representative samples of HE staining with 3100 magnification from
each group. (A) Normal tissue of the control group; (B) Fibrotic liver
of rat administered DEN for 8 weeks; (C) Pseudolobule formed after
DEN administration for 12 weeks; (D) HCC were observed after DEN
administration for 16 weeks.
Microscopy Research and Technique
674 Z. GANG ET AL.
Effects of Pathological Changes on Tissue
Mechanical Properties
Since liver tissue is heterogeneous at the microscopic
level, stress may present differently at different sites.
It is difficult to select a representative load-displace-
ment curve for a given sample, although the slope rate
of a typical curve (Fig. 2) may reflect the gross stiffness
of a sample. The data obtained from repeated multiple-
site measurements revealed the average modulus of
the sample, and statistical results showed that these
data fit a Gaussian distribution (Fig. 3).
The E value of control group samples sacrificed at
different times were not significantly different; the av-
erage E value for control samples was 0.18 6 0.04
MPa. The E increased throughout the first 8 weeks of
DEN-induced carcinoma, when fibrosis was aggra-
vated. The E values of samples within the fibrotic
(8 weeks of DEN) and cirrhotic (12 weeks of DEN)
groups were 0.25 6 0.06 MPa and 0.39 6 0.06 MPa,
respectively. The differences between the fibrotic and
control groups and between the fibrotic and cirrhotic
groups were both significant. The average elastic mod-
ulus of the cirrhotic liver was two-foldlarger than that
of the control liver, as measured by the AFM. However,
there was no significant difference between the E val-
ues of the cirrhotic liver and HCC (16 weeks DEN; E 5
Fig. 3. Histograms and corresponding Gaussian distribution curves for stiffness measurements
within the different groups. Histograms were based on all of the load-displacement curves within each
group.
Fig. 2. Typical load-displacement curve. X-axis represent the rela-
tive distance between the sample and probe. Y-axis represent cantile-
ver deflection. The cantilever did not deflect until the probe contacted
the sample. The increase in deflection is associated with the depth of
indention, which is manifested as the ascent part of the curve. The
red and blue curves represent the approaching and retracting force
curves, respectively. As evidence of the viscous nature of liver tissue,
the two curves did not coincide. The first 50–75% part of the
approaching curve was used to calculate indentation. The parameters
necessary for data analysis are depicted on the graph: the zero deflec-
tion (d0) and the range of analysis defined by the two data points
(d1,z1) and (d2,z2). [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
Microscopy Research and Technique
675MICROENVIRONMENT MECHANICAL STRESS BY AFM
0.42 6 0.07 MPa). Figure 4 shows the dynamic changes
of the elastic modulus during carcinogenesis.
DISCUSSION
We used AFM to detect changes in the liver tissue
stiffness during DNA-induced hepatocarcinogenesis in
rats. We found that while the E values of normal (0.18
6 0.04 MPa), fibrotic (0.25 6 0.06 MPa), and cirrhotic
(0.39 6 0.06 MPa) tissues were significantly different,
there was no significant difference between the E val-
ues of the cirrhotic and the HCC (0.42 6 0.07 MPa) tis-
sues. Our results may help elucidate the nature of liver
mechanical properties during tumor development and
provide a foundation for further study on the correla-
tion between hepatic cirrhosis and HCC.
The ECM is a complex molecular milieu that serves
as a reservoir of soluble and insoluble signaling mole-
cules. Additionally, the ECM provides cells with the
tensile scaffolding needed for their appropriate assem-
bly into three-dimensional macroscopic structures
(Comoglio and Trusolino, 2005). Cells interact with
other cells and the ECM to establish a dynamic stress
homeostasis. Cellular responses to cell-exerted forces
involve a feedback loop of inside-outside signaling
coupled to the ECM elasticity. The feedback of the local
matrix stiffness on the cell state likely has important
implications for cellular development, differentiation,
disease, and regeneration (Discher et al., 2005).
Although the molecular pathways involved are only
partially known (Vogel, 2006; Vogel et al., 2006), epi-
thelial cells and fibroblasts, muscle cells, neurons, and
many other tissue cells have been shown to sense sub-
strate stiffness (Engler et al., 2004, Yeung et al., 2005).
Moreover, a physical cue devoid of chemical specificity
may switch cells between normal and cancerous states,
perhaps by initiating a cascade of multiple switches
that simultaneously trigger the leap from one self-sta-
bilizing ‘‘attractor’’ state to another within the genome-
wide cell regulatory network (Huang and Ingber, 2005
Huang et al., 2005).
More than 85% of cases of HCC occur in countries
with high rates of chronic HBV infection. In the West-
ern world where HBV is not prevalent, cirrhosis is
present in 85–90% of HCC cases, usually in the setting
of other chronic liver diseases without HBV infection
(Robbins, 2005). Furthermore, research from Japan
indicates that 20% of patients with alcohol-induced
liver cirrhosis also had HCC (Horie et al., 2003).
Whether liver cirrhosis is merely a passive stage dur-
ing carcinogenesis or cirrhosis positively enhances the
cell malignant transformation is worth further discus-
sion. Therefore, we hypothesized that the altered ECM
stiffness in cirrhosis may cause hepatic cell malignant
transformation and thus contribute to hepatic carcino-
genesis. Our primary task was to identify the mechani-
cal changes of the ECM during hepatocarcinogenesis.
In this research, we established rat models for HCC
and developed a protocol for detecting the stiffness of
the liver tissue by AFM.
Drug-induced carcinoma and tumor inoculation can
be used to establish a rat hepatocellular carcinoma
model. Despite the easily controlled size and locality of
the tumor and the short induction period, absence of
cirrhosis in the transplanted tumor model is a vital
flaw. In contrast to previous models, the present model
reproduces the sequence of cirrhosis and HCC, as is
most often the case in human liver disease. Thus, our
model provides a unique tool for understanding the
pathogenesis and evolution of liver cancer, and for
identifying preventive treatments for HCC. When
injected i.p. for 16 weeks (Schiffer et al., 2005), DEN
caused the sequential formation of fibrosis, cirrhosis,
and HCC after 8, 12, and 16 weeks, respectively. The
model had a tumor induction rate of 100% and a low
mortality rate.
Until now, cellular mechanical properties have been
studied by several techniques, including optical tweez-
ers (Ashkin and Dziedzic, 1989), pipette suction (Evans
et al., 1995), ultrasound (Ziol and Handra-Luca, 2005),
surface force apparatuses (Mazza et al., 2007), and
others. AFM measurements in particular merit careful
treatment due to the need to infer the load-indentation
relationship from the position and deflection of the can-
tilever. AFM has proven to be a powerful tool for bio-
physical studies, such as those investigating ligand-re-
ceptor interactions (Florin et al., 1994) or cell or tissue
elasticity (Engler et al., 2007). Relative to other techni-
ques, the main advantage of working on biological sam-
ples with AFM is the ability to operate nondestruc-
tively at nanometer-level resolution in ambient air or
in fluid environments.
AFM has been used in fields from polymer to biomed-
ical sciences as a tool for characterizing the topographi-
cal and mechanical properties of biological and syn-
thetic materials. As such, various analytical methods
have been developed for extracting elastic properties,
namely the Young’s (E) or the shear (G) modulus, from
deflection-position data (Lin et al., 2006). These analy-
sis methods are easily simplified for use with force-dis-
Fig. 4. Elastic modulus of the different groups. Error bars indicate
6 SD. *Significance from the Bonferroni posthoc test (P < 0.05). The
E values of liver samples from the control groups harvested at differ-
ent times were not significantly different (P 5 0.85). The E values of
the normal, fibrotic and cirrhotictissues were significantly different,
there was no significant difference between the E values of the cir-
rhotic and the hepatic cell carcinoma tissues (P 5 0.37).
Microscopy Research and Technique
676 Z. GANG ET AL.
placement data from depth-sensing systems, and the
Hertziam model is most commonly used by researchers
for AFM processing.
Most biological tissues, including the liver, are visco-
elastic. When an AFM probe indents on such materials,
an adhesion force will be generated between the con-
tact surfaces, causing a large decrease in force sensitiv-
ity and an unstable cantilever oscillation (Butt et al.,
1995). In addition, the surface layer of a liver section
exposed to the air will dry and shrink rapidly. To main-
tain the physiological state of the liver tissue and to
ensure the accuracy and stability of the results, we per-
formed AFM measurements in the liquid phase. PBS
merely consist water and several kinds of necessary
salt, with low viscosity that may benefit to the experi-
ments. Low temperature will slow down metabolic rate
of the hepatocytes to someextent, thus benefit to keep
tissue viability during experiment. On the other hand,
low temperature have influence on the structure and
function of some proteins within cells (Al-Fageeh and
Smales, 2006), which may bring deviation to the
results. Therefore, it will be meaningful to optimize
experimental temperature during AFM detection in
advanced research.
The cytoskeleton that mechanically stabilizes a cell
is a tensed tensegrity framework composed of molecu-
lar struts, ropes, and cables on the nanometer scale.
Ingber (1997) first described it as a model of ‘‘tensegrity
architecture,’’ which originated from the building prin-
ciple. Cells bind to one another and the ECM through
various junctional and nonjunctional adhesive mecha-
nisms, allowing the cell and the microenvironment to
maintain a dynamic steady state. Whenever a probe
contacts a cell, collagen, or other tissue structure, it is
contacting these components at steady state. Multiple
measurements microscopically represent the stiffness
or stress of the integral structure, which we define as
the microenvironment mechanical stress. Based on
this perspective and on the particular method used in
this research, our study revealed not only the change
in stiffness, but also the stress that the cells and their
microenvironment are exposed to.
New instruments are now available to researchers
that can be used to reveal the stiffness of a tissue or
organ by echo technology. This technologies include
transient elastography (Castera et al., 2008) and mag-
netic resonance elastography (Yin et al., 2007), which
are based on common ultrasound and magnetic reso-
nance. These instruments are gradually being used in
clinical research; however, the results merely repre-
sent the stiffness of a tissue on the macroscopic level.
In addition, technical limitations mean that they have
limited application on experimental animals. There-
fore, AFM was the more suitable method for the goals
of the current study.
In the present research, correlations between the
elastic modulus and histological findings during DEN-
induced carcinogenesis were evaluated. The results
revealed that the E value coincides with clinical path-
ological features. Since the E value detected by AFM
at the nanometer level reflects the interactions
between the hepatic cell and ECM, this method pro-
vides a great tool for investigating the roles that
microenvironmental stresses play on liver cell malig-
nant transformation.
ACKNOWLEDGMENTS
The authors thank Dr. Yi Lin for her insightful com-
ments and assistance in AFMmeasurement.
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