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Formation of tethers from spreading cellular aggregates Gregory Beaune, Francoise M Winnik, and Françoise Brochard-Wyart Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b02785 • Publication Date (Web): 28 Oct 2015 Downloaded from http://pubs.acs.org on November 3, 2015

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Formation of tethers from spreading cellular aggregates

Grégory Beaune, † Françoise M. Winnik†, ǁ and Françoise Brochard-Wyart*,‡,§ †WPI International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044 Japan ǁDepartment of Chemistry and Faculty of Pharmacy, University of Montreal, CP 6128 Succursale Centre Ville, Montreal QC H3C3J7, Canada ‡UPMC Univ Paris 06, UMR 168, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France §CNRS, UMR 168, Institut Curie, 26 rue d’Ulm, 75248 Paris Cedex 05, France

Abstract

Membrane tubes are commonly extruded from cells and vesicles when a point-like force is applied on the membrane. We report here the unexpected formation of membrane tubes from Lymph Node Cancer Prostate (LNCaP) cell aggregates in the absence of external applied forces. The spreading of LNCaP aggregates deposited on adhesive glass substrates coated with fibronectin is very limited because cell-cell adhesion is stronger than cell-substrate adhesion. Some cells on the aggregate periphery are very motile, and try to escape from the aggregate, leading to the formation of membrane tubes. Tethered networks and exchange of

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cargos between cells were observed as well. Growth of the tubes is followed by either tube retraction or tube rupture. Hence, even very cohesive cells are successful in escaping aggregates, which may lead to epithelial mesenchymal transition and tumor metastasis. We interpret the dynamics of formation and retraction of tubes in the framework of membrane mechanics.

Introduction For many years, cellular aggregates have been used as model systems for cancer research and embryonic development. They result from the aggregation of a few thousands of cells assembled into spheroids to minimize their surface energy.1 Studies of cellular aggregates are critical to understand the rheology of model tissues, their spreading, and their mechanosensitivity.1,2 Cell aggregates are also reliable and cost-effective systems for drug screening to bridge the gap between cell-based assays and animal studies. They are more relevant to clinical chemotherapy studies, compared to single cells.3,4 They are also promising tools in cell therapy and tissue engineering, as they can serve as building blocks for organ reconstruction.5,6 Cell aggregates are readily are produced in vitro by various techniques, such as the agitation in an orbital shaker,1,7 the pendant drop technique,8,9 the hydrogel micro-well cell-culture method,10 the proliferation of a single cell entrapped in a microscopic capsule,11 or the assembly of cells cultured on a non-adhesive surface.12,13 The dynamics of tissue spreading result from the competition between cell-cell and cellsubstrate adhesion, as suggested by Ryan et al.14 Cell-cell adhesion is mediated by E-cadherin transmembrane proteins present on the cell surface and is characterized by WCC, the cell-cell adhesion energy per unit area. Cell-substrate adhesion is mediated by integrin proteins, with WCS the cell-substrate adhesion energy per unit area. The spreading of cell aggregates or “living droplets” can be conveniently described in the framework of wetting, using the


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analogy between cell aggregates and liquids that form spherical droplets to minimize the surface energy,15–18. The spreading coefficient “S”19 of an aggregate that spreads on a substrate is defined by S = WCS - WCC.7 Thus, if WCS < WCC (S < 0), at equilibrium a cellular aggregate will form a spherical cap with a finite contact angle, which corresponds to partial wetting. If WCS > WCC (S > 0), the aggregate will spread, surrounded by a precursor film consisting of a monolayer of cells, as in the case of complete wetting.7 In vivo, a decrease of cell-cell adhesion allows cells to escape from a primary tumor, leading to the metastatic progression of cancers. In human cancers, depletion of E-cadherin expression correlates to the mesenchymal epithelial transition leading to malignancy. Human prostate cancer cells are known to aggregate easily and to form colonies due to the expression of adhesive proteins similar to E-cadherin.20 The aggregation is believed to be irreversible.21,22 During the initial stages of the aggregation, isolated prostate cancer cells extrude nanotubes, or filopodia, in order to make contact with neighboring cells aggregates.23 We consider here another mechanism which invokes the formation of tubes produced by aggressive cells attempting to escape from an aggregate. In the case of a tumor, this event could lead to the release of individual cells and ultimately to cancer metastasis. Previous invitro studies of the strongly adhesive LNCaP22 prostate cancer cells aggregates have failed to detect the emergence of membrane tubes from the aggregates. Unexpectedly, we observed that the very aggressive LNCaP cells remain surprisingly motile after aggregation. They probed the surface of the substrate using membrane tubes to stay connected with the aggregates and to allow the exchange of cell components. Complex tubes structures like three-junctions and chains of tubes were observed as well. The cells dynamics observed are examined within the theoretical framework of liquid wetting and membrane dynamics and discussed in terms of their relevance to the onset of cancer metastasis.


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Experimental section Materials. Water (18.2 MΩ·cm) was deionized using a Milli-Q water purification system (Millipore). Fibronectin, fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich Co. Trypsin-EDTA, penicillin-streptomycin, and Roswell Park Memorial Institute Medium (RPMI) were obtained from Life Technologies Co. Cell culture and aggregates preparation. LNCaP cells, which are human prostate adenocarcinoma cells, were a generous gift of Dr. L. Xia (WPI International Center for Materials Nanoarchitectonics (MANA), National Institute for Materials Science (NIMS), Japan). Cells were cultured at 37 °C under a 95% air / 5% CO2 atmosphere in culture medium consisting of RPMI supplemented with 10% (vol/vol) Fetal Bovine Serum (FBS) and antibiotics (100 µm.mL-1 streptomycin and 100 U.mL-1 penicillin). Cells were cultured to reach confluence in culture medium. They were dissociated by addition of trypsin-EDTA (0.05 wt % in PBS). They were isolated by centrifugation (300 g, 3 min, room temperature), resuspended in trypsin-EDTA (0.05 wt %), and incubated for 5-10 min. The suspended cells were centrifuged at 300 g for 3 min. The recovered cells were diluted in culture medium to a cell concentration of ~ 2 × 104 cells.mL-1. Subsequently, LNCaP aggregates were prepared by the hanging droplets method,24 whereby droplets (15 µL) of the cell suspension in cell culture medium were deposited on the lid of a Petri dish. The lid was inverted and placed on top of a Petri dish filled with PBS, such that the droplets containing the cells in the medium and hanging from the lid were maintained under a high humidity atmosphere. After a 2-day incubation at 37 °C under a 95% air / 5% CO2 atmosphere, the cells formed aggregates ranging in diameter from 100 to 500 µm. Preparation of coated glass substrates for cellular assays. Circular glass coverslips (diameter: 25 mm) were sonicated in ethanol for 5 min, dried at room temperature, and exposed to deep UV for 5 min. They were coated with fibronectin using a drop of a solution


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of fibronectin in PBS (0.1 mg.mL-1, pH 7.4), squeezed between a parafilm and the coverslip. They were kept for 1 hr at room temperature, rinsed with PBS (pH 7.4), and used immediately. Aggregate spreading assays and optical microscopy. A cylindrical sample cell suitable for use with an inverted optical microscope (Zeiss Axiovert 100 equipped with a ×10 0.45 objective or Leica TIRF AF 6000LX, equipped with a ×10 0.30 objective) was fitted with a fibronectin-coated glass coverslip, which formed the bottom of the sample cell. The microscope cell was filled with CO2-equilibrated culture medium, consisting of RPMI supplemented with 10% (vol/vol) FBS and antibiotics (100 µm.mL-1 streptomycin and 100 U.mL-1 penicillin), maintained at 37 °C using a temperature-controlled platform. LNCaP cell aggregates were placed in the sample cell. Subsequently, the open surface was sealed with mineral oil to prevent water evaporation. Bright field images were taken at various time intervals ranging from 5 to 10 min. Videos were recorded with a CCD camera (Photometrics Cascade 512B, Roper Scientific) at an acquisition rate of 1 frame every 5 min. Images were exported from the instrument software in TIFF format and visualized using the ImageJ software package v.1.46r (National Institutes of Health, Bethesda, MD).

Theoretical treatments Aggregate spreading on a substrate. In the early stages of spreading, the spheroid flattens to form a contact zone of radius r with the substrate. The growth of the contact ( =

) is driven

by a capillary force.25,26 The capillary force per unit length of the contact line is given by = − − cos , where γ, γSO and γSC are the surface energies of, respectively, the tissue, the substrate, and the cell-substrate, and θ is the dynamic contact angle. The deformation of the aggregate is small ( ≅ ) leading to f ≅ − + =WCS using the relationship = + − .As shown in ref 7, the contact growth is described by the 5 ACS Paragon Plus Environment

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balance between the gain of surface energy, 2 , and the viscous dissipation associated to the flattening of an aggregate of radius R0. During the spreading the flow field is imposed by the deformation field as in the case of the spreading of viscous pastes.7 The energy balance

leads to ~ (where η and R0 are, respectively, the viscosity and the initial radius of

the aggregate) and after integration, we obtain: ⁄ = "# ∗ %⁄ & ⁄

(1)

where # ∗ = ' ⁄ is the characteristic velocity and α a numerical coefficient. This law describes the flattening of the spheroids, and holds for both partial and complete wetting. We have neglected the small contribution of cell division because it will not modify the dynamics of the flattening regime. We have shown with other cell types8 that cell division is hindered inside the aggregate because of the lack of free volume and divide at the surface

Membrane tube extrusion. In vitro, tubes can be pulled from cells and vesicles by applying on the membrane an external point line force F using micropipette aspiration27–29 together with optical tweezers,30,31 or wirh hydrodynamic flows.32–35 When using micropipette aspiration, the membrane tension σ is kept constant and is tuned by varying the aspiration pressure. Tubes are extruded from the vesicles by pulling on a small adhesive bead manipulated with optical tweezers. A tube is extruded if the force F is larger than a threshold force ( = 2√2*+, where κ is the curvature modulus of the membrane and σ the membrane tension. The dependence of FC versus σ has been verified experimentally.28 In the second type of experiment, a vesicle is attached to a microstick and submitted to a uniform flow of velocity U. The force acting on the vesicle is the Stokes friction force F = 6πηRU, where R is the vesicle radius. A tube is extruded if U > UC corresponding to F = FC = 6πηRUC. Because there is no reservoir of lipids, the extrusion of the tube leads to an increase of the membrane


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tension.29 The tube is extruded up to a stationary length Lmax, which leads to an increase of σ given by FC(σmax) = 6πηUR. The vesicle behaves like an elastic spring:36 F-FC = K Lmax

(2)

where K = 10-7 N.m-1.33 When tubes are extruded from a cell, rather than a vesicle, the membrane is linked to the cortex by specific protein adhesion, characterized by an adhesion energy W. Since the membrane must be detached from the cortex to form a tube, the force of tube extrusion becomes ( = 2,2*"+ + %.37 The radius of the tube rt is related to FC by the following equation: .

( = 4+ = 2,2*"+ + % = 2

(3)

where κ is the bending rigidity of the membrane of the cell, σ the tension of the membrane, W the membrane-cytoskeleton adhesion energy and rt the tube radius. Note that for cells in nonadhering conditions, W is orders of magnitude larger than σ, thus the increase of σ is masked entirely by W and the tube pulling force is governed by the cortex-membrane adhesion.37,38 In adhering motile cells, such as in keratocytes, σ and W are comparable39 and both contribute in pulling force. For LNCaP cells, an increase of σ with the tube length L is observed, therefore they represent conditions where W is small. Consequently, W will be neglected in the following. Membrane tube dynamics. When a motile cell escapes from an aggregate, a tube forms between the cell and the aggregate if the cell propulsion active force FP is larger than the threshold force FC. Forces of propulsion of cells can be measured by Fourier transform traction cytometry (FTTC) from the displacement of fluorescent particles in a soft substrate stretched by the cells. The largest values of FP reported are on the order of a few nN.40 The dynamics of a tube of length L, characterized by / = 0/ ⁄0& , can be derived from the balance between driving and friction forces associated to the flow of lipids from the cell membrane into the tube: 7 ACS Paragon Plus Environment

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where 233

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(4) (1 − ( = "233 + 45%/ -4 -5 -1 is the effective surface viscosity (~10 to ~ 10 Pa.s.m ) associated with the flow

of lipids,37,41 and k5 is the friction force associated to the slippage of the tethered cell, adhering to the substrate with a contact area 5. The friction coefficient k is on the order of ~ 108 N.s.m-3. 1 The growth of the tube is linear at early times and given by Equation (4) with FC = FC0 (σ = σ0). As the tube grows in length, the excess surface area of the cell decreases and its membrane tension σ increases. This slows down the extrusion of tube, which reaches a maximum length Lmax. The relative area extension ∆A/A is related to the membrane tension σ by:

67 7

89

>

= :;. ln > .36 For a tube length L, a tube radius rt and a cell radius R, we have ?@ =

2/ and @ = 4 , which leads to: ABC

8 9

>

D = :;. ln >

(5)

As L increases, σ increases and the tube growth eventually stops when ( "/EFG % = (1 . 8 H

I

D J Inserting FC into this equation, and assuming W < σ, leads to /EFG = (1 "";.% %ln I , which K

can be written as (1 -( = K Lmax where K = 10-7 N.m-1.33 The extrusion is followed by a tube retraction which corresponds to a passive state of the cell (FP = 0). The force balance equation becomes −( = "233 + 45%/

(6) 8 H

I

D J Using Equation (5) to derive the relationship between FC and L (/ = ( "";.% %ln I ) and K

Equation (6) leads to: C

/ = / L M N

(7) 8 H

I

D where τ, the time of retraction, is given by O = P233 + 45Q "";.% %ln I J . K

Results and discussion 8 ACS Paragon Plus Environment

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Spreading of LNCaP aggregates on fibronectin coated glass substrates. The very strong cohesion among LNCaP cells is reflected by the behavior of their aggregates upon deposition on: As observed in the micrograph shown in Figure 1A, which presents a LNCaP cell aggregate 7 hrs after deposition on a fibronectin coated substrate, LNCaP cell aggregates flatten, but do not spread, due to the stongly cohesive nature of LNCaP cells. Their behavior differs greatly from the spreading of S180 Ecad cells aggregates on the same substrate shown in Figure 1B. which presents a micrograph recorded 7 hrs after deposition of an S180 Ecad cells aggregate used in previous studies.7,42 The S180 Ecad aggregate spreads readily on the substrate forming a precursor film of cells surrounding the aggregates as it spreads. Within the cell aggregate/droplet analogy, LNCaP aggregates exhibit a case of partial wetting, whereas S180 Ecad aggregates undergo complete wetting. In other words, for S180 Ecad cells, the cell/substrate adhesion energy/unit area exceeds the cell/cell adhesion energy per unit area (WCS > WCC), whereas WCC for LNCaP cells is larger than WCS.


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Figure 1. Different wetting regimes observed for LNCap and S180 Ecad cell aggregates spreading on a rigid surface (glass cover slip coated with fibronectin). Experiments are performed with cell aggregates maintained in cell culture medium. Micrographs were recorded 7 hrs after aggregate deposition on the substrate. (A) The LNCaP cell aggregate (S < 0, partial wetting) forms a nearly spherical cap with a finite contact angle θE. (B) The S180 Ecad cell aggregate (S > 0, complete wetting) spreads with the formation of a precursor film of cells around the aggregate (θE = 0). The bottom sketches represent side views of the aggregates. The area covered by the aggregate in panel A is 52,500 µm2. In panel B, the surface area covered by the aggregate is 15,500 µm2 while the area covered by the aggregate and its precursor film is 66,500 µm2.

LNCaP aggregates deposited on the substrate were monitored by optical microscopy for up to 24 hrs following deposition. Their spreading rates were obtained by measuring the aggregate/substrate contact area as a function of time (Figure 2). Areas were normalized by /

to take into account variations in the size of the initial aggregates. The red circles in

Figure 2 represent the average of data collected over time for 12 aggregates. The solid curve is the fit using Equation (1) with V* = 4.8 × 10-9 m.s-1. Contrary to the case of Ecad cell aggregates, even after long spreading times (t > 24h), the precursor film did not appear, which indicates that the spreading coefficient S is negative, i.e. Wcc >Wcs. The low V* value V* implies that the viscosity of the aggregate is large due to the strong cohesion of LNCaP cells.


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Figure 2. Changes with time of the normalized area (A) of LNCaP aggregates. R0 is the initial radius of the aggregates. The circles correspond to experimental data (average values, n = 12) and the solid line is the fit of the data using Equation 1, with V* = 4.8 × 10-9 m.s-1.

Extrusion/retraction of membrane tubes from LNCaP aggregates. a) Observations by optical microscopy. In the course of their spreading, LNCaP aggregates deposited on fibronectin-coated glass substrates were often seen to form membrane tubes on their periphery. Cells scouted the substrate outside of the aggregate with the formation of a filopodia as seen in Figure 3B, t = 11 h). The shape of the cells changed with time. Subsequently, tethered cells started to venture away from the aggregate while remaining partly attached to it (Figure 3B, t = 12:15). Finally, the cells started to migrate on the substrate and continued to scout their surroundings, while remaining attached to the aggregate via a membrane tube (see schematic representation on Figure 3A) that increased in length over time (Figure 3B and 3C).


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Figure 3. (A) Schematic representation of a tube extruded from a motile cell (B, C) Series of optical micrographs showing the formation of membrane tubes between a cell and the cell aggregate recorded for various times after deposition (digits on the top left of the micrographs).


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LNCaP cells spread on the fibronectin coated substrate (WCS >> 0), although they are strongly cohesive (WCC >> 0). As extremely motile cells escape on the aggregate periphery, they tug neighboring cells such that forces are applied on adhesive intercellular cadherin patches, leading to the extrusion of membrane tubes. The formation of membrane tubes was observed in real time by bright field microscopy (Figure 3). Two cells into contact form tubes as they move apart from each other.43,44 Cells are trying to escape from the periphery of immobile aggregates, leading to the formation of membrane tethers that reach lengths L up to 150 μm. The tube pulling force is ~ 15 pN, as estimated from Fp = K Lmax with Lmax = 150 µm and the spring constant K = 10-7 N.m-1.33 Following membrane tube formation, one observes either tube retraction or tube rupture. Sequences of micrographs presenting membrane tubes retraction are presented in Figure 4. As a cell scouts further and further away from the aggregate, it adopts an elongated shape (Figure 4 A, left). The membrane tether linking it to the aggregate is under increasing tension. At this point, the tube length decreases and the cell recovers its spherical shape (Figure 4A, right). The cell is at rest and exerts no active pulling forces. After a few minutes, the retraction is complete, i.e. the cell is again in direct contact with the aggregate. Tube retraction is significantly faster than tube elongation.


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Figure 4. Micrographs of tube retraction sequences monitored for two different cell aggregates (A and B). In both sequences, one end of an extended tube is attached to the aggregate, the other to the cell, which moves towards the aggregate, “swallowing” its own tube until direct contact with the aggregate is achieved. b) Dynamics of membrane tubes formation and retraction. The evolution with time of the length of several tethers formed along the aggregate periphery (see white arrows) is presented in Figure 5A. Over the course of ~ 2 hrs, the tether length increases linearly with time,37 reaches a maximum value, and decreases or eventually ruptures (see below). Extrusion. The changes with time of tube lengths for six spontaneous tube extrusion/retraction processes are plotted in Figure 5A. The corresponding extrusion velocity Ue ranges from Ue ~ 4 to 12 × 10-3 μm.s-1 (Figure 5D) Retraction. A semi-log representation of the changes with time of the tube lengths during the retraction step of the processes in Figure 5A is given in Figure 5B. In all but one case, the membrane tubes retraction kinetics could be fitted using Equation (6), yielding a 14 ACS Paragon Plus Environment

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characteristic retraction time τ ~ 800 s. This time is much longer than the retraction times commonly observed for vesicles retraction (τ ~ 1 to 10 s), but it is typical for tube retractions monitored with cells.36 The slow relaxation rate observed for cells reflects the fact that the friction (ηeff + k5% associated with the extrusion of tubes from cells, where the membrane is attached to the cortex, is much larger than the Stokes friction acting on vesicles. In the case of cells, the value ηeff ranges from 10-4 to 10-8 N.s.m-1. We also noticed that the velocity of retraction is proportional to the length of the tether just before the retraction (Figure 5C). The extrusion force increases linearly with tether length (Equation (2)) and, as a result, the retraction velocity increases. The velocity of extrusion Ue (µm/s), the maximal length Lmax (µm) of tubes before retraction (or rupture), and the velocity of retraction Ur are indicated in Figure 5D. Inserting in Equation (6) the measured retraction velocity of tubes and the friction coefficient derived from τ, we find FP ~ 20 pN. This is very closed to the value previously obtained using Fp = K Lmax. From this value of FP ~ 20 pN and the retraction velocity 12 × 10-3 μm.s-1, we can estimate ηeff ~ 2.10-4 N.s.m-1.


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Figure 5. (A) Temporal evolution of tube lengths during tube formation and retraction. (B) Temporal evolution of tube lengths during the retraction process. Circles correspond to experimental data and solid lines are fits to the data using Equation (6). (C) Representation of the initial velocity of retraction as a function of tether length. Circles correspond to experimental data. The solid line is a guide to the eye. (D) Tube extrusion velocity Ue (left), tube critical length Lmax prior to retraction or rupture (middle), and retraction velocity in the case of rupture of tethers Ur (right) (see below). Blue and red columns correspond to the tubes that retract or break respectively.

Rupture of membrane tube. Sequences of membrane tube ruptures are shown in Figure 6. A tube ruptures if the pulling force is much larger than the force of extrusion. As the tube length increases, the membrane tension increases to a point where the tube ruptures and the tethered cell escapes. The escape of individual cells from the aggregate is the process known


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as epithelial-mesenchymal transition that leads to metastasis. Such a behavior was unexpected for prostate cancer cell lines known to form cohesive tissues. A sequence depicting the rupture of a membrane tube is presented in Figure 6 and in Movie S1 (see SI). As in the case of the retraction process, just before rupture, the membrane tube is under tension and the attached cell adopts an elongated shape. After the rupture, each part of the ruptured tube retracts. The free cell becomes spherical and spreads on the substrate. The changes with time of membrane tube lengths up to the point of rupture is shown in Figure 7 for six rupture events. Growth and rupture of the tubes last about 3-4 hrs. The corresponding velocity of extrusion Ue and critical length Lmax are given in Figure 5D. We note that the tether length at rupture is larger than the length at retraction Lmax (rupture) > Lmax (retraction). The membrane tension increases with the tube length leading to the rupture.

Figure 6. Pictures of a tube rupture sequence, each side of the tube pulling back to its own side (white arrows).


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Figure 7. Temporal evolution of the tube lengths before rupture for six different events. Cargos and Complex structures of tubes. In addition to tube elongation/retraction or elongation/rupture, cargo movement was detected either inside the membrane tubes (Figure 8A) or along their outer surface (Figure 8B). It was reported previously that cell components, such as vesicles or organelles, can be exchanged via tethers, and that bacteria can migrate along the surface of the tubes.45–47 This phenomenon can be explained in the framework of Marangoni flows. If the membrane tensions of two cells connected by a tube are not equal, a flow of membrane from the floppy cell toward the tense cell ensues, leading to an active transport of matter between the two cells.


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Figure 8. (A, B) Visualization of transfer of cargos along tubes (white signs). In most cases, tethers link a single cell to the aggregate. In some instances, several escaping cells are connected by trains of tubes or Y-junctions of tubes. Examples of such patterns are presented in Figure 9. Trains of 2, 3, or 4 cells linked by membrane tubes proceed straight or along a curved line. The trains do not escape from the aggregate: the caboose is always tethered to the aggregate. More complex cell junction patterns are presented in Figure 9B. Typically, three cells are linked. Two of them are tethered to the aggregate. The third cell is further removed from the aggregate, but reined-in by tubes connected to the two cells. Linear trains of cells can reach several hundreds of micrometers in length. Tubes with junction are much shorter, 20 to 30 μm in length, which may be taken as an indication that they are less stable than straight tubes, in agreement with previous reports.44


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Figure 9. (A) Formation of chains of tubes and (B) junctions from cell aggregates. Conclusion We have described the behavior of LNCaP cellular aggregates deposited on adhesive glass substrates. We have interpreted the spreading behavior in the framework of wetting of liquids. Unlike what can be observed with other cell lines, such as Ecad and CT26, the LNCaP aggregates flatten without the formation of a precursor film, which indicates that WCC > WCS. The growth dynamics of the cell/substrate contact area, A~&

T

obeys the same as that

observed for the spreading of a jelly particle.48 It shows that cellular aggregates behave like viscoelastic ultraviscous pastes. But for LNCaP cells the contact is very dynamics: cells try to escape, remaining tethered to the aggregate by long tubes of membrane which can form complex structures (chains of tubes or tubes junctions). This behavior (in Fig 3) is not a generic feature of cell clusters in partial wetting. We have studied previously the spreading of S180 aggregates on non-adhesive substrates7 and we didn’t observe cell escape at the contact 20 ACS Paragon Plus Environment

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line. We conclude that very aggressive cells can escape by tether extrusion. We have described the formation of these tubes and their dynamics in the framework of membrane mechanics. The pulling force of tube extrusion is the cell traction force Fp which vary from cell to cell. Two scenarios occur: (i) for small Fp, the tubes will elongate and reach a plateau value. Then the cell relaxes and Fp goes down to zero. The tube retracts and pulls the cell back toward the aggregate. (ii) For large Fp, the tube elongates to larger values and breaks. The cell escape successfully. This result is important as it indicate that cells can escape from very cohesive tissues characterized by strong cell-cell adhesion. This unexpected observation unveils a new mechanism for tumor proliferation that does not invoke a loss of adhesion (cadherin depletion) and could be observed with other types of very aggressive cells forming cohesive tumors.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +33 (0)1 56 24 67 78

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS LNCaP cells were a generous gift from Dr. Xia Li (WPI International Center for Materials Nanoarchitectonics (NIMS) National Institute for Materials Science (MANA), Japan). This study was supported by NIMS Molecule & Material Synthesis Platform in "Nanotechnology Platform Project" operated by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. REFERENCES

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Table of Contents Graphic


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