biochimica et biophysica acta - proteom-msu.org · the membranes were mounted to glass slides, and...

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Biochimica et Biophysica Acta xxx (2015) xxx–xxx

BBAMCR-17568; No. of pages: 9; 4C: 3, 4, 5

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bbamcr

Distinct impact of targeted actin cytoskeleton reorganization onmechanical properties of normal and malignant cells☆

FYu.M. Efremov ⁎, A.A. Dokrunova, A.V. Efremenko, M.P. Kirpichnikov, K.V. Shaitan, O.S. SokolovaM.V. Lomonosov Moscow State University, Faculty of Biology, 111991, 1/73 Leninskie Gory, Moscow, Russia
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☆ This article is part of a Special Issue entitled: Mechan⁎ Corresponding author at: M.V. Lomonosov Mosco

Biology, Department of Bioengineering, 11991, 1/73 LeTel./fax: +7 4959395738.

E-mail address: [email protected] (Y.M. Efremov

http://dx.doi.org/10.1016/j.bbamcr.2015.05.0080167-4889/© 2015 Published by Elsevier B.V.

Please cite this article as: Y.M. Efremov, et aland malignant cells, Biochim. Biophys. Acta

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Article history:Received 23 January 2015Received in revised form 23 April 2015Accepted 5 May 2015Available online xxxx

Keywords:AFMForce spectroscopyProstate cancerForminArp 2/3Cortical actin
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The actin cytoskeleton is substantially modified in cancer cells because of changes in actin-binding proteinabundance and functional activity. As a consequence, cancer cells have distinctive motility and mechanicalproperties, which are important for many processes, including invasion and metastasis. Here, we studied theeffects of actin cytoskeleton alterations induced by specific nucleation inhibitors (SMIFH2, CK-666), cytochalasinD, Y-27632 and detachment from the surface by trypsinization on themechanical properties of normal Vero andprostate cancer cell line DU145. The Young's modulus of Vero cells was 1300 ± 900 Pa, while the prostatecancer cell line DU145 exhibited significantly lower Young's moduli (600 ± 400 Pa). The Young's moduliexhibited a log-normal distribution for both cell lines. Unlike normal cells, cancer cells demonstrated diverseviscoelastic behavior and different responses to actin cytoskeleton reorganization. They were more resistant tospecific formin-dependent nucleation inhibition, and reinforced their cortical actin after detachment from thesubstrate. This article is part of a Special Issue entitled: Mechanobiology.

© 2015 Published by Elsevier B.V.

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One of the major characteristics of cancer cells is their ability to mi-grate to nearby tissues, blood or lymph vessels and to form secondarytumors at multiple distant sites. Tumor metastases are responsible forapproximately 90% of all cancer-related deaths [1]. The mechanism bywhich malignant cells develop such ability involves changes in theirmechanical properties and actin cytoskeleton dynamics. The ability tomove is a basic property of living cells. The movement of cells and celllayers organizes and determines the formation of organs in embryogen-esis, wound healing and the progress of inflammation. The actin cyto-skeleton, together with other factors (nucleation promoting factors,microtubules, intermediate filaments, lipids), is responsible for thesefunctions [2]. The dynamics of the cytoskeleton are regulated by actin-binding proteins (ABPs) promoting actin turnover, the key processin cellular motility [3]. The Arp2/3 complex is responsible for actinbranching [4]. Cofilin and the Srv2 complex coordinate actin cablesevering [5] and actin depolymerization [6,7]. Formin family proteinsregulate the polarized growth of cells, stress fiber formation, and actinnucleation [8–10]. They also coordinate interactions between actin fila-ments and microtubules [11]. Cytoplasmic formins are autoinhibited

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obiology.w State University, Faculty ofninskie Gory, Moscow, Russia.

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., Distinct impact of targeted(2015), http://dx.doi.org/10.1

[12] and activated upon interactionwith Rho GTPases [13]. After activa-tion, they processively nucleate long actin cables, thus generating lead-ing edge protrusions [14]. Recently, a significant increase of forminexpression in colorectal cancer [15–17] and leukemia [18] has beendemonstrated. It has been hypothesized that formins may act astumor suppressors [19], yet no such activity has been specificallyidentified.

Nucleation of the actin branch by the Arp2/3 complex occurs incombination with other proteins including the N-WASP/WAVE family.The activated complex binds to the side of the existing filament, alignsin a similar manner to an actin dimer and nucleates the formation ofa new filament, which extends from the side of the existingfilament at a70° angle. Dense branched networks generated by Arp2/3 activity areessential for mesenchymal invasion and the formation of lamellipodiaand invadopodia. Overexpression of WASP family proteins and theArp2/3 complex has been associated with malignant phenotypes ofseveral human cancers, including breast [20], colorectal [21], lung [22],and head and neck squamous cell carcinoma [23].

In cancer cells, the actin cytoskeleton is substantially modified,accompanied by changes in the expression and activity of ABPs [19,24,25]. As a result, cancer cells have distinctive mechanical propertiesfrom both normal and benign cells. With some exceptions [26–28], alow rigidity of cancer cells was reported in the majority of works[29–35]. It has been suggested that the elastic properties of cancercells play a major role in the metastatic process, and the degree ofdeformability of cells has been proposed as a marker for metastatic po-tential [36,37]. Hypothetically, more aggressive cancer cells should

actin cytoskeleton reorganization on mechanical properties of normal016/j.bbamcr.2015.05.008

http://dx.doi.org/10.1016/j.bbamcr.2015.05.008

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demonstrate enhanced mechanical deformability because these cellsundergo substantial mechanical deformation during tissue invasion,intra- and extravasation. Yet results from recent studies show allpossible types of correlation (direct, reversed, nonsignificant, and com-plicated) between the cell stiffness and the metastatic potential of can-cer cells [30,34,35,38–40], meaning that the independent mechanicalcharacterization is not sufficient to evaluate the metastatic potential ofcells. Further investigations related to mechanical aspects, such asmeasurements in different conditions and after different treatments,are required to understand the connection of mechanical compliancewith the metastatic potential.

Recently, several methods were suggested to characterize the me-chanical properties of cells including different types of microrheology[41], optical tweezers [42], micropipette aspiration [43], magnetictweezing cytometry [44], atomic force microscopy (AFM) [37,45,46]and others. Previously, we employed force spectroscopy, a commonlyused AFM technique, to investigate the elastic properties of non-transformed epithelial Vero cells and showed that this cell line may bea good experimental model for studying the effects of different treat-ments on cell mechanical properties [47]. Here, we examined themechanical properties (quasi-static and complex Young's moduli) ofprostate cancer cells DU145, and compared them to normal Vero cellsbefore and after different actin cytoskeleton reorganizations. Althoughthe studied cell lines have different origins (both are epithelial, butVero are from monkey healthy kidney and DU145 are from humanprostate cancer), we showed here that they share some common prop-erties with normal (Vero) and malignant (DU145) cells, which includecytoskeleton structure, migration abilities and mechanical parameters.The effects of inhibitors of the major actin nucleators, formin andArp2/3, Cytochalasin D (CytD) and Y-27632 (ROCK inhibitor) treat-ments, as well as the detachment of cells from the culture substrateby trypsinization, were studied.

2. Materials and methods

2.1. Cell cultivation

Human prostate cancer epithelial cell line DU145 with mediuminvasiveness was chosen for the experiments [48]. A healthy Africangreen monkey Cercopitecus aethiops kidney cell line (Vero) served as acontrol. Both cell lines were grown in DMEM (PanEco, Russia) with10% fetal bovine serum (FBS) (HyClone, USA). Cells were grown at37 °C in a humidified 5% CO2 atmosphere in a CO2-incubator (Sanyo,Japan) for 5–6 passages after being defrosted. For AFM imaging andforce spectroscopy, cells were harvested from subculture, seeded onsterile glass coverslips (Menzel-Gläser, Germany) in sterile 35 mmPetri dishes and placed into the incubator at the same conditions asstated above for 1–2 days.

The cells were treated with the following compounds: SMIFH2 – aninhibitor of formin-mediated actin assembly [49]; CK-666 – an inhibitorof the Arp2/3 complex-dependent pathway of actin nucleation [50];Cytochalasin D (all from Sigma-Aldrich, Germany) – an inhibitor oftotal actin polymerization; and Y-27632 (Abcam, UK) – inhibitor ofthe Rho kinase (ROCK) pathway of myosin light chain phosphorylation.Stock solutionswere prepared in DMSO, and then reagentswere dilutedto final concentrations in a serum-free medium. Trypsinization wasperformed by the replacement of the cell culture medium with 0.25%Trypsin–EDTA in HBSS (PanEco, Russia).

2.2. Confocal microscopy

For confocal fluorescence imaging, cells were seeded on sterile35 mm glass bottom Petri dishes (Greiner Bio-one, Germany) at a den-sity of 3 * 105 cells per dish and allowed to grow for the next 24 h. Then,the cells were fixed with 2% formaldehyde, permeabilized with 0.4%Triton-X100 in PBS, and stained. The filamentous actin (F-actin) was

Please cite this article as: Y.M. Efremov, et al., Distinct impact of targetedand malignant cells, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1

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labeled with fluorescent phalloidin conjugate (with Alexa Fluor® 488or Alexa Fluor®546, Life Technologies, Europe). Cell nucleiwere stainedwith DAPI (Life Technologies, Europe). The samples weremounted intothe SlowFade® medium (Life Technologies, Europe). Fluorescenceimages were taken using Zeiss LSM510 Meta or Zeiss LSM710 Metainverted confocal microscopes (Carl Zeiss, Germany) with a 63 × α(alpha)-Plan-Apochromat oil-immersion objective (NA = 1.46, CarlZeiss AG).

For quantitative F-actinmeasurements after different treatments thestaining procedure was slightly modified from the above. Control andtreated cells were cultivated separately on the same glass slide(Menzel-Gläser, Germany) with attached Flexiperm silicone wells(Greiner Bio-one, Germany). After removing the silicone wells, thecells were fixed and stained in an identical way on the same slide, sothe direct comparison of relative amounts of F-actin was possible.Fluorescence of Alexa Fluor® 546 phalloidin and DAPI were recordedusing a Z-stack regime. The Image J software (National Institutes ofHealth, USA) was used for processing the fluorescent images. At least40–50 cells were examined in each sample. The quantitative map ofthe Alexa Fluor® 546 phalloidin distribution was calculated for eachmeasured cell and processed with the Image J software to estimateaverage cytoplasmic fluorescence intensity.

2.3. In vitro Matrigel invasion assay

Cell invasion experiments were performed using Bio-Coat cellmigration chambers (BD Biosciences, USA) according to the manufac-turer's protocol. Inserts were composed of 8 μm pore size filters coatedwith the Matrigel basement membrane matrix (BD Biosciences, USA).Briefly, detached cells (2 * 105 in 500 μl of serum-free DMEM/RPMImedium) were added to each insert (upper chamber), and the chemo-attractant (5% FBS in DMEM/RPMI) was placed in each well of a 24-wellcompanion plate (lower chamber). After 14-h incubation at 37 °C in a5% CO2-incubator, the noninvasive cells from the upper surface of thefilter were thoroughly removed with a cotton swab. Invading cells onthe lower surface of the membrane were fixed with methanol andstained with 1% Toluidine blue (Fluka) in 1% borax. For quantification,the membranes were mounted to glass slides, and the cells from 5 ran-dom microscopic fields (×200 magnification) were counted. Assayswere performed in duplicate, and migration was expressed in terms ofcells/field.

2.4. Young's modulus measurements.

AFM measurements were performed using a commercial Solver Bioatomic force microscope (NT-MDT, Russia), combined with an invertedoptical microscope (Olympus, Japan) as described previously [47]. Cells,subjected to AFMmeasurements, were non-confluent (about 60%), andwell attached to the Petri dish surface. Typically, 3–5 force curves weretaken at a 2 μm/s rate from each of the studied cells near its center usingtipless AFM probes CSG11 (NT-MDT, Russia) modified with a 9 μmdiameter polystyrene bead. Hertz's contact model was applied for pro-cessing of the first 500 nmof indentations to calculate apparent Young'smodulus (E). Experiments were conducted during a 30–40 min period,at 37 °C, after replacing the growth medium with the DMEM HAM/F12mediumwith 15mMHEPES (Sigma, United States). Three independentexperiments were conducted for each cell line before and after treat-ment, with 10–25 cells analyzed in each experiment.

Viscoelastic behavior of cells was characterized by applying low-amplitude (10–15 nm) sinusoidal perturbations with a 0.3–230 Hzfrequency (in a random order) to scanner Z-electrodes, when thecantilever was at a ≈500 nm indentation. An E*(f) was computedwith correction for the tip-cell contact geometry (the first term of theTaylor expansion of the Hertz model was used) and for the hydrody-namic viscous drag [51]. At least 10 cells of each cell linewere analyzed.



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2.5. Statistical analysis

A non-parametric Mann–Whitney U test was used to determine thestatistically significant differences between the groups. Analysis wasperformed using the Statistica software, version 8.0 (StatSoft, USA).Since the Young's modulus values were distributed log-normally, theywere described as geometric mean */ multiplicative standard deviationand in standard form as arithmetic mean ± SD. N – total number ofcells measured in all experiments. Box-and-whisker plots are shownfor log-normally-distributed data, and the percentiles are 10%, 25%,50%, 75% and 90%.

3. Results

3.1. Morphology, cytoskeleton features, invasiveness and mechanicalproperties of normal and cancer cells

In confocal microscopy experiments the difference in cytoskeletonstructure and morphology of Vero and DU145 cell lines was revealed.Vero cells exhibited an epithelial-likemorphology, the F-actinwas orga-nized into prominent contractile bundles (stress fibers), locatedpredominantly at the basal part of the cell, just above the substrate(Fig. 1a). DU145 cells had a polygonal or elongated shape with a signif-icant number of small filopodia, and F-actin cables and bundles werethinner than in Vero cells. Thickness of F-actin bundles evaluated fromseveral (5–7) confocal images was 0.5 ± 0.1 for Vero and 0.3 ±0.1 μm for DU145 cells, but this is a rather qualitative evaluation, sincethese values are close to the resolution limit of the confocal microscope.Although it might be just the inherent characteristics of these cell lines,

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Fig. 1. Morphology, cytoskeleton features, invasiveness and mechanical properties of normal (images, scale bar 20 μm. Cells were stained with Alexa Fluor® 546-conjugated phalloidin and Dcorresponding images. Vero cells have thicker actin bundles than DU145 cells (wider peaks onstudied cells.


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the significant reduction in the thickness of F-actin cables and bundleswas previously reported for different types of cancer cells [34,35,45,46,52].

To study the invasiveness of normal and cancer cells, we used the 3Dtranswell Matrigel invasion assay (Fig. 1b). As expected, prostate cancercell line DU145 shows significant invasion abilities in comparison toVero cells. Moderate invasiveness of DU145 cells was observed previ-ously and it reflects the metastatic potential of these cells [53].

The Young's modulus measured by force spectroscopy for Vero cellswas 1300± 900 (geometric mean 1000*/1.9,N=324) Pa (Fig. 1c). TheYoung'smoduli of DU145 cellswere significantly (~60%, p b 0.01) lower,with values of 600 ± 400 (450*/2.0, N = 328) Pa, apparently due to aweaker cytoskeleton structure of DU145 cells. All of the studied celllines demonstrated log-normal distribution of Young's modulus valueswith close multiplicative standard deviation values [47].

3.2. Viscoelastic properties of Vero and DU145 cells

The components of the complex Young's modulus (E*) measured atdifferent frequencies for control Vero and cancer DU145 cells are shownin Fig. 2. At low frequencies (below 10 Hz) E′ was at least three timeshigher than E″ for both cell lines, so they demonstrated solid-like behav-ior (loss tangent ~0.3). The frequency dependence of E* of Vero andDU145 cells clearly exhibited a power–law behavior. Both average andsingle cell E*(f) data were fitted well with the power–law structuraldamping model [51,54].

E� fð Þ ¼ E0 1þ iηð Þ ff 0

� �α

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Vero) and prostate cancer (DU145) cells growing in vitro. (a) Actin cytoskeleton, confocalAPI to visualize F-actin and nuclei. Insets – fluorescence intensities across the lines on theline graphs). (b) Invasion of studied cells through the Matrigel. (c) Young's moduli of the



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Fig. 2.Viscoelastic properties of Vero andDU145 cells. A plot of the average Е′ and E″ of Vero andDU145 cells as a function of frequency. Solid lines represent the resultantfit by the power–law structural damping model, Eq. (3Q1 ). Error bars represent the standard deviation.


where η = tan(απ/2) is the hysteresivity or structural damping coeffi-cient of the model, and f0 is a scale factor for frequency (1 Hz in thiswork). DU145 had lower values of the scale factor for storage and lossmoduli E0 (600 ± 200 vs. 1000 ± 300 Pa in control cells) and highervalues of the power–law exponent α (0.18 ± 0.07 versus 0.14 ±0.04), while values of Newtonian viscosity μ were close in both celltypes (5 ± 2 and 7 ± 4 Pa s for DU145 and Vero cells, respectively).Such results correspond well with the data obtained from quasi-staticexperiments (force curves). When applying low indentation speedsduring force spectroscopy experiments, an elastic response is mostlyobtained, and the measured quasi-static Young's modulus for cancercells is lower than for normal cells due to the difference in E0. But athigher indentation speeds an observable decrease in effective Young'smodulus of cancer cells can be diminished due to differences in thepower–law exponent.

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properties of Vero and DU145 cells

As a positive control, we used Cytochalasin D (CytD), which causesthe non-specific inhibition of actin polymerization. CytD is a cell-permeable fungal toxin that affects intracellular actin cytoskeletaldynamics via multiple mechanisms [55]. After several (10–15) min ofCytD treatment (4 μM), almost complete disassembly of actin filamentsand a significant decrease in Young's moduli (80% for DU145, 90% forVero) of both cell lineswere observed (Fig. 3a). Young'smoduli of treat-ed cells measured after 15 min of incubation with CytD were around80–100 Pa. Residual F-actin in cells formed foci and small punctatestructures. The integral fluorescent signal of the labeled F-actin(Fig. 3b) reduced significantly for both cell lines, 65% for Vero and 40%for DU145 (p b 0.01). We did not observe any effects of DMSO (asolvent for actin polymerization inhibitors) addition to the medium oncell mechanical properties and the actin cytoskeleton structure at theworking concentrations (≈0.5% v/v) and times used (15 min–2 h,data not shown).

To study the differences in actin cytoskeleton structure upon formininactivation, the cells were incubated with a small molecule inhibitor offormin-mediated actin assembly, SMIFH2 [49], at a concentration of20 μM for 2 h. After treatment with SMIFH2, the number of thick actinfilaments (Fig. 3a) and integral fluorescent signal of labeled F-actinin the Vero cells decreased (Fig. 3b). The remaining filaments weregenerally located in the basal part of the cells. The Young's moduli ofVero cells were decreased by ~90% (p b 0.01), which is similar to resultsfrom the CytD treatment (Fig. 3c). No significant influence of formininhibitor SMIFH2 on the cytoskeleton morphology has been found inthe prostate cancer cell line DU145 (Fig. 3a). The integral fluorescent


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signal of labeled F-actin (Fig. 3b) and Young'smodulus (Fig. 3c) reducedslightly (~20%, p b 0.01).

The Arp2/3 complex-dependent pathway of actin nucleation wasinhibited using a small molecule inhibitor, CK-666. It is known thatthe Arp2/3-mediated actin assembly takes place on the leading edgeof moving cells [56].We did not observe any effects of Arp2/3 inhibitionwith CK-666 (100 μM, 2 h) on the actin cytoskeleton structure (Fig. 3a,B) and the mechanical properties of cells (Fig. 3c). As shown previouslyfor epithelial cells [57], CK-666mostly affects the quantity and structureof lamellipodia, thus impairing cell motility.

Inhibition of ROCK with Y-27632 (50 μM, 1 h) led to a significantdecrease in Young's moduli of both cell lines (Fig. 3c), which wasmore pronounced for Vero cells (~70% versus ~30% for DU145). ROCKregulates the formation of contractile actin stress fibers [25]. Most ofthick and long actin bundles in Y-27632-treated cells disappeared(Fig. 3a), although a reduction in the integral fluorescent signal of la-beled F-actin was observed only for DU145 cells (~50%, p b 0.01). Cellsoftening after ROCK inhibition could also be associated with decreasein the cortical tension [58,59].

Surprisingly, we observed a different response of Vero and DU145cells to trypsinization (Fig. 3c). After 20 min of trypsinization, cellswere rounded and only slightly attached to the surface. It should benoted, that the application of the simple Hertz model in such measure-ments should lead to relatively good estimations of the cell's Young'smodulus, since a distinct contact area with the substrate exists [60].Detachment leads to the destruction of focal adhesions, intracellular con-tacts and associated stress fibers. Since contractile stress fibers play amajor role in the determination of cell stiffness [45,46], their disassemblyshould lead to cell softening. We were able to observe such an effect forVero cells (reduction ~70%, p b 0.01). In contrast, the cancer cells dem-onstrated a significant increase in Young's moduli (~50%, p b 0.01). Onconfocal images (medium section) the cortical actin layer seemedthicker in DU145 cells (~1 μm versus ~0.7 μm in Vero cells), althoughthis is only a qualitative evaluation due to the limited resolution of theconfocal microscope. The integral fluorescent signal of labeled F-actinwas not calculated for these cells because of the large cell height andcomplicated F-actin distribution in them. The results of all treatmentsare summarized in Table 1. The difference between daily control exper-iments may be caused by subtle uncontrolled experimental factors, likethe difference in medium or FBS composition between batches,confluency level (between 50% and 70% in this work), quality of glasssubstrate, passage number and others.

4. Discussion

Mechanical properties of cancer cells play an important role in themetastatic process, when the activemigration of cells to the neighboring



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Fig. 3. Impact of actin cytoskeleton reorganizations onmechanical properties of Vero and DU145 cells. (a) Cytoskeleton reorganizations in Vero and DU145 cells after different treatments(CytD 4 μM 15 min, SMIFH2 20 μM 2 h, CK-666 100 μM 2 h, Y-27632 50 μM 1 h, trypsinization), confocal images, scale bar 10 μm. (b) F-actin level in the studied cells before and aftertreatments expressed as arbitrary fluorescence intensity. (c) Young's moduli calculated for Vero and DU145 cells before and after treatments.


and distant tissues takes place. These processes require an active changein the structure of the cytoskeleton [61]. It is well known that epithelialcells may undergo a so-called epithelial–mesenchymal transitionduring carcinogenesis [62], at which point intercellular contacts break,cells become more elongated, and can move alone, along with a


mesenchymal–amoeboid transition, which further increases cancercellmigration abilities [63]. These transitions greatly improve the abilityof an individual cell to migrate and invade into the neighboring tissues.Pathways that lead to restructuring of the actin cytoskeleton in cancercells remain largely unknown.



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t1:1 Table 1t1:2 Young'smoduli calculated for the cell lines (in Pa). Data are presented as geometricmeanst1:3 */ multiplicative standard deviation. Data for control cells obtained in the same day aret1:4 provided in brackets. The percent change from the control values is shown.

t1:5 Cell line CytD SMIFH2 CK-666 Y-27632 Trypsinization

t1:6 Vero 80*/1.7 130*/2.5 1200*/1.8 300*/1.6 240*/2.2t1:7 (710*/1.7) (1300*/1.8) (1300*/1.7) (1100*/1.8) (900*/2.2)t1:8 ~90% ~90% ~8% ~70% ~70%t1:9 p b 0.001 p b 0.001 p = 0.44 p b 0.01 p b 0.001t1:10 Du145 70*/1.6 340*/2.2 720*/1.8 250*/1.6 700*/2.3t1:11 (320*/2.1) (440*/1.8) (760*/1.7) (350*/1.7) (480*/1.8)t1:12 ~80% ~23% ~5% ~30% ~50%t1:13 p b 0.001 p b 0.001 p = 0.23 p b 0.01 p b 0.001


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Here we studied the cytoskeleton structure, mechanical propertiesand invasion ability of normal and cancer cells. We compared the Verokidney epithelial cell line with prostate cancer epithelial cell lineDU145 established from the brain metastases [48]. The DU145 cell linepossesses the medium invasive capability in accordance with previousresearch [64] and maintains tumorigenic properties [65], while Verocells normally do not [66]. It should be noted that these cell lines havedifferent tissue origin (kidney and prostate), so we cannot exclude thetissue-specific nature of effects observed in this work. However, asshown here, all common properties, including invasion abilities, cyto-skeleton structure and Young's modulus, which distinguish normaland cancer cells, may be observed in this cell system. Although thegenetic and molecular origin of malignancy is multifaceted, changes inthe mentioned parameters during the progression of disease are com-mon to many forms of cancer [33–35,45,46,52].

Actin polymerization and the formation of an entangled corticalnetwork facilitate cellmigration [67]. These processes lead to the chang-ing stiffness of migrating cells, which can be characterized by theYoung's modulus value. Measurement of the Young's modulus usingAFM has been suggested as a powerful method to study processes thatinvolve cytoskeleton reorganization [29,31,32,37]. In the current work,we used AFM cantilevers modified with microspheres in order toaccurately control the probe geometry and to rapidly obtain Young'smodulus values, averaged over a significant (~1 μm2) contact area, asdescribed earlier [47]. Representative force curves, which were obtain-ed on both cell lines, are shown in Fig. S1, accompanied with the resultsof the Hertz model fitting. Although other models for extraction of me-chanical properties from force curves are emerging [27,28], applicabilityof Hertzian contact mechanics was shown in the majority of work ondifferent cell samples [26,35,45,68]. Here, a good agreement betweenthe Hertz model fits and experimental data was also observed for bothcell lines before and after treatments (adjusted R2 ≈ 0.95–0.99).

The obtained Young's moduli for normal Vero cells were typically1.3 ± 0.9 kPa, which is consistent with the previous results for non-malignant cells [68], but higher than the values reported previously byour group [47]. This is apparently due to the usage of culture mediuminstead of PBS and a higher indentation speed (2 versus 1 μm/s) in thecurrent AFM experiments. DU145 cancer cells exhibited significantlylower Young's moduli (~40% of normal value). The E values obtainedfor DU145 cells differ from those obtained previously by other scientificgroups [32], who used sharp cantilevers for force spectroscopy. Ourresults were systematically lower, probably due to the application ofcantilevers with attached microspheres [69] or differences in passagenumbers. The basic difference in stiffness between normal and cancercells remains the same.

The viscoelastic behavior of Vero and DU145 cells was consistentwith the soft glassy rheology (SGR) model [54]. Accordingly, E*(f) datafitted well with the power–law structural damping model, which isconsidered to be governed by SGR. As expected from the model, softerDU145 cells had a lower value of power–law exponent α than Verocells. Similar results were obtained in the work [27], where an inversecorrelation between G0 (storage modulus scaling factor) and α was


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found for human breast benign and malignant cell lines. Since storagemodulus is much higher than loss modulus at low frequencies, quasi-static force spectroscopy experiments at low indentation speeds pro-vide a quite close estimation of cell Young's modulus. Such experimentswere conducted with Vero and DU145 cells after treatment withdifferent agents causing actin cytoskeleton reorganization.

Total inhibition of actin polymerization by CytD led to a rapid(b15 min) and significant reduction in Young's moduli of both Veroand DU145 cells (Fig. 3b). CytD at the concentration used (4 μM) notonly binds to the barbed ends of actin filaments, thus inhibiting boththe polymerization and depolymerization of actin subunits, but alsobinds to the actin monomers [55]. Although themechanism of CytD ac-tion is still not fully understood, it is probably linked to both interactionswith filament barbed ends and capping proteins, actin monomers andmonomer binding proteins. Except for a decrease in the integral intensi-ty of fluorescently labeled F-actin, CytD treatment modifies cytoskele-ton organization from an isotropic network to focal accumulations(Fig. 3a). It is not surprising that such alterations severely affect themechanical properties of a cell; this was also shown in previous workswith Cytochalasins D or B [46,70,71]. Such a result also proves themajor contribution of the actin cytoskeleton in determiningmechanicalproperties of the studied cells.

The two main actin nucleation pathways in the cell are formin-dependent and Arp2/3-dependent. After treatment with formin inhibi-tor SMIFH2 [49], the Young's moduli of normal Vero cells decreased by70% (Fig. 3b). The opposite effect – an increase in stiffness – was previ-ously shown for fibroblasts after transfection with a constitutivelyactive mutant of formin Dia1, which is in agreement with our data[72]. The actin polymerization inhibitor SMIFH2 acts by binding to thehighly conserved FH2 domain and breaking its interaction with F-actinusing an unknown mechanism [49]. These interactions are so strongand specific that SMIFH2 may displace formin from the barbed end ofthe pre-formed F-actin filaments in the absence of profilin. Consistently,the width and number of actin bundles were reduced in Vero cells(Fig. 3a). The overall intensity of labeled F-actin in treated cells was~40% lower (Fig. 3b).

Earlier it has been demonstrated that carcinoma cell line A549 wasmore resistant to SMIFH2 treatment than benign cells in terms of viabil-ity [49]. Here we showed that treatment of DU145 prostate cancer cellswith SMIFH2 was also ineffective. Unlike Vero, DU145 cell treatmentwith SMIFH2 poorly affects the cytoskeleton, since there were no pro-nounced actin bundles initially, and causes only a slight reduction instiffness (~20%). This observed softening might be partially attributedto cortex weakening. Indeed, formin-dependent actin nucleation wasshown to be important for cell cortex formation [73].

There is an increasing amount of evidence that shows that genesencoding formins are often lost in prostate, breast, hematopoieticand colorectal cancer cells [74–76]. On the other hand, an elevation offormin gene (FMNL2) expression in metastatic cancer cell lines [15]has been reported. Our results suggest that, in the DU145 prostatecancer cell line, formin is actually less active and may not be associatedwith F-actin cables. This is in agreement with recent findings of mDia1and mDia2, associated with cortical actin pools in epithelial cancercells [77].

Inhibition of Arp2/3-dependent polymerization by the small mole-cule CK-666 poorly affected the cytoskeleton structure and mechanicalproperties of the studied cell lines. Recently reported, the crystal struc-ture of the CK-666 bound to the Arp2/3 complex revealed that CK-666binds between the Arp2 and Arp3 subunits to stabilize the inactiveopen conformation of the complex [50]. As shownpreviously for epithe-lial cells [57], treatmentwith CK-666 at high concentrations (100 μm, asused here, and higher) caused the formation of clustered and bundledactin fibers. We did not observe such alterations in the current work.Probably, the cells studied here were more resistant to Arp2/3 in-hibition, like some other cell types [78]. It is worth mentioning thatCK-666 mostly affects the quantity and structure of lamellipodia, thus



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impairing cell motility [57]. Here, we mainly investigated cell bodymechanical properties, and therefore suppose that Arp2/3-inhibitionhas a poor effect on them. As recently shown, the Arp2/3 complexis also important for the generation of cortical actin [79]. However,apparently, inhibition of the Arp2/3 complex alone is not enough forthe cortex weakening.

Rho kinase regulates intracellular mechanics in a different waycompared to actin nucleators. While nucleators increase the total levelof polymerized actin in the cell, the activation of ROCK causes an in-crease in actomyosin contractility. Enhanced actomyosin contractilitymanifested, in particular in stronger stress fibers and focal adhesions,a higher cortical tension. ROCK induces these effects, mainly by phos-phorylating myosin light chains and the assembly of myosin II motors[25]. Contractility affects measured Young's modulus of cells. In general,cells with a higher contractility level should have higher Young'smoduli[80]. As previously shown, the inhibition of ROCK with Y-27632 or my-osin II with blebbistatin led to a decrease in cell stiffness [81–83]. Here,we observed the same effect, which was more pronounced in normalVero cells (~70% versus ~30% in DU145) and, apparently, associatedwith the disassembly of stress fibers. A decrease in cortical tension isalso possible for both cell lines.

Actin cytoskeleton reorganization, caused by cell detachment fromthe substrate by trypsinization, had an opposite impact on Vero andDU145 cells. Vero cells demonstrated a significant reduction in Young'smodulus, apparently, as in previous case, associated with the disassem-bly of stress fibers. At the same time, the Young's modulus of DU145cells increased (Fig. 3b). Initially DU145 had a less developed stressfiber system than Vero cells; therefore, during AFMmeasurements, weexpected the cortical actin to mainly contribute to DU145 cell mechan-ical properties. The actin cytoskeleton structure depends on signalscaught by receptors, which facilitate cell–cell (e.g. cadherins) and cell–substrate contact formation (e.g. integrins in focal adhesion structures).These receptors undergo partial proteolytic degradation at the extracel-lular domains by trypsinization and, as a consequence, the actin cyto-skeleton rearranges, due to the disruption of these signaling cascades.These rearrangements in the actin cytoskeleton of DU145 cells maylead to reinforcement of the cortical actin, increasing cortical tension,and thus in higher stiffness values. Cortical tension is generated by acto-myosin contractility, which is controlled by small GTPase RhoA, ROCKand myosin light chain kinase MLCK [84]. Rac1, another small GTPase,regulates Arp2/3-dependent actin polymerization in lamellipodia ofadherent cells. As demonstrated previously, cortical tension is higherin cells with higher RhoA-ROCK than Rac1 activity, in other words,when contractility dominates over actin polymerization [85]. ElevatedRhoA, ROCK and MLCK expressions have been detected in severaltypes of tumors and cancer cell lines (listed in [25]), which may leadto a rapid switch to activation of RhoA in cancer, but not in normalcells detached from the substrate.

The cortical actin plays a significant role in cancer cell migration andmechanics. Amoeboid-type movement of cancer cells is predominantlygenerated by cortical F-actin rearrangements and cortical actomyosincontractility [63]. Amoeboid cells are naturally representing at leastthe part of the population within DU145 and other aggressive prostatecancer cell lines [75]. Another aspect of cortical mechanics may be con-nected with the viability of cells in the circulation. During metastasis,cancer cells enter the circulation in order to gain access to distanttissues; fluid shear stress arises from hemodynamic shear forces, affect-ing cells in the vessels. Compared to non-transformed cells, cancer cellsare more resistant to fluid shear stress [59], which may be a conse-quence of altered actin cytoskeleton regulation.

The distinct impact of targeted actin cytoskeleton reorganization onnormal and malignant cells provides new information about malignantchanges and can serve as an additional diagnosticmarker. Future exper-iments with optimal control cell lines, cells from patients and compleximpacts of inhibitors or activators should clarify the molecular mecha-nisms of malignancy.


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5. Conclusions

The actin cytoskeleton is rearranged in cancer cells, promotinginvasion and the formation of metastases. Here, we demonstrate thatlarge actin bundles are absent in adherent prostate cancer DU145cells, making them more compliant, compared to normal Vero cells.The frequency dependences of the complex Young's modulus werefitted well with the power–law structural damping model for both celllines; DU145 cells demonstrated higher values of the power–law expo-nent. Cancer cell treatment with specific actin nucleator inhibitors(SMIFH2 and CK666) did not significantly affect their stiffness, whilethe inhibition of Rho kinase decreased it. On the other hand, detach-ment of cancer cells from the surface by trypsinization increased theirstiffness. The data reported here might evidence cortical actin playinga significant role in cancer cell mechanics and migration.

Transparency document

The Transparency document associated with this article can befound, in online version.

Acknowledgments

This work has been partially supported by the RFBR grant 13-04-01570-a, the CRDF-Global grant RUB1-7099-MO-13 (to O.S.) and theOPTEC (2013) grant (to Yu.E. and A.E.). The authors thank Ms. LisaTrifonova for proofreading the manuscript.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbamcr.2015.05.008.

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