Force spectroscopy on a cell drum: AFM measurements on the

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Biological and Medical Applications of Materials and Interfaces

Force spectroscopy on a cell drum: AFM measurements on the basolateral side of cells via inverted cell cultures Joo Hyoung Kim, Kristina Riehemann, and Harald Fuchs ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01990 • Publication Date (Web): 29 Mar 2018 Downloaded from http://pubs.acs.org on March 29, 2018

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ACS Applied Materials & Interfaces

Force Spectroscopy on a Cell Drum: AFM Measurements on the Basolateral Side of Cells via Inverted Cell Cultures Joo Hyoung Kim,a Kristina Riehemann a, * and Harald Fuchs,a a.

Physikalisches Institut, WWU Münster, D-48149 Münster, Germany and Center for

Nanotechnology (CeNTech) KEYWORDS cancer diagnostics, atomic force microcopy, scanning probe bottom-up approach, elasticity, inverted cell culture

ABSTRACT

The elasticity of a cell is one of the most critical measures of the difference between cancerous cells and healthy cells: cancer cells tend to be softer than healthy cells, and highly invasive cells tend to be more elastic than less aggressive cells. In this work, we present a novel ‘bottom-up’ cell force spectroscopy method for the biophysical characterization of cancer cells, in which an atomic force microscopy (AFM) tip approaches from the backside of a net-shaped culture substrate

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exposing the basolateral cell membrane drum, and compare it with the conventional ‘top-down’ AFM measurements. We used two different human pancreatic carcinoma cell lines, PaTu8988S and PaTu8988T. Our ‘bottom-up’ AFM tip approach provided a more statistically synchronized distribution of the measured elastic moduli of the cells, demonstrating its superior applicability for the clinical use of force spectroscopy, which is not attainable with the conventional ‘top-down’ AFM approach.

INTRODUCTION

According to the World Health Organization, 4.7 million people died from cancer in 2012 worldwide. This is almost one third of the mortalities on the planet, which indicates how hazardous the disease is to the human race. Similar to other fatal diseases, the early detection and diagnosis of carcinoma results in a higher likelihood of curing the patients.1, 2 Studies have revealed that the properties of the cytoskeleton are the key to a broad spectrum of processes in cells. Recently, the biophysical properties of cells have gained as much interest as their biochemical properties as factors that can be used to characterize cancer-related changes in cells.3-5 One of the most important processes for the development of a metastatic tumor is the epithelial– mesenchymal transition (EMT), which is one of the main characteristics of invasive cells.6 This process leads to a repression of e-cadherin-mediated cell-to-cell contact, which results in an enhanced mobility.7 These changes in the morphology of the cytoskeleton are also accompanied by EMT.8 Previous studies have shown that the elasticity of healthy cells is higher than the


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elasticity of cancerous cells, and more invasive cells are more deformable than less invasive cells.9, 10

In recent years, a wide range of experimental approaches have been used for the analysis of

living cells. Essentially, these methods can be categorized into three major streams,11-17 first, probing the cell membrane apically at the subcellular level (e.g., atomic force microscopy (AFM) or magnetic twisting cytometry), second, analyzing the cell behavior under the influence of a mechanical force on an entire cell (e.g., micropipette aspiration) and, third, simultaneously recording the response of a whole cell population under external stress (e.g., shear flow and stretching methods).

An overview with a detailed description of these techniques can be found in the literature.18 Recently, AFM has been shown to be an appropriate tool for quantifying the biomechanical properties of cells, 13, 14, 16, 17, 19, 20 membranes21-23, and tissues.24, 25 Among them, the AFM probe tip is used to deform the cell membrane from the top side (apical) of the cell with a varying force. This technique can be used to provide information on the elastic properties of the cytoskeleton and the mechanical compliance of the whole cell.21 Usually, these experiments are performed with cells attached on a substrate. However, owing to the cell shape and the dynamic nature of living cells, it is virtually impossible to precisely estimate the acting forces. The cell under measurement responds to the external measurement tool by reorganization of the cytoskeleton. Furthermore, the speed and the strength of the response process depend on many different cell characteristics (e.g., stage in cell cycle, cell morphology). Thus, the technique mentioned above yields different results for the elasticity even for the same cell line under the same conditions.12 Therefore, these techniques are hardly suitable for clinical applications (e.g., early detection of invasive cancer).


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Here, we present an inverted cell culture method followed by a novel bottom-up AFM tip approach for the physical characterization of cells, which avoids the influence of the dynamic responses of cells under measurement. To reduce the side effects described above, our approach is performed by measuring the cell elasticity from the basolateral side by using AFM. The results were compared with cell elasticity measurements from the apical side of the cell, both performed with a spherical AFM tip to obtain an overall documentation of the cell stiffness by AFM. We used the pancreatic cancer cell lines PaTu8988T and PaTu8988S as reference samples. These cell lines were first isolated in 1985 from liver metastases of a human pancreas adenocarcinoma and characterized by several groups.27-29 It was shown that PaTu8988T presents a lower degree of differentiation with regard to the cytoskeleton as well as a faster and more disordered growth behavior29. Thus, they differ significantly in their cytoskeleton structure, and consequently, in their elasticity. To establish our new method, the two cell lines were chosen because they were isolated from the same tumor; therefore, they offer comparable cell systems, which are suitable for studies focusing on establishing methods. For the apical setup, the cells were grown on a coverslip as a substrate and were directly approached from the top side. For the basolateral setup, the cells were grown on TEM grids as substrates with a well-defined grid-hole size (ATHENE 2000 mesh thin (5–7 µm) bar, gold coated, 5 µm bar, 2000 pores, Plano GmbH; Wetzlar, Germany). For the measurement, the substrate was inverted by 180 degrees and the cell was approached by the AFM probe from the bottom side of the substrate through the holes of the grids. In this way, the membrane of the adherently grown cells span over the grid holes of the substrate like a skin on a drum and the determination of the cell elasticity should be more reproducible owing to less degree of freedom by restriction of the template (more uniform membrane shape) compared to the infinite free space for the apical cases


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(more degree of freedom in the membrane shape during contact with the AFM probe). This technique enabled us to analyze the cells without (or with reduced) reaction forces against the measuring tool. Furthermore, measurement of the elasticity in this way circumvents any effect of the underlying substrate, particularly in the case of large tip indentations into the cell membrane. Even though there have been several attempts to extract mechanical information of basal part of cells from apical AFM measurements in an indirect way, such as stiffness tomography,30-32 these approaches have a limited indentation depth (less than 1 μm), lack in fine resolution,30 and still have all the side effects listed above because these are ‘apical’ measurements. However, in our case, as the tip approaches the basolateral side of cell directly, we could obtain wholesome basolateral properties, avoiding the disadvantages gained, e.g., from cellular topography present in apical force spectroscopy.

(a)

(b)

Figure 1. Experimental setup for the apical (a) and basolateral (b) measurement using AFM. (a) Cells are grown on a coverslip and (b) cells are grown on a grid which is turned upside down for measuring and approached through the pores.


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The human pancreatic carcinoma cell lines PaTu8988S and PaTu8988T (DSMZ; Braunschweig, Germany) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L D-Glucose without phenol red, 5% fetal bovine serum, 5% donor horse serum, and 2 mM L-Glutamine (Biochrom, Berlin, Germany) at 37°C in a humidified atmosphere of 7% CO2 content in air. For the basolateral measurements, the cells were grown for 48 h on a poly-L-lysine (PLL)-coated gold TEM grid with a well-defined pore size. The additional PLL coating was necessary to ensure the cell adhesion on the gold-coated nets, and this treatment was sufficient to sustain a tight contact of cells to the grids when the substrate was turned upside down for the basolateral measurements. Then, the cell was approached by the AFM probe from the bottom side of the substrate through the pores (Figure 1). In this way, the membrane of the adherently grown cells span over the pores. As described above, this technique realizes the analyses of the cells without (or with reduced) reaction forces against the probe itself. For the apical measurement, the cells were grown for 48 h on 15 mm glass coverslips, which were sterilized overnight in 70% ethanol before use. As a comparison, another group of cells were grown for the same period of time on the same PLL-coated gold grids as the basolateral measurements, but the substrate was not upside down. For these cases, the cells were approached from the top. Gold is biocompatible under the experimental conditions and the grid acted as a supporting template for adherently growing cells. The ratio between the bars and the pores of the mesh was chosen to maximize the contact between the cells and the substrate, by simultaneously ensuring that the pores of the mesh were large enough for the AFM probe tip to fit through. The individual steps for the sample preparation for the basolateral measurement are illustrated in Figure S1. For better handling and a higher stability, the substrate was glued onto a stainless-steel plate. This prevented the floating of the grid on/in the cell culture media and, therefore, the


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unwanted cell growth on the ‘basolateral’ side of the grid (step 1). After drying for 24 h, the grid was coated with PLL for 24 h. This enhanced the adhesion between the cells and the grid (step 2). Afterwards, the substrate was turned upside down in a 12 well-plate and overlaid with a medium containing 6×104 cells per square centimeter and incubated for 48 h in an incubator under the conditions described above (step 3). After growing for 48 h, the sample was ready for measurement (step 4) and thus, rotated by 180 degrees. For the apical setup, the 15 mm glass coverslips were used directly after evaporating of the ethanol, in which they were sterilized overnight. The control apical group with the gold grid was prepared in the same way as that of the basolateral case, but without rotating the substrate. To confirm the functioning of the cytoskeleton for the basolateral setup, we compared experiments using cytochalasin D (Sigma-Aldrich, St. Louis, USA), one of the most famous mycotoxins known as an inhibitor of actin polymerization, by treating it (1 μg/mL for 1 h) before AFM measurements for cells in the apical experiment groups, both on the coverslips and on the gold grids. The experiments were performed in the measurement media (medium 2 in Figure S1; cultivation media with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)). To avoid the signal loss of the laser, the media without phenol red was chosen, whereas HEPES was added to maintain the pH of the media during the measurement without a controlled CO2 environment. As shown by the light microscopy pictures in Figure S2 taken just before the AFM measurements, each cell line showed a characteristic growth behavior. PaTu8988S grew in an associated cell layer whereas PaTu8988T grew as single cells. The dark shadow in the right parts of the images indicates the position of the probe.


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I(a)

I(b)

I(c)

II(a)

II(b)

II(c)

Figure 2. PaTu8988T (I) and PaTu8988S (II) growing on grids for the basolateral measurement (a; b) and on coverslips for the apical measurement (c). The nuclei are presented in blue (4′,6Diamidin-2-phenylindol (DAPI)), the actin cytoskeleton in green (Alexa Fluor 488 phalloidin), and the gold grid reflects in red.

For high quality fluorescent measurements, the cells were washed with PBS containing Ca2+ and Mg2+ (Biochrom; Berlin, Germany) after the incubation period of 48 h, fixated for 15 min at room temperature (RT) with 4 % paraformaldehyde (Sigma-Aldrich; St. Louis, USA) in PBS, and


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washed again. Subsequently, they were permeabilized for 10 min at RT with 0.5% Triton X-100 (Sigma-Aldrich; St. Louis, USA) and 1% BSA (Roth; Karlsruhe, Germany) in PBS, washed with PBS again, and blocked with 1% BSA in PBS for 30 min at RT and rinsed. The actin cytoskeleton was stained with Alexa Fluor 488 coupled phalloidin (Life Technologies; Carlsbad, USA) for 20 min at RT at a final concentration of 165 nM with 1% BSA in PBS. The nucleus was stained with at RT. The coverslips were washed again and mounted with Fluoromount (Sigma-Aldrich, St. Louis, USA) on a slide and dried overnight at 4°C. Samples were examined with a Nikon Eclipse TE2000-U (Nikon Instruments; Chiyoda, Japan). We performed fluorescent staining of the cells for two purposes. First, to visualize the cells attached on the nets and grown over the pores ready for basolateral measurements (Figure 2-I and II, both a and b). Second, for a comparison of the cell growth on the net and on the coverslip (Figure 2-Ic and IIc). The staining with Alexa Fluor 488 phalloidin revealed the distribution of the actin cytoskeleton through the cell, whereas the DAPI staining visualized the nucleus. These figures confirmed the differences in the morphology of the two cell lines that were already observed in the light microscopy pictures (Figure S2), but show no alteration of the cytoskeleton distribution between nets and coverslips. An AFM MFP-3D (Asylum Research; Santa Barbara, USA) was used for the apical and basolateral measurements. The probes consisted of spherical CP-PNPL-SiO2C-5 particles (Nano and More; Wetzlar, Germany) with typical parameters of 200 µm length for the cantilever, a spring constant of 32 nN/pm, 17 kHz resonance frequency in air, and 6.62 µm diameter of the spherical tip. This tip diameter was smaller than the pore size of the grid, but larger than the pore thickness, which was suitable for the basolateral measurements of cells without interference from the grid. The force measurements were performed within a short period of time from 30 min to 1 h to ensure


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that most of the cells were alive during the spectroscopic experiments. At least 100 cells were measured for each cell type and experimental setup. Prior to the measurements, the spring constants of the probes were determined by analyzing the thermal noise spectra of the measurement media. During the measuring processes with the grids, it was hardly observable that the cells pressed by the AFM tip were detached from the grids. All force–distance (F–D) curves were analyzed using the Hertz–Sneddon model. Because the indention depth was approximately 10% of the cell height (Figure S3), the parabolic model was adopted instead of the spherical model. The indentation depths of about 0.5 µm can be taken from Figure S4, while as presented by Kemper et al., the height of the two cell lines is between 6.5 and 20 µm.33 Previously, AFM contact models for suspended lipid membranes on porous substrates have been suggested,21-23 here we applied the classical Hertz–Sneddon model. This is justified, first, because restoring forces of cells mainly come from the elasticity of the cytoskeleton and the osmotic pressure of the cells,21, 34 and second, an indentation depth of approximately 10% of the cell height was observed, which was a plausible scale for regarding the cells as an infinite half space underneath a tip. Representative raw AFM data traces as well as their fits are presented in Figure S4. For the apical measurement, the cells were grown directly onto glass coverslips and gold grids. Therefore, it was possible to see the cells directly with the integrated optical microscope of the AFM and to have an intended location of the tip on a cell of interest. For the PaTu8988S, the apical measurements show an elastic modulus of E = (1612 ± 520) Pa, and E = (1170 ± 335) Pa for basolateral measurements. For the PaTu8988T with the apical approach, E = (652 ± 213) Pa, and E = (731 ± 150) Pa were found for basolateral cases, respectively (Figure 3a and b, Table 1 and S1).


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(a)

(b)

(c)

(d)

Figure 3. Distribution of the elastic moduli when measured on the apical side of the cell for PaTu8988T (a) and PaTu8988S (b), and the basolateral side of the cell for PaTu8988T (c) and PaTu8988S (d).

The basis for a successful detection of cell elasticity from the basolateral side is the ability to recognize whether the cantilever hits the substrate or the holes with a cell below it. This is necessary because the resolution of the camera was not high enough to directly see through the mesh. In each experiment, two types of F–D curves were recorded: one on a bar of the grid and the other one through a mesh under which a cell is located. Both curves were recorded in one cycle of measurements (For detailed information, See Figure S3).


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Table 1. Overview of the E-moduli of apical and basolateral measurements of PaTu8988S and PaTu8988T cells with relative errors. Approach direction Basolateral

Apical

Cell type

Value [Pa]

Error [Pa]

relative Error [%]

PaTu8988S

1445

176

12

PaTu8988T

582

97

17

PaTu8988S

1612

520

32

PaTu8988T

652

213

33

For the PaTu8988S, the basolateral measurement shows an elastic modulus of E = (1445 ± 176) Pa. For the PaTu8988T, E = (582 ± 97) Pa. A comparison of the results between the apical and basolateral measurement is shown in Table 1 and S1. It is clear that the approach from the basolateral side through a grid results in a significantly lower measurement error. Finally, apical control groups treated with actin disruptor cytochalasin D showed a total decrease in their elastic moduli of E = (634 ± 211) Pa for PaTu8988S, and E = (558 ± 180) Pa for PaTu8988T on coverslips, whereas similar results were observed for the experiments on gold grids, as shown by E = (442 ± 204) Pa for PaTu8988S and E = (370 ± 182) Pa for PaTu8988T (Table S1). Compared to the abrupt decrease in the elasticity for the PaTu8988S cell line after the inhibition treatment, PaTu8988T showed a relatively small change, which might be attributed to its original soft nature, resulting in a lower actin disruption effect. Despite this, the difference in the elasticity observed after actin polymerization inhibition proved that the actin cytoskeleton still had an important role for the basolateral cell culture setup. In agreement with our expectations, PaTu8988S was more than two times harder compared to the PaTu8988T cell line. This result agreed with previous studies, which predict a higher


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invasiveness of the PaTu8988T cells based on an indirect correlation of invasiveness and cell stiffness. Because we measured certain points of cells randomly, there could not be an inherent difference in the probing area for both the basolateral and apical approach cases, which implied that these smaller standard deviations for the basolateral measurements do not come from the smaller probing area, although the values of the basolateral forces vary over a wider range. This can be described in terms of the different reactions of cells when an F–D measurement is performed without the glass surface, on which the cells are grown when measured from the apical side. Another possible explanation is that the part of the cell that is measured cannot be identified when the measurement is performed on the basolateral side of the cells. Different studies have shown that the elastic moduli of the cellular surface differ by orders of magnitude. In the case of an F–D measurement on the actin network filament and the intermediate filaments, the elasticity was significantly higher than that of microtubules.35 It is also reasonable to say that further statistical analyses such as normal distribution fitting or noise analysis are not needed because the elastic moduli of different experimental groups were well separated, which indicated that the measurement noise cannot interfere in the distinction of these two different cell groups in a substantial degree.

CONCLUSION

Apical and basolateral force spectroscopy was performed on two different human pancreatic carcinoma cell lines, PaTu8988S and PaTu8988T. For the apical measurements, fitting of the F– D curves by AFM revealed an elasticity of the PaTu8988S of E = (1612 ± 520) Pa on cover slips


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and E = (1170 ± 335) Pa on grids, and for the PaTu8988T an elasticity of E = (652 ± 213) Pa on cover slips and E = (731 ± 150) Pa on grids. When measured on the basolateral side, the cells were grown on scanning electron microscopy grids and an AFM was used to record F–D curves through the holes of the mesh; the Young’s modulus was E = (1445 ± 176) Pa for the PaTu8988S and E = (582 ± 97) Pa for the PaTu8988T. Both experiments distinguish the difference in elasticity between the two cell lines as well as the lower elasticity of the more aggressive cell line PaTu8988T. Interestingly, the measurement from the basolateral side of the cells grown on a grid resulted in a distinctly lower error in the AFM measurements. According to the findings of this work, we believe that this technique is a suitable tool for more precise in vitro investigation for the disease related biomechanical characterizations of cells.


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(a)

(b)

Figure 1. Experimental setup for the apical (a) and basolateral (b) measurement using AFM. (a) cells are grown on a coverslip (b) cells are grown on a grid which was turned upside down for measuring and approached through the pores.


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I(a)

I(b)

I(c)

II(a)

II(b)

II(c)

Figure 2. PaTu8988T (I) and PaTu8988S (II) growing on grids for the basolateral measurement (a; b) and on coverslips for apical measurement (c). The nuclei are presented in blue (DAPI), the actin cytoskeleton in green (Alexa Fluor 488 phalloidin) and the gold grid reflects in red.


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(a)

(b)

(c)

(d)

Figure 3. Distribution of the elastic moduli when measured on the apical side of the cell for PaTu8988T (a) and PaTu8988S (b), basolateral side of the cell (c) and (d), likewise.


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Table 1. Overview of the E-moduli of apical and basolateral measurements of PaTu8988S and PaTu8988T cells with relative errors.

Approach direction Basolateral

Apical

Cell type

Value [Pa]

Error [Pa]

relative Error [%]

PaTu8988S

1445

176

12

PaTu8988T

582

97

17

PaTu8988S

1612

520

32

PaTu8988T

652

213

33

ASSOCIATED CONTENT Supporting Information. Schematics to the individual steps of sample preparation for basolateral measurement, light microscopy images of cancer cells, force-displacement curves recorded within one cycle during basolateral measurements, raw AFM data traces as well as their fits, average of the E-moduli of apical measurements on various substrates with and without treatment of cytochalasin D. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *K. [email protected]. Funding Sources Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT The authors thank Jana Salich (Physikalisches Institut/CeNTech, Münster) for excellent technical support. REFERENCES (1) International Agency for Research on Cancer (under World Health Organization), GLOBCAN 2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide 2012, http://globocan.iarc.fr/Pages/fact_sheets_population.aspx (accessed July 2017) (2) WHO, Early Detection of Cancer, http://www.who.int/cancer/detection/en/ (accessed October 2016). (3) Eghiaian, F; Schaap, I. A. T. Structural and Dynamic Characterization of Biochemical Processes by Atomic Force Microscopy. Methods Mol. Biol. 2011, 778, 71-95. (4) Cross, S. E; Jin, Y. S.; Rao, J.; Gimzewski, J. K. Nanomechanical Analysis of Cells from Cancer Patients. Nat. Nanotechnol., 2007, 2, 780-783. (5) Rebelo, L. M.; Sousa, J. S. de; Mendes, J.; Radmacher, M. Comparison of the Viscoelastic Properties of Cells from Different Kidney Cancer Phenotypes Measured with Atomic Force Microscopy. Nanotechnology, 2013, 24, 055102. (6) Radisky, D. C. Epithelial-Mesenchymal Transition. J. Cell Sci. 2005, 118, 4325-4326. (7) Liu, Y; Liu, Y.; Lee, H.; Hsu, Y; Chen, J. Activated Androgen Receptor Downregulates E-Cadherin Gene Expression and Promotes Tumor Metastasis. J. Mol. Cell. Biol., 2008, 28, 7096-7108.


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Force spectroscopy on a cell drum: AFM measurements on the

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