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Review TRENDS in Biotechnology Vol.25 No.3
Review
TRENDS in Biotechnology
Vol.25 No.3

Biomechanics approaches to studying human diseases

Gabriel Y.H. Lee 2 and Chwee T. Lim 1 , 2 , 3

1 Division of Bioengineering and Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, Singapore 2 Singapore–MIT Alliance, 4 Engineering Drive 3, Singapore 117576, Singapore 3 NUS Nanoscience & Nanotechnology Initiative, 9 Engineering Drive 1, Singapore 117576, Singapore

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 pro- gression, because diseased cells differ physically from healthy ones, but has also provided important knowl- edge 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.

Nanobiomechanics and its connections to human diseases Human disease can be defined as a condition, state or process occurring in our body that not only impairs our bodily structures and functions but also threatens our health and well-being. Every disease is unique and can vary in symptoms, signs and outcomes. Disease not only causes biological and functional alterations but also results in abnormalities in the physical and structural characteristics of cells. Current research on diseases mainly focuses on the molecular, microbiological, immunological and pathological aspects, rather than on the mechanical basis, which might make direct contributions to the symptoms and pathophy- siological outcomes. Recent studies on the pathophysiology of a wide range of human diseases have suggested that their etiologymight have resulted from deviation in the structural and mechanical properties of cells as well as from abnormal mechanotransduction (see Glossary) [1,2]. This not only gives rise to the breakdown of physiological functions in diseased states, it also disrupts or deregulates the molecular mechanisms by which cells sense mechanical signals and convert them into biochemical responses. Some examples of such diseases are arthritis, asthma, cancer, elliptocytosis, malaria, sickle cell anemia and spherocytosis (Table 1). Nanobiomechanics – an emerging topic of research interest –– involves the study of the mechanics of living cells and biomolecules and their connections to human diseases [3] . One reason for the emergence of this research

Corresponding author: Lim, C.T. ( ctlim@nus.edu.sg). Available online 25 January 2007.

is the availability of biophysical and nanotechnological tools that can mechanically probe cells and biomolecules in their physiological states at forces and displacement resolutions at the piconewton and nanometer scales, respectively. Such measurements were previously not achievable. Some of the recently developed biophysical and nanotechnological tools and experimental techniques include atomic force microscopy (AFM), molecular force spectroscopy, cytoindenter, flow cytometry, magnetic twisting cytometry, microfluidics, magnetic tweezers, microplate manipulation, optical tweezers or laser traps, and optical stretcher (see [4,5] for reviews). This has facilitated quantitative experimental and computational studies at the nano- and microscale on how the mechanical properties of a cell can be altered by the organizational or molecular changes occurring within the cell that can lead to or can arise from human diseases [1] ( Table 1). Studying human diseases from a biomechanics perspective can lead to a better understanding of the pathophysiology and pathogenesis of a variety of human diseases because changes occurring at the molecular and cellular levels will affect, and can be correlated to, changes occurring at the macroscopic level. This will provide an alternative and better approach to assess the onset or progression of diseases as well as to identify targets for therapeutic interventions. This review article will high- light some of the research in cell and molecular biomecha- nics being performed to study three types of diseases that

Glossary

Biorheological: relating to the deformation and flow of cells and biological fluids in a physiological environment. Creep behavior: a time-dependent deformation behavior of a body as it attempts to relieve itself of an applied stress. Displacement resolution: smallest division of distance measurable. Mechanotransduction: mechanism by which cells convert mechanical stimuli into biochemical responses. Merozoites: daughter protozoan cells that result from the asexual division of parasitic sporozoan cells during their life cycle. Rheoscope: instrument using fluid sheer stress to measure the deformability of RBCs. Shear elastic modulus: parameter that provides a measure of the resistance of a body to shearing or twisting. Schizonts: late-stage malaria-infected RBCs. Trophozoites: mid-stage malaria-infected RBCs. Young’s modulus : also known as elastic modulus, it is a parameter that provides a measure of the resistance of a material to elongation or compression.

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0167-7799/$ – see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2007.01.005

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manifest structural and mechanical property changes:

malaria, sickle cell anemia and cancer.

Role of mechanics in the pathophysiology of human diseases Malaria Healthy red blood cells (RBCs) are highly deformable – they transport oxygen to various parts of the body by squeezing their way through narrow capillaries. From a mechanistic perspective, two important outcomes in the pathophysiology of malaria (Box 1) are the increased rigidity and cytoadher- ence (stickiness) of infected RBCs. These not only cause serious impairment of blood flow but also result in severe anemia, coma or even death. Thus, investigating how an infected RBC undergoes extensive molecular and structural changes and how this eventually contributes towards the increased stiffness and cytoadherence will be important in the understanding of the pathophysiology of malaria [6]. Research into the biomechanics of malaria was first performed in 1971 by Miller et al., who studied suspensions of monkey RBCs infected with two different strains of the malarial parasites, using a viscometer and cell-filtration technique [7,8] . Their results were the first to indicate that infection by the Plasmodium parasites could impair the deformability of RBCs and, hence, the biorheological prop- erties of blood. Progressing from looking at a suspension of cells to observing the deformability of an individual cell, Cranston et al. [9] , in 1984, used a rheoscope to study the deformability of individual malaria-infected RBCs at different stages of infection. Similarly, Suwanarusk et al. [10] used a laminar–shear flow system to evaluate the deformability of RBCs at different stages of infection caused by the Plasmodium falciparum and Plasmodium vivax malarial parasites. The use of the micropipette aspiration technique [4] ( Figure 1) and optical tweezers [4] ( Figure 2 ) enables more accurate probing of the mechanical response of single infected cells down to piconewton and micrometer resol- utions. Nash et al. [11] , in 1989, were the first to use micropipette aspiration to study the abnormalities of RBCs infected with Plasmodium falciparum at different stages of infection. Subsequently, the micropipette aspiration tech- nique was used in 1993 to evaluate the effect of different strains of the malarial parasite on the deformability of

Box 1. Malaria

Malaria is one of the most severe parasitic diseases on earth. It dwarfs all other infectious diseases in that it infects 350– 500 million and results in 1.3–3 million deaths each year [54]. Malaria arises from the protozoan vertebrate blood parasites of the genus Plasmodium and is transmitted by the female Anopheles mosquito. Of the four Plasmodium strains of the malarial parasite ( falciparum , vivax , ovalae and malariae ), the Plasmodium falcipar- um strain accounts for 80% of all known cases and 95% of the malaria-associated mortalities [55]. In the event of an infection by the malarial parasite, the following changes take place in a RBC: decreased deformability and mechan- ical and rheological property changes; export of parasite proteins to the surface membrane; development of cytoadherent and rosetting properties, resulting in the sequestration of RBCs containing late- stage trophozoites and schizonts in deep vascular beds; and digestion of hemoglobin. When a malarial parasite invades and matures within a RBC, the cell becomes stiff and cytoadherent (sticky). The erythrocytic stage of malaria affects human RBCs and is associated with the pathophysiology and pathogenesis of the disease. The erythrocytic developmental stage of the parasite begins with the ring stage at 30 min after invasion of a parasite into a RBC [56–58]. At 20 h after invasion, it develops into a trophozoite, where the parasite continues to grow inside the RBC while exporting parasite proteins to the surface membrane of the cell. At 25 to 40 h after invasion, it finally develops into a late-stage schizont, where the nuclear division of the parasite results in the proliferation of between 12 to 20 (or more) merozoites as well as further export of parasite proteins to the cell surface membrane. As a result, distortion of the cell cytoskeleton and membrane occurs, and the infected RBC becomes more spherical than biconcave. At the end of the schizont stage, which is 48 h after invasion, the merozoites break out of the cell and begin to invade other healthy RBCs. Owing to the extensive cell modification caused by the parasite, as well as the direct specific interaction of the exported parasite proteins with the membrane and spectrin network of the RBC, the cell becomes stiff and sticky [56]. This contributes to the pathophysiology of malaria, which can result in blood clogging, anemia, coma or even death [58].

infected RBCs [12] . Two proteins – the knob-associated histidine rich protein (KAHRP) and the Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) – are known to have been exported from the parasite to the RBC membrane during infection. In 2002, Glenister et al. [13] used micropipette aspiration to study the change in the shear elastic modulus of RBC membranes infected with transgenic parasites (with either of the KAHRP or PfEMP3 genes deleted) to determine the contribution of these

Table 1. Some human diseases and the accompanying biophysical techniques and tools for detecting them

Human disease

Pathophysiological outcomes

Biophysical techniques and tools

Refs

Arthritis

Chondrocyte stiffening and increased viscosity

Micropipette aspiration

[65]

Asian ovalocytosis

Erythrocyte stiffening

Flow cytometry

[66]

Asthma

Airway smooth-muscle cell stiffening and contracting

Optical magnetic twisting cytometry, traction microscopy

[67]

Cancer

Epithelial and fibroblast cells softening and metastasis

Atomic force microscopy, optical stretcher, scanning acoustic microscopy

[35–37,47,68]

Cryohydrocytosis

Erythrocyte stiffening

Flow cytometry

[66]

Elliptocytosis

Erythrocyte stiffening and increased adherence

Dynamic flow adhesion assay

[69]

Malaria

Erythrocyte stiffening and cytoadherence

Viscometer, rheoscope, laminar flow assay, micropipette aspiration, optical or laser tweezers, microfluidics

[4,7–15,18,19]

Sickle cell anemia

Erythrocyte stiffening and increased viscosity

Optical or laser tweezers, micropipette aspiration

[27–30]

Spherocytosis

Erythrocyte stiffening and increased adherence

Laser diffraction viscometer

[70]

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Review TRENDS in Biotechnology Vol.25 No.3 113 Figure 1 . (a) Schematic diagram of the micropipette

Figure 1. (a) Schematic diagram of the micropipette aspiration of a cell. This technique uses a suction pressure, DP, to aspirate a single cell, wholly or partially, into a micropipette of inner radius, R P , and measures the cell elongation, L, as a function of suction pressure to determine the elastic shear modulus, G, of the cell membrane using the relation.

L

R P

¼ k DP

G

where k is a constant whose value depends on the mechanical model used in analyzing the experimental results. (b) An optical image showing the micropipette aspiration of a RBC. Figures reprinted from [4], Copyright 2006, with permission from Elsevier.

proteins to the increased rigidity of infected RBCs. Results showed that the removal of either protein reduced the rigidity of the membrane of the parasitized cell. This experiment demonstrates how the contribution of specific parasite-exported proteins to the stiffening of Plasmodium

falciparum -harbored RBCs can be determined quantitat- ively. Certainly, the effects of other parasite-exported proteins, as well as the mechanisms by which these proteins stiffen the cell membrane, still need to be further studied. In addition, optical tweezers were used, in 2004, to

In addition, optical tweezers were used, in 2004, to Figure 2 . (a) Schematic diagram showing

Figure 2. (a) Schematic diagram showing an example of the stretching of a single RBC using an optical tweezers setup. (b) Two silica microbeads are non-specifically attached diametrically across the RBC, as shown in the optical image, with the left bead anchored to the glass slide. (c) A laser beam is used to trap the right bead as the radiation pressure from a focused beam of laser gives rise to a force that could physically trap, control and manipulate small particles such as microbeads. The origin of this force is due to gradients in light intensity. The RBC is subsequently stretched by moving the glass slide to the left. The optical image shows the stretching of a healthy RBC to large deformation. The optical tweezers technique is capable of applying forces in the piconewton range. Figures in (b) and (c) were reprinted from [17], Copyright 2004, with permission from Elsevier.

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114 Review TRENDS in Biotechnology Vol.25 No.3 Figure 3 . (a) Schematic diagram showing the geometry

Figure 3. (a) Schematic diagram showing the geometry of a elastomeric microchannel used to study the flow behaviour of different stage malaria- infected RBCs with the width (w) of the microchannel sized at 2, 4, 6 and 8 mm, and height of the microchannel fixed at 2 mm. The arrow points to the entrance of the microchannel and indicates the flow direction in general. (b) An optical image of a healthy RBC squeezing through the entrance of the elastomeric microchannel depicted in (a), with w = 2 mm. (c) The optical image shows the stiffer late-stage schizonts clogging the entrance of the elastomeric microchannel depicted in (a) with w = 2 mm.

measure the mechanical property differences between healthy and infected RBCs at different stages of infection [14,15]. The stiffness of the infected RBCs was found to increase as the disease progresses. In all of these works, the researchers have focused on the changes in the shear elastic modulus of the membrane of infected RBCs, based on a RBC membrane model [16,17]. More recently, Shelby et al. [18] and Cheng et al. [19] were among the first to use microfluidics to investigate whether the rigidified infected RBCs can block capillaries that have diameters smaller than those of the infected RBCs ( Figure 3). Microfluidics enables the observation of the rheological behavior of individual cells flowing through microfluidic devices with micrometer-scale channels and chambers [20] . In 2003, Shelby et al. qualitatively showed that late-stage schizonts can cause blockages in narrow elastomeric channels, which were used to mimic the narrow capillaries found in the human body. Although much research has been focused on investigating the stiffening of malaria-infected RBCs, relatively less research has been done to quantify the physical adhesion involved in cytoadherence (stickiness). Studies have revealed that knob-like structures mani- fested on the infected cell membrane contain parasite- exported proteins that form the focal adherent points, onto which the infected cell sticks to the endothelial cells lining the blood vessels and capillaries. Some ligand–receptor pairs involved in cytoadherence have been identified [21] , but the contributions of each specific cytoadherent binding-prot ein has not been well quanti- fied. Almost all cytoadherence is mediated by the inter- action of the parasite protein PfEMP1 [22] with various endothelial receptor molecules, such as CD36 [23,24] , intercellular-adhesion molecule 1 (ICAM-1) [25] , E-selec- tin, and vascular cell-adhesion molecule 1 (VCAM-1) [26] . Thus, more quantitative measurements of the interaction force between the cytoadherent binding-proteins and the receptor molecules of the endothelial cells need to be performed. These studies demonstrate the relevance of biomechanics in studying malaria. With a better under- standing of malaria pathophysiology from a biomechanics

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perspective, we can provide useful information to clinicians on how they can better reduce parasite virulence. This will also assist in developing testing strategies that can quan- titatively evaluate the effectiveness of drugs being devel- oped to prevent or inhibit stiffening and cytoadherence of infected RBCs.

Sickle cell anemia Sickle cell anemia is a hereditary blood disorder that gives rise to blood circulatory problems due to an alteration in the molecular structure of hemoglobin (Box 2). Affected RBCs take the shape of a curved sickle and become more rigid and sticky. The mechanical properties of sickle-shaped RBCs have been probed using optical tweezers and micropipette aspiration. RBCs obtained from patients with sickle cell anemia are found to be stiffer and more viscous compared with healthy RBCs [27–30]: instead of moving through the bloodstream with great ease, these stiffer sickle-shaped RBCs end up clogging blood vessels and hence deprive tissues and organs of oxygen. According to Branda˜ o et al. [27] and Itoh et al. [31], because sickle cell anemia affects individual RBCs, the key advantage of using the micropip- ette aspiration and optical tweezers techniques is that they enable the study of individual diseased cells as compared with the average cell mechanical properties obtained from a cell population. This enables direct comparisons between a healthy and a diseased cell. In addition, Ballas et al. [32] and Branda˜ o et al. [27] studied the effects of hydroxyurea, an anti-tumor drug commonly used in the treatment of sickle cell disease. They found that the deformability of RBCs in sickle cell anemia patients undergoing hydroxyurea treatment for at least six months is almost identical to that of healthy RBCs. This suggests that cellular elasticity might also be used as a

Box 2. Sickle cell anemia

Sickle cell anemia, an inherited blood disorder, is a result of a genetic error occurring in the hemoglobin molecules. An autosomal recessive gene causes the formation of an abnormal hemoglobin (hemoglobin S [HbS]) that results in deformed RBCs. Major changes in cellular mechanics occur upon deoxygenation, when the HbS polymerizes. The deformed RBCs are rigid and come in the form of a curved sickle or crescent shape. Because these rigid sickle-shaped RBCs have difficulty flowing through the small blood vessels and capillaries in the body, this gives rise to blood circulatory problems, which deprive tissues and organs of oxygenated blood. The RBCs also rupture more easily and are removed from circulation by the spleen. Millions of people throughout the world are affected by sickle cell anemia. Each year, an estimated 120 000 infants are born with the disease in Africa (http://www.cwru.edu/med/epidbio/mphp439/ Sickle_Cell_Disease.htm), whereas in the USA 72,000 people are affected (http://www.nhlbi.nih.gov/health/dci/Diseases/Sca/SCA_ Summary.html). Some serious consequences of sickle cell anemia include stroke and infection. Pneumonia, however, remains the leading cause of death in children born with this disease; hence, early screening and diagnosis in infancy is vital for preventing death in children born with this type of hereditary blood disorder. Nevertheless, a person with the sickle cell trait (carrier) is known to demonstrate an increased resistance to malaria. First, in such a carrier, the malarial parasite can easily rupture the RBC and is, therefore, unable to reproduce. Second, owing to the polymeriza- tion of hemoglobin the parasite is unable to digest it. Therefore, in malaria-prone areas, the chance of surviving is actually higher for people carrying the sickle cell trait.

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Box 3. Cancer

Cancer is currently one of the leading causes of death worldwide, with >11 million cases and 7 million deaths attributed to it each year (http://www.who.int/cancer/en/). It is estimated that by 2020 there will be 15 million new cancer cases expected anually [59]. Cancer is a disease that results from a rapid, unrestricted and uncontrolled proliferation of abnormal cells, which possess increased deform- ability and adhesion. Cancer occurs when the genes responsible for cellular growth and repair undergo changes. To effectively treat or cure for cancer, there is a need to be able to detect the presence of cancer cells at the earliest stage possible. This will help to prevent them from spreading to other parts of the body in a process called metastasis (Box 4). When cancer is detected at an early stage, the odds of curing it are normally high. Although current diagnostic approaches have enabled the detec- tion of cancer, they are still not sensitive enough to quickly, reliably and accurately detect the presence of cancer at an early stage [60]. In most cases, cancer is not diagnosed or detected until the cancer cells have begun to metastasize or migrate; and metastasis is the major cause of death due to cancer.

potential drug-response indicator to monitor the effective- ness of drugs used in the treatment of diseases. Never- theless, for carriers and patients, early diagnosis is still important in providing preventive care or proper treatment for some of the devastating complications aris- ing from this disease.

Cancer Cancer can be characterized by the uncontrolled division of abnormal cells, which spread to various parts of the body and infiltrate and destroy healthy body tissue (Box 3). In the fight against cancer, complications in surgical treatment, and the inefficient and non-specific action, as well as the unwanted side effects, of chemotherapeutic drugs have been major hurdles for progress [33]. One pathophysiological outcome of cancer is that the affected cells are more deform- able than non-malignant cells [34–36]; therefore, the differ- ence in cell deformability can be exploited to distinguish cancer cells from healthy cells. Another outcome is the capacity of malignant cancer cells and tumors to infiltrate, invade or metastasize to distant sites. Currently, there is little understanding of how changes in the biomechanical properties of cancer cells and tumors can contribute to cancer metastasis. The use of AFM in cell and molecular biology is emerging as a powerful nanobiomechanical probe because of its ability to function as a high-resolution topographical imaging tool and a force sensor with piconewton resolution [4]. One key advantage of the AFM is its inherent ability to carry out real- time measurements on biological samples in their physio- logical conditions. Lekka et al. [37] used the AFM, in 1999, to determine the elasticity of normal and cancerous human bladder epithelial cells; they found the Young’s modulus of cancer cells to be about one-tenth that of healthy cells. The optical stretcher is a recently developed tool to study the deformation of single suspended cells [36,38,39] (Figure 4). One advantage of this technique is that the entire cell surface is acted upon by a distribution of the laser-induced forces, thus revealing the overall mech- anical property of the cell [36] . This is in direct contrast to some of the other techniques, such as the micropipette aspiration [40,41], optical tweezers [4] and AFM [42–45],

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tweezers [4] and AFM [42–45] , www.sciencedirect.com Figure 4 . Schematic diagram showing the axial stretching

Figure 4. Schematic diagram showing the axial stretching of a cell using an optical stretcher. The cell is trapped by the optical forces from two divergent laser beams, and is subsequently stretched along the laser beam axis when a higher laser power is applied. The optical stretcher makes use of the fact that the total force acting on a dielectric object is zero, with surface forces being additive when it is placed between two divergent laser beams. This results in a force pulling on either side of the cell, thus stretching it. Figure adapted from [38], Copyright 2001, with permission from the Biophysical Society.

which involve the application of forces at localized areas of the cell surface. However, because the optical stretcher enables the testing of cells in suspension, adherent cell types are not tested in their physiological state. In 2005, Wottawah et al. [36] studied the creep behavior of individual suspended fibroblasts using the optical stretcher, and found that cancerous mouse fibroblast cells were 50% more deformable than healthy cells. This increase in deformability is consistent with the results of earlier works carried out on cancer cells using micropipette aspiration and AFM techniques [37,46] . In a separate study, Lincoln et al. [47] used the optical stretcher to investigate the malignant transformation of human breast epithelial cells. They found that cancer cells stretch approximately five times more than healthy cells, and metastatic cancer cells stretch about twice as much as non-metastatic cancer cells. Moreover, the optical stretcher technique can be easily incorporated into a micro- fluidic device to produce a high-throughput flow-cytometric system that is capable of sorting and trapping cells without intruding into their underlying biological functions so that further analysis and therapeutic measurements can be made. The optical stretcher is an example of a biomecha- nical characterization tool that might be useful in the diagnosis of cancer because it can potentially detect the disease by measuring how deformable cells are, given the fact that cancer cells are, in general, more deformable than their non-cancerous counterparts. Metastasis ( Box 4) is the process whereby cancer cells spread from their primary site to other parts of the body. It involves the breaking away of cancer cells from a primary tumor, penetration into blood vessels and then settling and growing in a normal tissue at a distant site (metas- tasize) in the body ( Figure I ). Because metastasis is the predominant cause of death due to cancer, it is important to understand the detailed mechanisms involved so that strategies can be developed to keep the disease under control. Although some work on the mechanics of tumor cell migration has been done by Dong et al. [48,49] , more work is still needed to better elucidate the detailed mech- anisms involved in the metastasis of cancer cells. These include the abnormal receptor-mediated adhesion of cancer cells to the extracellular matrix, breaching of

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Box 4. Invasion and metastasis in cancer

During metastasis, cancer cells penetrate into blood vessels, lympha- tics and body cavities, thus enabling the cancer to spread (Figure I). Metastasis is initiated by the down-regulation of E-cadherin expres- sion: E-cadherins are adhesion molecules that keep epithelial cells together. The cancer cells then lose their ability to adhere to each other, become detached from the primary tumor, and begin to invade the surrounding tissues. Hence, at the site of the primary tumor, a carcinoma will first breach the underlying basement membrane, traverse the interstitial connective tissue and penetrate the vascular basement membrane, before gaining access to circulation. To penetrate into the surrounding extracellular matrix (ECM), tumor cells must be able to adhere to it. Some studies indicate that receptor- mediated attachment of tumor cells to laminin and fibronectin is necessary for invasion and metastasis. Normal (polarized) epithelial cells are known to express receptors with high-affinity for the basement membrane laminin on their basal surface. However, some cancer cells express many more receptors and integrins, which are distributed throughout the cell membrane and serve as receptors for the various components of the ECM. In fact, there seems to be a correlation between the density of these receptors and the invasive- ness of cancer [61]. After attaching to components of the basement

membrane or interstitial ECM, tumor cells then create passageways and migrate towards the vascular basement membrane. Once the vascular basement membrane is breached, and cancer cells intrava- sate into the circulation, some tumor cells will aggregate and travel in clumps as emboli. For extravasation of tumor cell emboli (egress of cancer cells through the vascular basement membrane) to occur at distant sites, tumor cells must first be arrested at and adhere to the capillary beds. During the extravasation process, this will, again, involve adhesion molecules such as integrins and laminin receptors and proteolytic enzymes. Normally, these tumor cells might contain adhesion molecules, the ligands of which are expressed preferentially on the endothelial cells of the distant target organs [62]. Alternatively, chemokines can also have an important role in determining the target sites. For example, cancer cells express the chemokine receptors CXCR4 and CCR7, which have affinity for the chemokines that are highly expressed in the tissues and sites where breast cancer commonly metastasizes [63]. Finally, chemoattractants might also be released by some target organs; these subsequently recruit cancer cells to their sites. At the new site, tumor cells will begin to proliferate and develop a vascular supply while evading the host defences [64].

a vascular supply while evading the host defences [64] . Figure I . Cancer metastasis. Schematic

Figure I. Cancer metastasis. Schematic diagram showing the different stages whereby cancer cells spread from a primary tumor site to a distant site in the body. Figure modified from [61], Copyright 2005, with permission from Elsevier.

basement membranes, intravasation of cancer cells into the circulation, interaction with lymphoid cells and plate- lets to form tumor-cell emboli, rheology of the tumor-cell emboli in the circulation, arrest and adhesion of tumor

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cells in capillary beds at distant sites, and extravasation through the vascular basem ent membrane. Specific and effective therapies can then be developed to target these migratory cancer cells or disrupt the metastatic process.

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Potential role in the detection and diagnosis of human diseases The research work reviewed here suggests that biomechanics can, indeed, play an important role in better understanding the pathophysiology of a variety of diseases. The knowledge obtained can also be useful in the development of new and improved assays and diagnostic devices and techniques that are not only sensitive enough in the early detection of diseases but are also highly accurate, even when the symptoms or signs of the diseases are hardly discernable. This is particularly needed for diseases where early diagnosis and detection are crucial for their prevention and control. For example, cell deform- ability, which can be determined using appropriate mech- anical probes, could be used as a potential biological marker in the detection and diagnosis of human diseases because the change in the cellular mechanical properties can quantitatively reflect their diseased states. This emer- ging field of research can also assist in the development of suitable testing strategies that can evaluate the efficacy of certain agents and drugs being developed to prevent or treat some of the diseases.

Future directions The use of biomechanics approaches to studying human diseases is still in its infancy, and there are many out- standing questions that need to be addressed. For example, human diseases can be caused by hereditary factors, changes in the internal physiological condition or invasion by foreign organisms such as viruses and parasites; there- fore, how do these factors induce the cellular and molecular changes that eventually lead to structural and biomecha- nical property changes in the cells, such as cell adhesion, cell elasticity, motility and rheology? Furthermore, how do changes in the structural and biomechanical properties in cells eventually lead to diseases? To address the above questions, newer and novel techniques need to be developed. Although powerful state-of-the-art tools exist to probe human diseases at the cellular and molecular levels, as described in this article, they are, nevertheless, tedious and difficult to use. There is a need to develop techniques and devices that can mechanically characterize not only individual cells but also populations of cells more rapidly and with enhanced sensitivity and accuracy. Also, because cells are frequently subjected to multiple cues in addition to bio- chemical and mechanical stimuli in their physiological environment, devices and systems must be designed so that they can also present cells with these cues and stimuli in a controlled and reproducible way. Thus, an integrated high-throughput approach will be needed that must be able to perform the following tasks: accurately manipulate and handle small sample volumes of bodily fluids and cells; provide the necessary controlled stimuli; and incorporate high-resolution techniques that can analyze the functions and gene expression in these cells. Emerging technologies, such as laboratory-on-chip or micro total analysis system ( mTAS) [50–52], could be used to integrate and automate all the necessary processes, to provide for the rapid, sensi- tive and effective analysis of diseased cells in their phys- iological environment. These will not only lead to the

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development of new, novel and inexpensive medical diag- nostic devices and techniques but also enable the minia- turization and integration of sophisticated tools and complex processes on a chip; hence, making them accessible to the developing world, where some of these diseases are rampant [53] . Finally, for such research to succeed, we will need to develop close multi-disciplinary collaborations not only among biologists, biochemists, life scientists and clinicians but also with engineers and physicists. Ultimately, it is hoped that this effort will lead to a better understanding of diseases, provide alternative means to assess their onset or progression and assist in developing better treatment, or even aid in their prevention.

Acknowledgements

The support provided by the Singapore-MIT Alliance is gratefully acknowledged.

References

1 Lim, C.T. et al. (2006) Mechanical models for living cells – a review. J. Biomech. 39, 195–216

2 Ingber, D.E. (2003) Mechanobiology and diseases of mechanotransduction. Ann. Med. 35, 564–577

3 Fitzgerald, M. (2006) Ten emerging technologies: nanobiomechanics. Technol. Rev. 109, 67

4 Lim, C.T. et al. (2006) Experimental techniques for single cell and single molecule biomechanics. Mat. Sci. Eng. C 26, 1278–1288

5 Van Vliet, K.J. et al. (2003) The biomechanics toolbox: experimental approaches for living cells and biomolecules. Acta Mater. 51, 5881–5905

6 Lim, C.T. (2006) Single cell mechanics study of the human disease malaria. J. Biomech. Sci. & Engrg. 1, 82–92

7 Miller, L.H. et al. (1971) Alteration in the rheologic properties of Plasmodium knowlesi -infected red cells. A possible mechanism for capillary obstruction. J. Clin. Invest. 50, 1451–1455

8 Miller, L.H. et al. (1972) Decreased deformability of Plasmodium coatneyi -infected red cells and its possible relation to cerebral malaria. Am. J. Trop. Med. Hyg. 21, 133–137

9 Cranston, H.A. et al. (1984) Plasmodium falciparum maturation abolishes physiologic red cell deformability. Science 223, 400–403

10 Suwanarusk, R. et al. (2004) The deformability of red blood cells parasitized by Plasmodium falciparum and P. vivax . J. Infect. Dis. 189, 190–194

11 Nash, G.B. et al. (1989) Abnormalities in the mechanical properties of red blood-cells caused by Plasmodium falciparum . Blood 74, 855–861

12 Paulitschke, M. and Nash, G.B. (1993) Membrane rigidity of red blood cells parasitized by different strains of Plasmodium falciparum . J. Lab. Clin. Med. 122, 581–589

13 Glenister, F.K. et al. (2002) Contribution of parasite proteins to altered mechanical properties of malaria-infected red blood cells. Blood 99,

1060–1063

14 Mills, J.P. et al. (2004) Nonlinear elastic and viscoelastic deformation of the human red blood cell with optical tweezers. Mech. Chem. Biosyst. 1,

169–180

15 Suresh, S. et al. (2005) Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria. Acta Biomater. 1, 15–30

16 Dao, M. et al. (2003) Mechanics of the human red blood cell deformed using optical tweezers. J. Mech. & Phy. Solids 51, 2259–2280 (Also see Errata at J. Mech. & Phy. Solids 53, 493–494 (2005).)

17 Lim, C.T. et al. (2004) Large deformation of living cells using laser traps. Acta Mater. 52 (7), 1837–1845 (Also see Corrigendum at Acta Mater. 52, 4065–4066 (2004).)

18 Shelby, J.P. et al. (2003) A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum -infected erythrocytes. Proc. Natl. Acad. Sci. U. S. A. 100, 14618–14622

19 Cheng, T.M. et al. (2005) Microrheology study of malaria infected erythrocytes flowing through microfluidic channel. Proceedings of the 12th International Conference on Biomedical Engineering , p. 184

118

Review

TRENDS in Biotechnology

Vol.25 No.3

20 Huang, H. et al. (2004) Cell mechanics and mechanotransduction:

pathways, probes, and physiology. Am. J. Physiol. Cell Physiol. 287,

1–11

21 Cooke, B. et al. (2000) Falciparum malaria: sticking up, standing out and out-standing. Parasitol. Today 16, 416–420

22 Magowan, C. et al. (1988) Cytoadherence by Plasmodium falciparum -

infected erythrocytes is correlated with the expression of a family of variable proteins on infected erythrocytes. J. Exp. Med. 168, 1307–

1320

23 Barnwell, J.W. et al. (1989) A human 88 kD membrane glycoprotein (CD36) functions in vitro as a receptor for a cytoadherence ligand on

Plasmodium falciparum -infected erythrocytes. J. Clin. Invest. 84, 765–

772

24 Ockenhouse, C.F. et al. (1989) Identification of a platelet membrane glycoprotein as a falciparum malaria sequestration receptor. Science 243, 1469–1471

25 Berendt, A.R. (1989) Intercellular-adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum . Nature 341, 57–59

26 Ockenhouse, C.F. et al. (1992) Human vascular endothelial cell adhesion receptors for Plasmodium falciparum-infected erythrocytes: roles for endothelial leukocyte adhesion molecule 1 and vascular cell adhesion molecule 1. J. Exp. Med. 176, 1183–1189

27 Branda˜ o, M.M. et al. (2003) Optical tweezers for measuring red blood cell elasticity: application to the study of drug response in sickle cell disease. Eur. J. Hematol. 70, 207–211

28 Evans, E.A. and La Celle, P.L. (1975) Intrinsic material properties of the erythrocyte membrane indicated by mechanical analysis of deformation. Blood 45, 29–43

29 Hochmuth, R.M. et al. (1979) Red cell extensional recovery and the determination of membrane viscosity. Biophys. J. 26, 101–114

30 Nash, G.B. et al. (1984) Mechanical properties of oxygenated red blood cells in sickle cell disease. Blood 63, 73–82

31 Itoh, T. et al. (1995) Effects of hemoglobin concentration on deformability of individual sickle cells after deoxygenation. Blood 85, 2245–2253

32 Ballas, S.K. et al. (1989) Effect of hydroxyurea on the rheological properties of sickle erythrocytes in vivo . Am. J. Hematol. 32, 104–

111

33 Lundstrom, K. and Boulikas, T. (2002) Breakthrough in cancer therapy: encapsulation of drugs and viruses. Curr. Drug Discov. 19– 23 November

34 Gast, F.U. et al. (2006) The microscopy cell (MicCell), a versatile modular flowthrough system for cell biology, biomaterial research, and nanotechnology. Microfluid. Nanofluid. 2, 21–36

35 Guck, J. et al. (2005) Optical deformability as inherent cell marker for testing malignant transformation and metastatic competence. Biophys. J. 88, 3689–3698

36 Wottawah, F. et al. (2005) Characterizing single suspended cells by optorheology. Acta Biomater. 1, 263–271

37 Lekka, M. et al. (1999) Elasticity of normal and cancerous human bladder cells studied by scanning force microscopy. Eur. Biophys. J. 28,

312–316

38 Guck, J. et al. (2001) The optical stretcher: a novel laser tool to micromanipulate cells. Biophys. J. 81, 767–784

39 Guck, J. et al. (2000) Optical deformability of soft biological dielectrics. Phys. Rev. Lett. 84, 5451–5454

40 Discher, D.E. et al. (1994) Molecular maps of red cell deformation:

hidden elasticity and in situ connectivity. Science 266, 1032–

1035

41 Hochmuth, R.M. (2000) Micropipette aspiration of living cells. J. Biomech. 33, 15–22

42 Mahaffy, R.E. et al. (2000) Scanning probe-based frequency-dependent microrheology of polymer gels and biological cells. Phys. Rev. Lett. 85,

880–883

www.sciencedirect.com

43 Mahaffy, R.E. et al. (2004) Quantitative analysis of the viscoelastic properties of thin regions of fibroblasts using atomic force microscopy. Biophys. J. 86, 1777–1793

44 Rotsch, C. et al. (1999) Dimensional and mechanical dynamics of active and stable edges in motile fibroblasts investigated by using atomic force microscopy. Proc. Natl. Acad. Sci. U. S. A. 96, 921–926

45 Rotsch, C. et al. (2000) Drug-induced changes of cytoskeletal structure and mechanics in fibroblasts: an atomic force microscopy study. Biophys. J. 78, 520–535

46 Ward, K.A. et al. (1991) Viscoelastic properties of transformed cells:

role in tumor cell progression and metastasis formation. Biorheology 28, 301–313

47 Lincoln, B. et al. (2004) Deformability-based flow cytometry. Cytometry A 59A, 203–209

48 Dong, C. et al. (2002) In vitro characterization and micromechanics of tumor cell chemotactic protrusion, locomotion, and extravasation. Ann. Biomed. Eng. 30, 344–355

49 Dong, C. et al. (2005) Micromechanics of tumor cell adhesion and migration under dynamic flow conditions. Front. Biosci. 10, 379–384

50 Daw, R. et al. (2006) Lab on a chip. Nature 442, 367

51 Whitesides, G.M. (2006) The origins and the future of microfluidics. Nature 442, 368–373

52 El-Ali, J. et al. (2006) Cell on chips. Nature 442, 403–411

53 Yager, P. et al. (2006) Microfluidic diagnostic technologies for global public health. Nature 442, 412–418

54 Campbell, N.A. and Reece, J.B. (2005) Biology, Addison Wesley Longman, Inc.

55 Engwerda, C.R. et al. (2005) The importance of the spleen in malaria. Trends Parasitol. 21, 75–80

56 Bannister, L.H. et al. (2000) A brief illustrated guide to the ultrastructure of Plasmodium falciparum asexual blood stages. Parasitol. Today 16, 427–433

57 Miller, L.H. et al. (2002) The pathogenic basis of malaria. Nature 415,

673–679

58 Cooke, B.M. et al. (2001) The malaria-infected red blood cell: structural and functional changes. Adv. Parasitol. 50, 1–86

59 International Agency for Research on Cancer (2003) World Cancer Report (B.W. Stewart and P. Kleihues, eds), World Health Organization.

60 Wulfkuhle, J.D. et al. (2003) Proteomic applications for the early detection of cancer. Nat. Rev. Cancer 3, 267–275

61 Kumar, V. et al. (2005) Robbins and Cotran Pathologic Basis of Disease, (7th edn), Elsevier Saunders

62 Ruoslahti, E. (2002) Specialization of tumor vasculature. Nat. Rev. Cancer 2, 83–90

63 Muller, A. et al. (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410, 50–56

64 Fidler, I.J. (2003) The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited. Nat. Rev. Cancer 3, 453–458

65 Trickey, W.R. et al. (2000) Viscoelastic properties of chondrocytes from normal and osteoarthritic human cartilage. J. Orthop. Res. 18, 891–898

66 King, M.J. et al. (2000) Rapid flow-cytometric test for the diagnosis of membrane cytoskeleton-associated haemolytic anaemia. Br. J. Haematol. 111, 924–933

67 An, S.S. et al. (2006) Do biophysical properties of the airway smooth muscle in culture predict airway hyperresponsiveness? Am. J. Respir. Cell Mol. Biol. 35, 55–64

68 Lemons, R.A. and Quate, C.F. (1975) Acoustic microscopy: biomedical applications. Science 188, 905–911

69 Wandersee, N.J. et al. (2004) Increased erythrocyte adhesion in mice and humans with hereditary spherocytosis and hereditary elliptocytosis. Blood 103, 710–716

70 Clark, M.R. et al. (1983) Osmotic-gradient ektocytometry:

comprehensive characterization of red cell volume and surface maintenance. Blood 61, 899–910