Journal of Biomechanics 47 (2014) 3373–3379
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Atomic force microscope-based single cell force spectroscopy of breast cancer cell lines: An approach for evaluating cellular invasion Ramin Omidvar a, Mohammad Tafazzoli-shadpour a,n, Mohammad Ali Shokrgozar b, Mostafa Rostami a a b
Faculty of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
art ic l e i nf o
a b s t r a c t
Article history: Accepted 1 August 2014
The adhesiveness of cancerous cells to their neighboring cells significantly contributes to tumor progression and metastasis. The single-cell force spectroscopy (SCFS) approach was implemented to survey the cell–cell adhesion force between cancerous cells in three cancerous breast cell lines (MCF-7, T47D, and MDA-MB-231). The gene expression levels of two dominant cell adhesion markers (E-cadherin and N-cadherin) were quantified by real-time PCR. Additionally, the local stiffness of the cell bodies was measured by atomic force microscopy (AFM), and the actin cytoskeletal organization was examined by confocal microscopy. Results indicated that the adhesion force between cells was conversely correlated with their invasion potential. The highest adhesion force was observed in the MCF-7 cells. A reduction in cell–cell adhesion, which is required for the detachment of cells from the main tumor during metastasis, is partly due to the loss of E-cadherin expression and the enhanced expression of N-cadherins. The reduced adhesion was accompanied by the softening of cells, as described by the rearrangement of actin filaments through confocal microscopy observations. The softening of the cell body and the reduced cellular adhesiveness are two adaptive mechanisms through which malignant cells achieve the increased deformability, motility, and strong metastasis potential necessary for passage through endothelial junctions and positioning in host tissue. This study presented application of SCFS to survey cell phenotype transformation during cancer progression. The results can be implemented as a platform for further investigations that target the manipulation of cellular adhesiveness and stiffness as a therapeutic choice. & 2014 Elsevier Ltd. All rights reserved.
Keywords: Atomic force microscopy Single cell force spectroscopy Cellular adhesion force Breast cancer Cellular invasion
1. Introduction Epithelium carcinoma describes a chronological pattern of uncontrolled cell division, formation of blood vessels (angiogenesis), detachment of cells from the main tumor, invasion to the surrounding tissue, and finally migration to distant biological sites (metastasis) (Suresh, 2007). The physical and mechanical properties of malignant cells have been observed to differ from those of normal cells in such a way to assist invasion. Hence, the quantification of the changes in such properties is a significant indicator of cancer progression and even a potential diagnostic tool (Cross et al., 2008; Suresh, 2007). Cytoskeleton reorganization and cell–cell disassociation are vital for epithelial tumor cells to achieve higher motility and invasiveness (Radisky, 2005; Takeichi, 1993).
n Corresponding author at: Faculty of Biomedical Engineering, Amirkabir University of Technology (Tehran Polytechnic), PO Box 15875-4413, No. 424, Hafez Ave, Tehran, Iran. Tel.: þ 98 21 6654 2385; fax: þ 98 21 6646 8186. E-mail address: [email protected] (M. Tafazzoli-shadpour).
http://dx.doi.org/10.1016/j.jbiomech.2014.08.002 0021-9290/& 2014 Elsevier Ltd. All rights reserved.
Various studies have characterized the mechanical behavior of cells under healthy and pathological conditions, such as cancer, mostly using the atomic force microscopy (AFM). A pioneering study (Lekka et al., 1999) showed that Young’s modulus of cancerous human bladder cell lines is lower than that of normal cells. The stiffness of metastatic cancer cells extracted from a patient’s body was seventy percent lower than that of benign cells (Cross et al., 2007). Li et al. (2008) focused on cancerous (MCF-7) and non-cancerous (MCF-10A) human breast cell lines and reported a smaller Young’s modulus for MCF-7 compared with MCF-10A cells. They also found that the extent and organization of the actin cytoskeleton are directly correlated with the mechanical properties of cell bodies. A recent study used Young’s modulus as an indicator for the detection of cancer cells in tissue samples (Lekka et al., 2012). Furthermore, AFM is capable of measuring the elastic and viscoelastic properties of biological cells through indentation (Darling et al., 2008; Kelly et al., 2011). High sensitivity, easy sample preparation, operation in different environments (air, liquid and vacuum) makes AFM versatile as a force spectroscope
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(Butt et al., 2005; Sokolov, 2007). Additionally, AFM can provide a new approach for directly probing cellular adhesion. In this approach, which is called AFM-based single cell force spectroscopy (SCFS), an individual cell is attached to the apex of a tipless cantilever, and its adhesiveness with another cell or a substrate (tissue or functionalized surface) can be investigated (Helenius et al., 2008). Compared to other adhesion assays, AFM-based SCFS can detect a broad range of adhesion forces by applying precise controllable movement and contact time (Friedrichs et al., 2013; Helenius et al., 2008). This method can be used to measure unbinding forces between individual molecules, i.e., singlemolecule force spectroscopy (Benoit et al., 2000; Panorchan et al., 2006). In general, the adhesion between normal epithelial cells is strong and stable. During metastasis, such tight association of tumorigenic cells need to be disrupted (Takeichi, 1993). The epithelial–mesenchymal transition (EMT) hypothesis, which has been one of the leading theories for the initiation of metastasis for two past decades, argues that epithelial cells undergo phenotype transformation toward mesenchymal cells (Radisky, 2005; Tomaskovic-Crook et al., 2009). This transition enables cells to free themselves from neighboring cells and start their journey within the body. At the molecular level, the downregulation of E-cadherin expression accompanied by the high expression of mesenchymal markers, such as N-cadherin and vimentin, detected in breast cancer supports the EMT theory (Huber et al., 2005; Tomaskovic-Crook et al., 2009). Epithelial cadherin (E-cadherin), as a cell adhesion molecule, is responsible for providing firm adhesion between two epithelial cells; hence, the loss of E-cadherin facilitates cell disconnection and invasion of malignant cells (Oka et al., 1993; Okegawa et al., 2004). Panorchan et al. (2006) utilized SCFS to reveal that the unbinding force between two E-cadherin molecules is much higher than that of two N-cadherin molecules. This result was also confirmed using micropipette aspiration when two cells were held connected for two time periods of four and thirty minutes (Chu et al., 2004). Such quantitative evidences confirm the EMT hypothesis through the fact that the low expression of E-cadherin and its replacement by N-cadherin generates lower cell–cell adhesiveness and assists the detachment of cancerous cells from their primary tumor. In this study, we implemented AFM-based single-cell force spectroscopy to evaluate the cell–cell adhesion forces among three different cancerous human breast cell lines to compare their cellular invasion with their adhesion levels. A quantitative realtime PCR assay was also performed to analyze the expression
levels of E-cadherin and N-cadherin genes. Due to the correlation between cytoskeletal filaments and cell–cell adhesion, the organization of actin filaments was investigated by confocal microscopy, and Young’s modulus of cancerous cells was assessed by atomic force microscopy.
2. Materials and methods 2.1. Materials Biotin-BSA (Sigma, Germany; Cat. No. A6043), streptavidin (Sigma, Germany; Cat. No. S4762), biotin-conjugated concanavalin A (Sigma, Germany; Cat. No. C2272), RPMI culture medium (Gibco, USA), Tipless Arrow-TL1 (Nanoworld, Switzerland), HYDRA6R-200NG (APPNANO, USA), phosphate buffer saline (Gibco, USA), phalloidin-FITC (Sigma, Germany; Cat. No. P5282), and E and N-cadherin primers were used in this study.
2.2. Cell preparation and culture We harvested cell lines of MCF-7 (non-invasive), T47D (slightly invasive), and MDA-MB-231 (highly invasive) provided from the National Cell Bank of Iran (Pasteur Institute of Iran, Tehran, Iran). Cells were cultured in RPMI medium containing 10% fetal bovine serum. After reaching a confluence of approximately 80% in T25 flasks, cells were moved to 60-mm Petri dishes one day before SCFS experiments.
2.3. Functionalization of AFM cantilevers According to the published protocol (Friedrichs et al., 2010), tipless Arrow-TL1 cantilevers with a spring constant of 30 7 2 mN/m were coated with concanavalin A. First, they were cleaned by UV-radiation for 45 min and then incubated in 50 μl of biotin-conjugated bovine serum albumin (0.5 mg/ml solution in NaHCO3). After overnight incubation, the cantilevers were washed with PBS three times to remove unbounded biotin molecules and were then incubated in 50 μl of streptavidin (0.5 mg/ml solution in PBS) for 30 min. After three times washes with PBS, they were placed in 50 μl of biotin-labeled concanavalin A (0.4 mg/ml solution in PBS) for 30 min and washed three times with PBS.
2.4. Single-cell force spectroscopy experiments The SCFS experiments were performed using a Cellhesion 200 device (JPK Instruments AG, Germany). Luminal A MCF-7 and T47D cells and basal-like MDAMB-231 cells were selected as nominees from both subtypes of breast cell lines (Neve et al., 2006). To prepare the probe cells, suspended cells in culture medium were incubated with 0.5% trypsin-EDTA (Gibco, USA) for approximately 2 min at 37 1C in T25 flasks (Friedrichs et al., 2010). The suspension was centrifuged at 1400 rpm for 5 min to form a cell pellet. The supernatant above the pellet was replaced by trypsin-free medium. The cell pellet was resuspended in the medium, and approximately 1–2 104 cells were added to a Petri dish. Following several
Fig. 1. Cantilever-bound cells for using in single cell force spectroscopy tests. A T47D cell has been attached to the concanavalin-A coated cantilever to be used as a probe for acquiring the cellular adhesion force between two T47D cells (A). Depiction of a single cell force spectroscopy procedure. (i) The cantilever-bound cell was brought into contact with the cultured cell (ii) After a contact time the cantilever was withdrawn from the cultured cell. (iii) The retraction movement of cantilever was continued until two cells were completely separated from each other (iv) (B).
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minutes to allow the cells to reach a relatively equilibrated state, round and healthy cells were ready to be captured for the experiments. After placing the sample on the stage and the functionalized cantilever on the head of the device, the laser spot was aligned onto the apex of the cantilever within the culture medium. The cantilever was left for at least 10 min to reach a thermal equilibration condition. It was then moved downward towards the spotted cell, and the cell was attached through retract cycles with a speed of less than 5 mm/s (Friedrichs et al., 2010). After cell attachment, at least a 10-min period of recovery was allowed (Fig. 1A). The cell-attached cantilever was then placed above the test sample and moved downward to contact the cell membrane with a pre-set force of 2.5 nN. After a predefined contact time interval, the cantilever was retracted from the cultured cell until complete separation (Fig. 1B). During this procedure, the vertical deflection of the cantilever was plotted with respect to the displacement of the head. The technical parameters of the SCFS tests are listed in Table 1. To assure complete separation of two cells, the head was set to vertically move approximately 90 mm (Friedrichs et al., 2010), considering the fact that the maximum vertical displacement of the Cellhesion 200 head was 100 mm. The tests were performed using two different contact time periods (Helenius et al., 2008): a short contact time of 10 s and a long contact time of 120 s. During the contact time, the control system of AFM was set to keep the vertical deflection (Force) of AFM cantilever constant (constant force mode). In this case, the approach and retract curves have a similar set-point and there is a shift in vertical displacement due to viscoelastic relaxation of cells and squeezing each other. For the short contact time, a maximum of 25 tests were conducted for each cell, and for the long contact time, 10 tests were performed (Friedrichs et al., 2010). For each cell type, approximately 200 correct adhesion SCFS experiments were analyzed for short contact time and 250 adhesion SCFS experiments were analyzed for long contact time. 2.5. Evaluation of mechanical properties of cells To measure the cell elasticity, the fresh HYDRA6R-200NG cantilever (tetrahedral tip shape) was interacted with a single cultured cell in a Petri dish using Nanowizard2 AFM (JPK Instruments). The vertical deflection of the cantilever, which is directly proportional to the tip-cell interaction forces, was recorded against the indentation depth. The vertical deflection of the cantilever was converted into the force by multiplying two cantilever physical properties called sensitivity and spring constant. (Butt et al., 2005; Hutter and Bechhoefer, 1993). The average spring constant of the cantilevers (0.03 7 0.015 N/m) was evaluated prior to the experiments. The temperature of the culture medium was maintained at 37 1C (PetriDishHeater, JPK Instruments). The maximum indentation and force were set to 0.5 μm and 1.0 nN, respectively. 2.6. Data analysis of mechanical properties of cells
Table 2 Primer sequences. F stands for forward and R stands for reverse.
A typical force-indentation plot is shown in Fig. 2A. Due to similarity of tetrahedral shape to quadrilateral pyramid, the modified Hertz model for a quadrilateral pyramid tip (Bilodeau, 1992; Lin et al., 2007) was selected to calculate Young’s modulus of the cell ðEcell Þ as F¼
1:4906Ecell tan ϕ 2 δ 2ð1 ν2cell Þ
ð1Þ
where F is the force, δ is the indentation depth, and ϕ is the half angle of the pyramid tip, which was set to 17.51. Poisson’s ratio ðνcell Þ of all cells was set to 0.5 assuming an incompressible material property for the cells (Lekka et al., 2012; Nikkhah et al., 2011). According to a previously reported algorithm (Guo and Akhremitchev, 2006; Nikkhah et al., 2011), Young’s modulus of cells was defined from the linear version of δ in Hertz’s model. The indentation depth ðδÞ was defined by subtracting the piezo position ðzÞ values from the cantilever deflection ðdÞ. In this case, Eq. (1) can be exchanged to a linear format in δ to yield " F 1=2 ¼
Fig. 2. Evaluation of mechanical properties of individual cells by AFM. A typical force-displacement resulted from AFM indentation (A). HYDRA6R-200NG Cantilever over the nuclei of a T47D cells is ready to perform the AFM indentation test (scale bar is 10 mm) (B).
#1=2 " #1=2 1:4906Ecell tan ϕ 1:4906Ecell tan ϕ ðz dÞ ðz0 d0 Þ 2 2 2ð1 νcell Þ 2ð1 νcell Þ
ð2Þ
where z0 and d0 are the piezo position and cantilever deflection at the contact point of the tip and cell, respectively. Young’s modulus ðEcell Þ can be directly calculated from the first bracket in Eq. (2), which is the slope of the linear polynomial curve of ðF 1=2 ; z dÞ. The indentation points were chosen over the cell nucleus (Fig. 2B).
Gene GAPDH E-cadherin N-cadherin
Sequence F: 5 QUOTE -ACACCCACTCCTCCACCTTTG-30 R: 50 -TCCACCACCCTGTTGCTGTAG-30 F: 50 -CCATTAACAGGAACACAGGAGTCA-30 R: 50 -GGAGGATTATCGTTGGTGTCAGT-30 F: 50 -CATCATCATCCTGCTTATCCTTGT-30 R: 50 -TTCTCCTCCACCTTCTTCATCATA-30
2.7. Real-time PCR assay To quantify the gene expression levels of E-cadherin and N-cadherin, the total RNA from three cell lines was extracted with the RNeasy plus mini kit according to manufacturer’s instructions (Qiagen, USA) and stored in RNase-free water at 80 1C. The optical density of the RNA was determined, and 1000 ng of RNA was reversely transcribed to cDNA (QuantiTect, USA). SYBR green primers (forward and reverse) were designed using the Primer Express software v3.0.1 (Applied Biosystems, USA) and are presented in Table 2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene, and the comparative cycle threshold method was used for the quantification of the expression levels.
2.8. Confocal microscopy of actin cytoskeleton
Table 1 Set of parameters for the single cell force spectroscopy test. Parameter
Value
Vertical displacement of head (cantilever) Approach/retract speed Contact time Contact feedback mode
90 mm 4 mm/s 10 and 120 s Constant force
To analyze the structure of the actin cytoskeleton, 5 103 cells were cultured over collagen-coated coverslips for two days and then fixed in 4% formaldehyde in PBS for approximately 20 min. After washing three times with PBS, Triton-X100 (Sigma, Germany) (0.1% in PBS) was added for 5 min to increase the permeability of the cells. The cells were again washed three times with PBS and then incubated at room temperature with phalloidin-FITC for 1 h. Before imaging, cells were further washed with PBS. The images were acquired by confocal microscopy using a TCS SP5 microscope (Leica Microsystem GmbH, Germany).
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Fig. 3. Result of Single cell force spectroscopy and real-time PCR analysis of three cancerous human breast cell lines. A typical curve of the short-contact-time test describing approaching and retracting. The detachment force was measured as the minimum value of the retract curve (A). Mean value of detachment forces among three various types of cancerous breast cells (MCF-7, T47D and MDA-MB-231) for 10 s and 120 s contact times (B). The levels of gene expression of E-cadherin (C) and N-cadherin (D) in three cell lines analyzed by real-time PCR test.
Table 3 Cell–cell detachment forces of cancerous cells for short and long contact time (mean value 7standard deviation). Type of cells
Detachment force (nN) Contact time 10 s
Detachment force (nN) Contact time 120 s
MCF-7 T47D MDA-MB-231
2.217 0.44 1.92 7 0.48 1.717 0.61
13.477 2.25 5.777 0.56 4.60 7 0.57
not allow the establishment of the entire number of bonds, the differences among the mean values of the detachment forces of the three types of cell lines were somehow lower yet statistically significant. According to the t-test and the one-way ANOVA test, the detachment forces for the long contact time (120 s) were statistically different from those for the short contact time (10 s) (po0.05). The MCF-7 cells exhibited the highest detachment forces among the observed cell lines. The basal-like MDA-MB-231 cells ranked the lowest.
3. Results
3.2. Gene expression
3.1. Cell adhesion force
The results indicate that the MCF-7 and T47D cells do not express N-cadherin, whereas the MDA-MB-231 cells did not express E-cadherin. The MDA-MB-231 cell line expressed N-cadherin at approximately the 30th cycle of each real-time test. The level of E-cadherin expression in the T47D cells was higher than that found in the MCF-7 cells (Fig. 3C and D).
For each SCFS run, a force-distance plot was acquired. A typical force–distance (F–D) curve, contains two main parts, namely Approach and Retract (Fig. 3A). During the retraction, the minimum force value can be interpreted as the detachment force, which is the overall force required for the separation of two cells (Helenius et al., 2008). For short (10 s) and long (120 s) contact times, the MCF-7 cells showed the highest level of cellular detachment force compared with the T47D and MDA-MB-231 cells (Table 3). The detachment forces in the short-contact-time-mode experiments were 2.2170.44, 1.9270.48, and 1.7170.61 nN (mean7SD) for MCF-7, T47D, and MDA-MB-231 cells, respectively. There were distinct differences between detachment forces of the cell lines for the long contact time (120 s) (Fig. 3B). Because the short contact time does
3.3. Mechanical properties The basal-like subtype of breast cancer showed the lowest Young’s modulus, and among the two luminal subtypes, the MCF-7 cells presented a higher Young’s modulus than the T47D cells. Around 250 force–distance curves (30 points in 1.5 1.5 mm areas for each cell) were analyzed for each group of cells. The average values of Young’s modulus were calculated to be 1.04, 0.94, and
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Fig. 4. The histograms of Young’s modulus of cells and confocal microscopy images of actin filaments. Histograms of Young’s modulus measured by atomic force spectroscopy for three cell lines (A)–(C). The actin filaments of MCF-7, T47D and MDA-MB-231 cells were stained by phalloidin-FITC and observed under a confocal microscope (D)–(F). Localization of actin filaments in MDA-MB-231 cells are marked by red arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
0.62 kPa for the MCF-7, T47D, and MDA-MB-231 cells, respectively (Fig. 4A–C). The statistical analysis revealed significant difference among the elastic moduli of three cell lines (P-value o0.05). The results indicated a strong positive correlation between the detachment force and Young’s modulus among test groups. The Pearson product moment correlation coefficients (r) between Young’s modulus and the detachment force were 0.98 and 0.91 for short and long contact times, respectively. Hence, our results showed a strong positive correlation between cell stiffness and cell adhesion. Fig. 4D–F shows images of phalloidin-labeled actin filaments that were obtained through confocal microscopy. Although the cytoskeletal filaments of the MDA-MB-231 cells are localized
(red arrows in Fig. 4F), these are monotonously distributed around the circumference of the MCF-7 and T47D cells.
4. Discussion There is an opposite relation between cell–cell adhesion and the cell power of invasion. The cellular adhesiveness of malignant cells decreases, and they tend to detach from the main tumor and metastasize (Radisky, 2005; Suresh, 2007). Cancerous cells alter their structural properties, such as stiffness and adhesiveness, with their surroundings to be able to fulfill their tasks of invasion,
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migration, and metastasis (Cross et al., 2008; Suresh, 2007). The changes in the adhesiveness of such cells are partly by means of reductions/alterations in the level of the expression of markers responsible for cell adhesion (Radisky, 2005; Takeichi, 1993; Tomaskovic-Crook et al., 2009). The reduction of cellular stiffness increases the deformability of cancerous cells, which assists them with their invasion to neighboring tissues and their transmission through intercellular junctions of endothelial cells in their attempt to find a new residence (Cross et al., 2007, 2008; Suresh, 2007). Using a Boyden chamber invasion assay on various breast cell lines, it has been shown that the basal subtype of breast cell lines have a higher tendency to invade compared with luminal-type cells (Neve et al., 2006). In fact, among the luminal subcategory of cancerous cells, T47D cells exhibited higher invasiveness potential than MCF-7 cells, and it is thus expected that MCF-7 cells should show stronger cell–cell adhesion than T47D cells (Neve et al., 2006). Our SCFS results confirmed that the overall adhesion force of MCF-7 cells is significantly higher than that of T47D cells for both contact times (Fig. 3B). Similarly, MDA-MB-231 cells exhibited a significant invasive potential in the Boyden chamber invasion assay (Neve et al., 2006), which is correlated with the lowest value of cell–cell adhesion force derived by the SCFS tests performed in this research study. Our results indicate that only the basal-like MDA-MB-231 cells express N-cadherin, whereas the two other types of cells do not express this marker (Fig. 3D). The single-molecule force spectroscopy (Panorchan et al., 2006) and micropipette aspiration (Chu et al., 2004) methods revealed that the adhesion force between N– N cadherins are far weaker than that of between E–E cadherins. Hence, cells expressing N-cadherin (MDA-MB-231) should exhibit a lower cell–cell adhesion compared with cells that express E-cadherin (T47D and MCF-7). Although the level of E-cadherin expression was higher in T47D cells compared to MCF-7 cells, the SCFS experiments unexpectedly indicated a stronger cell–cell adhesion force for MCF-7 than T47D cells. The level of E-cadherin expression among some breast cell lines has been reported in recent studies by real-time PCR (Holen et al., 2012) and immunoblotting analysis (Arima et al., 2012; Yan et al., 2010). These previous results also confirmed that the level of E-cadherin expression in T47D cells is higher than that found in MCF-7 cells. This difference may be related to the intricacy of the cell–cell adhesion phenomenon, which is controlled by a complex of molecules from the membrane to the cytoplasm. It is believed that cytoplasmic proteins and the actin cytoskeleton play indispensable roles in the functions of the cadherin family (Takeichi, 1993). Hence, the expression of cell adhesion markers is not the only determinant of the overall cellular adhesion. In general, the tendency of cancer cells to invade is accompanied by a decrease in cell adhesion through alterations in the number and/or function of adhesive proteins in the cell membrane and not necessarily associated to the extent of gene expression in the nuclei of cells. When cancer cells shift to the metastasis phase, E-cadherins are less abundant, and in some cases, weaker N-cadherins play important roles (Fig. 3D). Simultaneously, cells become more distensible (Fig. 4C) through alterations in the cytoskeletal structure (Fig. 4F) to be ready to interact with endothelial cells when highly deformable bodies are required to pass through endothelial junctions to produce a colony of cells within the new host tissue. In this study, we compared the differences in cellular elasticity between three groups of cells and sought a relationship between cellular elasticity and the detachment force. Using a small indentation depth (500 nm), a low setpoint force (approximately 1 nN) and an approaching velocity of approximately 1 mm/s with a rectangularshaped silicon nitride cantilever, a strong positive correlation (40.91) between cell elasticity and overall adhesion force was found. Fig. 5 describes the reduction in cell–cell adhesion accompanied by cell
Fig. 5. Cellular adhesion force and elasticity. The softening of the cell body (Reduction in Cellular Elasticity, right axis in kPa) accompanied by reduced cellular adhesiveness (left axis in nN) are two correlative mechanisms which malignant cells implement to increase their motility and achieve strong potential of invasiveness.
softening when the degree of invasiveness is elevated in the three cancer cell lines. These highly correlated phenomena are two adaptive mechanisms through which malignant cells achieve increased motility and strong metastasis potential, i.e. reduction in adhesive protein bonds and alterations in the cytoskeletal arrangement. The peripheral actin filaments in MCF-7 cells have been observed previously (Leporatti et al., 2009), in accordance with our confocal microscopy observations. A similar arrangement of actin filaments was detected in the T47D cells, whereas the filaments were found to be localized in MDA-MB-231 cells (Fig. 4D and F). Similarly, it has been shown that actin filaments are branched and localized in leading edges of migrating cells (Le Clainche and Carlier, 2008). It might be due to actin filaments reassembly with local adhesion molecule for better migration. Migrating cells have shown localized actin filaments in their leading edges. The results of this study can be implemented as a platform for further investigations that target the manipulation of cellular adhesiveness and stiffness as a therapeutic choice. The function of designed antibodies for cancer treatment can be examined in vitro through the AFM-based single cell force spectroscopy approach, particularly for the manipulation of cell adhesion. Although this approach is currently suffering from some drawbacks, such as being time consuming, having limited contact time (o10 min), and demanding a reliable amount of acquired data for statistical analysis, its distinguished advantages make it a useful tool in cancer research. In addition to cell–cell adhesion, this method can be employed for the study of interactions of cancerous cells with biomaterials.
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Journal of Biomechanics journal homepage: www.elsevier.com/locate/jbiomech www.JBiomech.com
Atomic force microscope-based single cell force spectroscopy of breast cancer cell lines: An approach for evaluating cellular invasion Ramin Omidvar a, Mohammad Tafazzoli-shadpour a,n, Mohammad Ali Shokrgozar b, Mostafa Rostami a a b
Faculty of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
art ic l e i nf o
a b s t r a c t
Article history: Accepted 1 August 2014
The adhesiveness of cancerous cells to their neighboring cells significantly contributes to tumor progression and metastasis. The single-cell force spectroscopy (SCFS) approach was implemented to survey the cell–cell adhesion force between cancerous cells in three cancerous breast cell lines (MCF-7, T47D, and MDA-MB-231). The gene expression levels of two dominant cell adhesion markers (E-cadherin and N-cadherin) were quantified by real-time PCR. Additionally, the local stiffness of the cell bodies was measured by atomic force microscopy (AFM), and the actin cytoskeletal organization was examined by confocal microscopy. Results indicated that the adhesion force between cells was conversely correlated with their invasion potential. The highest adhesion force was observed in the MCF-7 cells. A reduction in cell–cell adhesion, which is required for the detachment of cells from the main tumor during metastasis, is partly due to the loss of E-cadherin expression and the enhanced expression of N-cadherins. The reduced adhesion was accompanied by the softening of cells, as described by the rearrangement of actin filaments through confocal microscopy observations. The softening of the cell body and the reduced cellular adhesiveness are two adaptive mechanisms through which malignant cells achieve the increased deformability, motility, and strong metastasis potential necessary for passage through endothelial junctions and positioning in host tissue. This study presented application of SCFS to survey cell phenotype transformation during cancer progression. The results can be implemented as a platform for further investigations that target the manipulation of cellular adhesiveness and stiffness as a therapeutic choice. & 2014 Elsevier Ltd. All rights reserved.
Keywords: Atomic force microscopy Single cell force spectroscopy Cellular adhesion force Breast cancer Cellular invasion
1. Introduction Epithelium carcinoma describes a chronological pattern of uncontrolled cell division, formation of blood vessels (angiogenesis), detachment of cells from the main tumor, invasion to the surrounding tissue, and finally migration to distant biological sites (metastasis) (Suresh, 2007). The physical and mechanical properties of malignant cells have been observed to differ from those of normal cells in such a way to assist invasion. Hence, the quantification of the changes in such properties is a significant indicator of cancer progression and even a potential diagnostic tool (Cross et al., 2008; Suresh, 2007). Cytoskeleton reorganization and cell–cell disassociation are vital for epithelial tumor cells to achieve higher motility and invasiveness (Radisky, 2005; Takeichi, 1993).
n Corresponding author at: Faculty of Biomedical Engineering, Amirkabir University of Technology (Tehran Polytechnic), PO Box 15875-4413, No. 424, Hafez Ave, Tehran, Iran. Tel.: þ 98 21 6654 2385; fax: þ 98 21 6646 8186. E-mail address: [email protected] (M. Tafazzoli-shadpour).
http://dx.doi.org/10.1016/j.jbiomech.2014.08.002 0021-9290/& 2014 Elsevier Ltd. All rights reserved.
Various studies have characterized the mechanical behavior of cells under healthy and pathological conditions, such as cancer, mostly using the atomic force microscopy (AFM). A pioneering study (Lekka et al., 1999) showed that Young’s modulus of cancerous human bladder cell lines is lower than that of normal cells. The stiffness of metastatic cancer cells extracted from a patient’s body was seventy percent lower than that of benign cells (Cross et al., 2007). Li et al. (2008) focused on cancerous (MCF-7) and non-cancerous (MCF-10A) human breast cell lines and reported a smaller Young’s modulus for MCF-7 compared with MCF-10A cells. They also found that the extent and organization of the actin cytoskeleton are directly correlated with the mechanical properties of cell bodies. A recent study used Young’s modulus as an indicator for the detection of cancer cells in tissue samples (Lekka et al., 2012). Furthermore, AFM is capable of measuring the elastic and viscoelastic properties of biological cells through indentation (Darling et al., 2008; Kelly et al., 2011). High sensitivity, easy sample preparation, operation in different environments (air, liquid and vacuum) makes AFM versatile as a force spectroscope
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(Butt et al., 2005; Sokolov, 2007). Additionally, AFM can provide a new approach for directly probing cellular adhesion. In this approach, which is called AFM-based single cell force spectroscopy (SCFS), an individual cell is attached to the apex of a tipless cantilever, and its adhesiveness with another cell or a substrate (tissue or functionalized surface) can be investigated (Helenius et al., 2008). Compared to other adhesion assays, AFM-based SCFS can detect a broad range of adhesion forces by applying precise controllable movement and contact time (Friedrichs et al., 2013; Helenius et al., 2008). This method can be used to measure unbinding forces between individual molecules, i.e., singlemolecule force spectroscopy (Benoit et al., 2000; Panorchan et al., 2006). In general, the adhesion between normal epithelial cells is strong and stable. During metastasis, such tight association of tumorigenic cells need to be disrupted (Takeichi, 1993). The epithelial–mesenchymal transition (EMT) hypothesis, which has been one of the leading theories for the initiation of metastasis for two past decades, argues that epithelial cells undergo phenotype transformation toward mesenchymal cells (Radisky, 2005; Tomaskovic-Crook et al., 2009). This transition enables cells to free themselves from neighboring cells and start their journey within the body. At the molecular level, the downregulation of E-cadherin expression accompanied by the high expression of mesenchymal markers, such as N-cadherin and vimentin, detected in breast cancer supports the EMT theory (Huber et al., 2005; Tomaskovic-Crook et al., 2009). Epithelial cadherin (E-cadherin), as a cell adhesion molecule, is responsible for providing firm adhesion between two epithelial cells; hence, the loss of E-cadherin facilitates cell disconnection and invasion of malignant cells (Oka et al., 1993; Okegawa et al., 2004). Panorchan et al. (2006) utilized SCFS to reveal that the unbinding force between two E-cadherin molecules is much higher than that of two N-cadherin molecules. This result was also confirmed using micropipette aspiration when two cells were held connected for two time periods of four and thirty minutes (Chu et al., 2004). Such quantitative evidences confirm the EMT hypothesis through the fact that the low expression of E-cadherin and its replacement by N-cadherin generates lower cell–cell adhesiveness and assists the detachment of cancerous cells from their primary tumor. In this study, we implemented AFM-based single-cell force spectroscopy to evaluate the cell–cell adhesion forces among three different cancerous human breast cell lines to compare their cellular invasion with their adhesion levels. A quantitative realtime PCR assay was also performed to analyze the expression
levels of E-cadherin and N-cadherin genes. Due to the correlation between cytoskeletal filaments and cell–cell adhesion, the organization of actin filaments was investigated by confocal microscopy, and Young’s modulus of cancerous cells was assessed by atomic force microscopy.
2. Materials and methods 2.1. Materials Biotin-BSA (Sigma, Germany; Cat. No. A6043), streptavidin (Sigma, Germany; Cat. No. S4762), biotin-conjugated concanavalin A (Sigma, Germany; Cat. No. C2272), RPMI culture medium (Gibco, USA), Tipless Arrow-TL1 (Nanoworld, Switzerland), HYDRA6R-200NG (APPNANO, USA), phosphate buffer saline (Gibco, USA), phalloidin-FITC (Sigma, Germany; Cat. No. P5282), and E and N-cadherin primers were used in this study.
2.2. Cell preparation and culture We harvested cell lines of MCF-7 (non-invasive), T47D (slightly invasive), and MDA-MB-231 (highly invasive) provided from the National Cell Bank of Iran (Pasteur Institute of Iran, Tehran, Iran). Cells were cultured in RPMI medium containing 10% fetal bovine serum. After reaching a confluence of approximately 80% in T25 flasks, cells were moved to 60-mm Petri dishes one day before SCFS experiments.
2.3. Functionalization of AFM cantilevers According to the published protocol (Friedrichs et al., 2010), tipless Arrow-TL1 cantilevers with a spring constant of 30 7 2 mN/m were coated with concanavalin A. First, they were cleaned by UV-radiation for 45 min and then incubated in 50 μl of biotin-conjugated bovine serum albumin (0.5 mg/ml solution in NaHCO3). After overnight incubation, the cantilevers were washed with PBS three times to remove unbounded biotin molecules and were then incubated in 50 μl of streptavidin (0.5 mg/ml solution in PBS) for 30 min. After three times washes with PBS, they were placed in 50 μl of biotin-labeled concanavalin A (0.4 mg/ml solution in PBS) for 30 min and washed three times with PBS.
2.4. Single-cell force spectroscopy experiments The SCFS experiments were performed using a Cellhesion 200 device (JPK Instruments AG, Germany). Luminal A MCF-7 and T47D cells and basal-like MDAMB-231 cells were selected as nominees from both subtypes of breast cell lines (Neve et al., 2006). To prepare the probe cells, suspended cells in culture medium were incubated with 0.5% trypsin-EDTA (Gibco, USA) for approximately 2 min at 37 1C in T25 flasks (Friedrichs et al., 2010). The suspension was centrifuged at 1400 rpm for 5 min to form a cell pellet. The supernatant above the pellet was replaced by trypsin-free medium. The cell pellet was resuspended in the medium, and approximately 1–2 104 cells were added to a Petri dish. Following several
Fig. 1. Cantilever-bound cells for using in single cell force spectroscopy tests. A T47D cell has been attached to the concanavalin-A coated cantilever to be used as a probe for acquiring the cellular adhesion force between two T47D cells (A). Depiction of a single cell force spectroscopy procedure. (i) The cantilever-bound cell was brought into contact with the cultured cell (ii) After a contact time the cantilever was withdrawn from the cultured cell. (iii) The retraction movement of cantilever was continued until two cells were completely separated from each other (iv) (B).
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minutes to allow the cells to reach a relatively equilibrated state, round and healthy cells were ready to be captured for the experiments. After placing the sample on the stage and the functionalized cantilever on the head of the device, the laser spot was aligned onto the apex of the cantilever within the culture medium. The cantilever was left for at least 10 min to reach a thermal equilibration condition. It was then moved downward towards the spotted cell, and the cell was attached through retract cycles with a speed of less than 5 mm/s (Friedrichs et al., 2010). After cell attachment, at least a 10-min period of recovery was allowed (Fig. 1A). The cell-attached cantilever was then placed above the test sample and moved downward to contact the cell membrane with a pre-set force of 2.5 nN. After a predefined contact time interval, the cantilever was retracted from the cultured cell until complete separation (Fig. 1B). During this procedure, the vertical deflection of the cantilever was plotted with respect to the displacement of the head. The technical parameters of the SCFS tests are listed in Table 1. To assure complete separation of two cells, the head was set to vertically move approximately 90 mm (Friedrichs et al., 2010), considering the fact that the maximum vertical displacement of the Cellhesion 200 head was 100 mm. The tests were performed using two different contact time periods (Helenius et al., 2008): a short contact time of 10 s and a long contact time of 120 s. During the contact time, the control system of AFM was set to keep the vertical deflection (Force) of AFM cantilever constant (constant force mode). In this case, the approach and retract curves have a similar set-point and there is a shift in vertical displacement due to viscoelastic relaxation of cells and squeezing each other. For the short contact time, a maximum of 25 tests were conducted for each cell, and for the long contact time, 10 tests were performed (Friedrichs et al., 2010). For each cell type, approximately 200 correct adhesion SCFS experiments were analyzed for short contact time and 250 adhesion SCFS experiments were analyzed for long contact time. 2.5. Evaluation of mechanical properties of cells To measure the cell elasticity, the fresh HYDRA6R-200NG cantilever (tetrahedral tip shape) was interacted with a single cultured cell in a Petri dish using Nanowizard2 AFM (JPK Instruments). The vertical deflection of the cantilever, which is directly proportional to the tip-cell interaction forces, was recorded against the indentation depth. The vertical deflection of the cantilever was converted into the force by multiplying two cantilever physical properties called sensitivity and spring constant. (Butt et al., 2005; Hutter and Bechhoefer, 1993). The average spring constant of the cantilevers (0.03 7 0.015 N/m) was evaluated prior to the experiments. The temperature of the culture medium was maintained at 37 1C (PetriDishHeater, JPK Instruments). The maximum indentation and force were set to 0.5 μm and 1.0 nN, respectively. 2.6. Data analysis of mechanical properties of cells
Table 2 Primer sequences. F stands for forward and R stands for reverse.
A typical force-indentation plot is shown in Fig. 2A. Due to similarity of tetrahedral shape to quadrilateral pyramid, the modified Hertz model for a quadrilateral pyramid tip (Bilodeau, 1992; Lin et al., 2007) was selected to calculate Young’s modulus of the cell ðEcell Þ as F¼
1:4906Ecell tan ϕ 2 δ 2ð1 ν2cell Þ
ð1Þ
where F is the force, δ is the indentation depth, and ϕ is the half angle of the pyramid tip, which was set to 17.51. Poisson’s ratio ðνcell Þ of all cells was set to 0.5 assuming an incompressible material property for the cells (Lekka et al., 2012; Nikkhah et al., 2011). According to a previously reported algorithm (Guo and Akhremitchev, 2006; Nikkhah et al., 2011), Young’s modulus of cells was defined from the linear version of δ in Hertz’s model. The indentation depth ðδÞ was defined by subtracting the piezo position ðzÞ values from the cantilever deflection ðdÞ. In this case, Eq. (1) can be exchanged to a linear format in δ to yield " F 1=2 ¼
Fig. 2. Evaluation of mechanical properties of individual cells by AFM. A typical force-displacement resulted from AFM indentation (A). HYDRA6R-200NG Cantilever over the nuclei of a T47D cells is ready to perform the AFM indentation test (scale bar is 10 mm) (B).
#1=2 " #1=2 1:4906Ecell tan ϕ 1:4906Ecell tan ϕ ðz dÞ ðz0 d0 Þ 2 2 2ð1 νcell Þ 2ð1 νcell Þ
ð2Þ
where z0 and d0 are the piezo position and cantilever deflection at the contact point of the tip and cell, respectively. Young’s modulus ðEcell Þ can be directly calculated from the first bracket in Eq. (2), which is the slope of the linear polynomial curve of ðF 1=2 ; z dÞ. The indentation points were chosen over the cell nucleus (Fig. 2B).
Gene GAPDH E-cadherin N-cadherin
Sequence F: 5 QUOTE -ACACCCACTCCTCCACCTTTG-30 R: 50 -TCCACCACCCTGTTGCTGTAG-30 F: 50 -CCATTAACAGGAACACAGGAGTCA-30 R: 50 -GGAGGATTATCGTTGGTGTCAGT-30 F: 50 -CATCATCATCCTGCTTATCCTTGT-30 R: 50 -TTCTCCTCCACCTTCTTCATCATA-30
2.7. Real-time PCR assay To quantify the gene expression levels of E-cadherin and N-cadherin, the total RNA from three cell lines was extracted with the RNeasy plus mini kit according to manufacturer’s instructions (Qiagen, USA) and stored in RNase-free water at 80 1C. The optical density of the RNA was determined, and 1000 ng of RNA was reversely transcribed to cDNA (QuantiTect, USA). SYBR green primers (forward and reverse) were designed using the Primer Express software v3.0.1 (Applied Biosystems, USA) and are presented in Table 2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene, and the comparative cycle threshold method was used for the quantification of the expression levels.
2.8. Confocal microscopy of actin cytoskeleton
Table 1 Set of parameters for the single cell force spectroscopy test. Parameter
Value
Vertical displacement of head (cantilever) Approach/retract speed Contact time Contact feedback mode
90 mm 4 mm/s 10 and 120 s Constant force
To analyze the structure of the actin cytoskeleton, 5 103 cells were cultured over collagen-coated coverslips for two days and then fixed in 4% formaldehyde in PBS for approximately 20 min. After washing three times with PBS, Triton-X100 (Sigma, Germany) (0.1% in PBS) was added for 5 min to increase the permeability of the cells. The cells were again washed three times with PBS and then incubated at room temperature with phalloidin-FITC for 1 h. Before imaging, cells were further washed with PBS. The images were acquired by confocal microscopy using a TCS SP5 microscope (Leica Microsystem GmbH, Germany).
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Fig. 3. Result of Single cell force spectroscopy and real-time PCR analysis of three cancerous human breast cell lines. A typical curve of the short-contact-time test describing approaching and retracting. The detachment force was measured as the minimum value of the retract curve (A). Mean value of detachment forces among three various types of cancerous breast cells (MCF-7, T47D and MDA-MB-231) for 10 s and 120 s contact times (B). The levels of gene expression of E-cadherin (C) and N-cadherin (D) in three cell lines analyzed by real-time PCR test.
Table 3 Cell–cell detachment forces of cancerous cells for short and long contact time (mean value 7standard deviation). Type of cells
Detachment force (nN) Contact time 10 s
Detachment force (nN) Contact time 120 s
MCF-7 T47D MDA-MB-231
2.217 0.44 1.92 7 0.48 1.717 0.61
13.477 2.25 5.777 0.56 4.60 7 0.57
not allow the establishment of the entire number of bonds, the differences among the mean values of the detachment forces of the three types of cell lines were somehow lower yet statistically significant. According to the t-test and the one-way ANOVA test, the detachment forces for the long contact time (120 s) were statistically different from those for the short contact time (10 s) (po0.05). The MCF-7 cells exhibited the highest detachment forces among the observed cell lines. The basal-like MDA-MB-231 cells ranked the lowest.
3. Results
3.2. Gene expression
3.1. Cell adhesion force
The results indicate that the MCF-7 and T47D cells do not express N-cadherin, whereas the MDA-MB-231 cells did not express E-cadherin. The MDA-MB-231 cell line expressed N-cadherin at approximately the 30th cycle of each real-time test. The level of E-cadherin expression in the T47D cells was higher than that found in the MCF-7 cells (Fig. 3C and D).
For each SCFS run, a force-distance plot was acquired. A typical force–distance (F–D) curve, contains two main parts, namely Approach and Retract (Fig. 3A). During the retraction, the minimum force value can be interpreted as the detachment force, which is the overall force required for the separation of two cells (Helenius et al., 2008). For short (10 s) and long (120 s) contact times, the MCF-7 cells showed the highest level of cellular detachment force compared with the T47D and MDA-MB-231 cells (Table 3). The detachment forces in the short-contact-time-mode experiments were 2.2170.44, 1.9270.48, and 1.7170.61 nN (mean7SD) for MCF-7, T47D, and MDA-MB-231 cells, respectively. There were distinct differences between detachment forces of the cell lines for the long contact time (120 s) (Fig. 3B). Because the short contact time does
3.3. Mechanical properties The basal-like subtype of breast cancer showed the lowest Young’s modulus, and among the two luminal subtypes, the MCF-7 cells presented a higher Young’s modulus than the T47D cells. Around 250 force–distance curves (30 points in 1.5 1.5 mm areas for each cell) were analyzed for each group of cells. The average values of Young’s modulus were calculated to be 1.04, 0.94, and
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Fig. 4. The histograms of Young’s modulus of cells and confocal microscopy images of actin filaments. Histograms of Young’s modulus measured by atomic force spectroscopy for three cell lines (A)–(C). The actin filaments of MCF-7, T47D and MDA-MB-231 cells were stained by phalloidin-FITC and observed under a confocal microscope (D)–(F). Localization of actin filaments in MDA-MB-231 cells are marked by red arrows. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
0.62 kPa for the MCF-7, T47D, and MDA-MB-231 cells, respectively (Fig. 4A–C). The statistical analysis revealed significant difference among the elastic moduli of three cell lines (P-value o0.05). The results indicated a strong positive correlation between the detachment force and Young’s modulus among test groups. The Pearson product moment correlation coefficients (r) between Young’s modulus and the detachment force were 0.98 and 0.91 for short and long contact times, respectively. Hence, our results showed a strong positive correlation between cell stiffness and cell adhesion. Fig. 4D–F shows images of phalloidin-labeled actin filaments that were obtained through confocal microscopy. Although the cytoskeletal filaments of the MDA-MB-231 cells are localized
(red arrows in Fig. 4F), these are monotonously distributed around the circumference of the MCF-7 and T47D cells.
4. Discussion There is an opposite relation between cell–cell adhesion and the cell power of invasion. The cellular adhesiveness of malignant cells decreases, and they tend to detach from the main tumor and metastasize (Radisky, 2005; Suresh, 2007). Cancerous cells alter their structural properties, such as stiffness and adhesiveness, with their surroundings to be able to fulfill their tasks of invasion,
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migration, and metastasis (Cross et al., 2008; Suresh, 2007). The changes in the adhesiveness of such cells are partly by means of reductions/alterations in the level of the expression of markers responsible for cell adhesion (Radisky, 2005; Takeichi, 1993; Tomaskovic-Crook et al., 2009). The reduction of cellular stiffness increases the deformability of cancerous cells, which assists them with their invasion to neighboring tissues and their transmission through intercellular junctions of endothelial cells in their attempt to find a new residence (Cross et al., 2007, 2008; Suresh, 2007). Using a Boyden chamber invasion assay on various breast cell lines, it has been shown that the basal subtype of breast cell lines have a higher tendency to invade compared with luminal-type cells (Neve et al., 2006). In fact, among the luminal subcategory of cancerous cells, T47D cells exhibited higher invasiveness potential than MCF-7 cells, and it is thus expected that MCF-7 cells should show stronger cell–cell adhesion than T47D cells (Neve et al., 2006). Our SCFS results confirmed that the overall adhesion force of MCF-7 cells is significantly higher than that of T47D cells for both contact times (Fig. 3B). Similarly, MDA-MB-231 cells exhibited a significant invasive potential in the Boyden chamber invasion assay (Neve et al., 2006), which is correlated with the lowest value of cell–cell adhesion force derived by the SCFS tests performed in this research study. Our results indicate that only the basal-like MDA-MB-231 cells express N-cadherin, whereas the two other types of cells do not express this marker (Fig. 3D). The single-molecule force spectroscopy (Panorchan et al., 2006) and micropipette aspiration (Chu et al., 2004) methods revealed that the adhesion force between N– N cadherins are far weaker than that of between E–E cadherins. Hence, cells expressing N-cadherin (MDA-MB-231) should exhibit a lower cell–cell adhesion compared with cells that express E-cadherin (T47D and MCF-7). Although the level of E-cadherin expression was higher in T47D cells compared to MCF-7 cells, the SCFS experiments unexpectedly indicated a stronger cell–cell adhesion force for MCF-7 than T47D cells. The level of E-cadherin expression among some breast cell lines has been reported in recent studies by real-time PCR (Holen et al., 2012) and immunoblotting analysis (Arima et al., 2012; Yan et al., 2010). These previous results also confirmed that the level of E-cadherin expression in T47D cells is higher than that found in MCF-7 cells. This difference may be related to the intricacy of the cell–cell adhesion phenomenon, which is controlled by a complex of molecules from the membrane to the cytoplasm. It is believed that cytoplasmic proteins and the actin cytoskeleton play indispensable roles in the functions of the cadherin family (Takeichi, 1993). Hence, the expression of cell adhesion markers is not the only determinant of the overall cellular adhesion. In general, the tendency of cancer cells to invade is accompanied by a decrease in cell adhesion through alterations in the number and/or function of adhesive proteins in the cell membrane and not necessarily associated to the extent of gene expression in the nuclei of cells. When cancer cells shift to the metastasis phase, E-cadherins are less abundant, and in some cases, weaker N-cadherins play important roles (Fig. 3D). Simultaneously, cells become more distensible (Fig. 4C) through alterations in the cytoskeletal structure (Fig. 4F) to be ready to interact with endothelial cells when highly deformable bodies are required to pass through endothelial junctions to produce a colony of cells within the new host tissue. In this study, we compared the differences in cellular elasticity between three groups of cells and sought a relationship between cellular elasticity and the detachment force. Using a small indentation depth (500 nm), a low setpoint force (approximately 1 nN) and an approaching velocity of approximately 1 mm/s with a rectangularshaped silicon nitride cantilever, a strong positive correlation (40.91) between cell elasticity and overall adhesion force was found. Fig. 5 describes the reduction in cell–cell adhesion accompanied by cell
Fig. 5. Cellular adhesion force and elasticity. The softening of the cell body (Reduction in Cellular Elasticity, right axis in kPa) accompanied by reduced cellular adhesiveness (left axis in nN) are two correlative mechanisms which malignant cells implement to increase their motility and achieve strong potential of invasiveness.
softening when the degree of invasiveness is elevated in the three cancer cell lines. These highly correlated phenomena are two adaptive mechanisms through which malignant cells achieve increased motility and strong metastasis potential, i.e. reduction in adhesive protein bonds and alterations in the cytoskeletal arrangement. The peripheral actin filaments in MCF-7 cells have been observed previously (Leporatti et al., 2009), in accordance with our confocal microscopy observations. A similar arrangement of actin filaments was detected in the T47D cells, whereas the filaments were found to be localized in MDA-MB-231 cells (Fig. 4D and F). Similarly, it has been shown that actin filaments are branched and localized in leading edges of migrating cells (Le Clainche and Carlier, 2008). It might be due to actin filaments reassembly with local adhesion molecule for better migration. Migrating cells have shown localized actin filaments in their leading edges. The results of this study can be implemented as a platform for further investigations that target the manipulation of cellular adhesiveness and stiffness as a therapeutic choice. The function of designed antibodies for cancer treatment can be examined in vitro through the AFM-based single cell force spectroscopy approach, particularly for the manipulation of cell adhesion. Although this approach is currently suffering from some drawbacks, such as being time consuming, having limited contact time (o10 min), and demanding a reliable amount of acquired data for statistical analysis, its distinguished advantages make it a useful tool in cancer research. In addition to cell–cell adhesion, this method can be employed for the study of interactions of cancerous cells with biomaterials.
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