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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. 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