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RESEARCH ARTICLE
Fibronectin Modulates Cell Adhesion and
Signaling to Promote Single Cell Migration of
Highly Invasive Oral Squamous Cell
Carcinoma
Grasieli de Oliveira Ramos
1,2
, Lisiane Bernardi
1
, Isabel Lauxen
1
, Manoel Sant’Ana Filho
1,2
,
Alan Rick Horwitz
3
, Marcelo Lazzaron Lamers
1,2,4
*
1Basic Research Center, Dentistry School, Federal University of Rio Grande of Sul, Porto Alegre, Rio
Grande do Sul, Brazil, 2Hospital de Clínicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil,
3Department of Cell Biology, University of Virginia, Charlottesville, Virginia, United States of America,
4Department of Morphological Sciences, Institute of Basic Health Sciences, Federal University of Rio
Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
*marcelo.lamers@ufrgs.br
Abstract
Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins
and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to acto-
myosin organization. During invasion, epithelial-derived tumor cells switch from laminin-
enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this
switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates
the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory
properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (L
inv
/H
E-cad
)
or a highly invasive cell line with low e-cadherin levels (H
inv
/L
E-cad
), plated on different ECM
components. Compared to laminin, fibronectin induced non-directional collective migration
and decreased RhoA activity in L
inv
/H
E-cad
OSCC. For H
inv
/L
E-cad
OSCC, fibronectin
increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migra-
tion in both 2D and 3D environments. Consistent with these observations, human OSCC
biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of
the tumor, where cells encounter fibronectin. Our results indicate that ECM composition
might induce a switch from collective to single cell migration according to tumor invasiveness
due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomy-
osin organization.
Introduction
Oral squamous cell carcinoma (OSCC) is an epithelial neoplasm found in 80–90% of head and
neck cancer [1]. OSCC can occur at several sites of the oral mucosa and is originated from
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 1/18
OPEN ACCESS
Citation: Ramos GdO, Bernardi L, Lauxen I,
Sant’Ana Filho M, Horwitz AR, Lamers ML (2016)
Fibronectin Modulates Cell Adhesion and Signaling to
Promote Single Cell Migration of Highly Invasive Oral
Squamous Cell Carcinoma. PLoS ONE 11(3):
e0151338. doi:10.1371/journal.pone.0151338
Editor: Thomas Abraham, Pennsylvania State
Hershey College of Medicine, UNITED STATES
Received: March 5, 2015
Accepted: February 27, 2016
Published: March 15, 2016
Copyright: © 2016 Ramos et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This work was supported by Coordenação
de Pessoal de Nível Superior (CAPES, Brazil, #9748-
13-0, #202188/2014-0), Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq,
Brazil, #479527/2012-1, #443699/2014-3) and
National Institutes of Health (NIH, GM23244). FIPE/
HCPA - Fundo de Incentivo à Pesquisa e Eventos /
Hospital de Clínicas de Porto Alegre. The funders
had no role in study design, data collection and
genetically altered keratinocytes arising from exposure to a wide range of mutagenic agents [2].
Histopathologically, OSCC lesions are characterized by the presence of different degrees of
squamous differentiation, keratin production, nuclear pleomorphisms, mitotic activity, invasive
growth and metastasis. Despite advances in treatment, the OSCC prognosis remains poor with a
5 year survival rate of around 50%. This prognosis has not improved over the past several years
due to the development of distant metastasis, local recurrences and new tumors [1,3,4].
The ability of tumor cells to invade connective tissue is essential for them to access blood
vessels and ultimately promote distant metastasis. Both events, tissue invasion and metastasis,
are highly heterogeneous processes [5], requiring tumor cell adaptation to new environments
that alter the migratory mode. Depending on the tumor origin, differentiation level, and tumor
microenvironment, cancer cells migrate either as collective or single cells [6]. Amoeboid- and
mesenchymal-like single cell migration involve the coordinated interaction of structural and
signaling molecules that results in polymerization of actin at the leading edge, adhesion to the
extracellular matrix (ECM) through integrins, contraction of the cell cortex and detachment of
adhesions at the cell rear [7,8], whereas cluster or strand like collective cell migration involves
the single cell migration steps associated with the presence of cell-cell contacts, mainly medi-
ated by cadherin family members [6,9]. Rho family GTPases orchestrates changes in actomyo-
sin organization that drive these key events in cell migration. For example, Rac1 regulates actin
filament nucleation associated with nascent adhesion formation, and RhoA controls cell con-
tractility, actin elongation and adhesion maturation [7,10]. Changes in RhoGTPase activation
levels interfere with the balance between cell-cell and cell-ECM adhesions and likely influences
collective vs single cell migration [10–13].
Tumor formation is sensitive to the microenvironment, which varies by the region of the
tumor. The tumor microenvironment is characterized by intense angiogenesis, high concentra-
tions of growth factors and inflammatory cytokines, and ECM remodeling [14,15]. An abrupt
adaptation occurs during invasion of epithelial-derived tumors when they move from the basal
membrane, a laminin enriched environment, to the connective tissue region, which is rich in
collagen and fibronectin [16,17]. Oral squamous cell carcinoma biopsies exhibit decreased
laminin content and increased fibronectin, depending on the aggressiveness and the location of
the tumor [18,19]. It is likely that the characteristics of the tumor microenvironment, such as
the composition of the extracellular matrix, influence metastatic and invasive behavior due to
biochemical or physical activation of migration-related proteins and signaling pathways.
In this study, we report that the change from a laminin- to a fibronectin-rich environment
has a differential effect on the migration properties of OSCCs. In high invasive and low E-cad-
herin expressing OSCC cells (H
inv
/L
E-cad
), fibronectin induced a fast single cell migration phe-
notype that is associated with increased Rac1 activation levels and small cell-ECM adhesions;
in low invasive and high E-cadherin OSCC cells (L
inv
/H
E-cad
), fibronectin produces a collective,
non-directional migration, with high RhoA activity and altered cell-ECM adhesion. Consistent
with these results, human OSCC biopsies also demonstrated changes in cell-ECM and cell-cell
adhesion according to the tumor region. Together, these data show that the composition of the
extracellular matrix differentially affects cell-ECM adhesion, cell migration signaling pathways
and the migratory output of OSCC cells and that these effects vary according to the differentia-
tion level of the tumor.
Material and Methods
Human Biopsies and OSCC Cell Culture
The experimental design and the informed consent procedures were approved by the Ethical
Committee of Federal University of Rio Grande do Sul—Brazil and of Hospital de Clínicas de
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 2/18
analysis, decision to publish, or preparation of the
manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
Porto Alegre—Brazil (CAE#06397313.7.0000.5347) and all patients in this study provided writ-
ten informed consent. Patients (n = 10) with oral lesions were interviewed and submitted to
surgery; OSCC diagnosis was confirmed histopathologically by a pathologist and fragments
from regions corresponding to the center of the tumor and the carcinoma edge tissue, named
as tumor adjacent epithelia (TAE) were collected. OSCC cell lines were obtained from the Tis-
sue Culture Facility at School of Medicine of University of Virginia and checked for myco-
plasma by this facility. Cal27 cells (ATCC
1
CRL-2095™) were cultivated in DMEM high
glucose (Gibco) supplemented with 10% Fetal Bovine Serum (FBS) (Gibco) while SCC25 cells
(ATCC
1
CRL-1628™) in DMEM/F12 with 15mM HEPES and 0.5mM sodium pyruvate
(Gibco) supplemented with FBS 10% and hydrocortisone (400ng/ml, Sigma), and cells were
maintained in incubator (37°C, 5% CO
2
). Cal27 cells are considered low invasive OSCC cells
[20] with high E-cadherin levels (L
inv
/H
E-cad
), while SCC25 cells are highly invasive with low
E-cadherin levels (H
inv
/L
E-cad
). Spheroids were performed plating 5x10
4
cells in a 96 wells dish
covered with 1.5% agarose and, after 3 days, spheroids were gently collected and used for
experiments. For Total Internal Reflectance Fluorescence (TIRF) microscopy, cell lines (1x10
6
)
were nucleofected 24h before the experiment with 0.2μg Paxillin-GFP plasmid [21], using
Amaxa Nucleofection System (Lonza).
Experimental Conditions
Unless stated otherwise, all reagents were purchased from Sigma Aldrich. For 2D imaging
experiments, cells were trypsinized, washed and plated in glass-bottomed dishes covered with
fibronectin (2μg/ml), laminin (poly-l-lysine (1mg/ml) + laminin (2μg/ml)) or Matrigel
1
(50μl/
cm
2
, BD Bioscience) in the presence of CCM1 media (Hyclone, Thermo Scientific). For 3D
imaging experiments, it was used collagen (1.2mg/ml, rat tail collagen) matrices assembled
according to the manufacturer (Gibco) in the presence/absence of fibronectin (10μg/ml) or
laminin (10μg/ml). For each condition, a thin layer of the respective collagen matrix was ini-
tially plated at the surface of the glass-bottomed dishes. After polymerization, 3x10
4
cells or
spheroids were embedded in a new collagen matrix and, after 3h, imaged using CCM1 media.
To ensure that cells were in the 3D matrix, it was verified the lower and the upper focus with
detectable cells and it was always selected cells for imaging at an intermediate focus position.
Immunoblots
Antibodies were purchased from Cell Signaling (E-cadherin, N-cadherin, Integrins α4, α5, αv,
β1, β3), BD-Transduction (Paxillin, FAK) and Sigma (β-Tubulin, Vinculin). Cells (1x10
6
) were
trypsinized, washed and lysed in RIPA Buffer (25mM Tris-HCL pH 7.6, 150mM NaCl, 1% NP-
40, 1% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors cock-
tails. Cell lysates (20μg) were separated in 4–20% SDS Gels (Biorad) and proteins transferred
to PVDF membranes, blocked (4% BSA) and immunoassayed for E-cadherin, FAK, Paxilllin,
β-Tubulin, Vinculin or integrins (α4, α5, αv, β1, β3) using Pierce ECL Western Blotting Sub-
strate (Thermo Scientific). Densitometry of the bands was performed using ImageJ software
(http://rsb.info.nih.gov/ij), and values for each protein were normalized to the loading control.
Immunofluorescence
For tumor staining, human biopsies were fixed immediately after collection (4% formaldehyde,
4h, 4°C), cryoprotected with increasing sucrose concentrations (10–30%, 4°C), embedded in
OCT compound, frozen (-20°C), cut using cryostat and seven μm-thick slices were collected in
gelatin-covered slides. For cell lines staining, L
inv
/H
E-cad
and H
inv
/L
E-cad
were plated in cover-
slips covered with fibronectin (2μg/ml) or poly-l-lysine (1mg/ml) + laminin (2μg/ml) in the
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 3/18
presence of CCM1 media. After 3h, cells were washed (PBS) and fixed (formaldehyde, 4%,
10min, RT). Fixed cells or human biopsies were permeabilized (Triton X-100 0.3%, RT
10 min), blocked (10% normal goat serum, RT, 1h), incubated with antibodies for E-cadherin,
FAK, Paxillin, Vinculin or Fibronectin (ON, 4°C), washed (PBS) and incubated (2h, RT) with
the corresponding secondary antibodies containing Alexa488 dye (Molecular Probes, Oregon,
USA). Actin filaments were stained with phalloidin toxin conjugated to rhodamine (Molecular
Probes, Oregon, USA) for 2h (RT). Samples were washed (PBS) and mounted with antifade
medium (Vectashield, VectorLab, Burlingame, CA). Images were obtained in confocal micro-
scope (Olympus Fluoview 1000, Tokyo, Japan) with a 63x objective (UPlanSApo x63, 1.20 NA,
oil immersion objective) using FV-1000 ASW Fluoview software (Olympus, Tokyo, Japan).
Alexa488 was excited with the 488nm laser line of an Argon ion laser (Melles Griot, Albuquer-
que, NM), while rhodamine with the 543nm laser line of a Helium-Neon laser (Melles Griot,
Albuquerque, NM). Z-stacks were obtained from cells (0.1μm step size) and biopsy slices
(0.5μm step size) with or without digital zoom (3x for cell lines; 5x for biopsies). In order to
analyze the whole adhesion and avoid image background, 3 confocal-obtained slices were
merged using the “Z-stack/maximum projection”tool from the ImageJ software. This new
merged image corresponds to an equivalent 0.3μm or 1.5μm thick slice of the cell lines or the
biopsy samples, respectively. Besides brightness/contrast corrections, no further image editing
was performed and figures were prepared using Adobe Photoshop
1
7 software.
RhoGTPase Activity
For analysis of RhoGTPase activation, pull down assays [22] were performed. L
inv
/H
E-cad
and
H
inv
/L
E-cad
cells were plated in plastic dishes covered with fibronectin (2μg/ml) or poly-l-lysine
(1mg/ml) + laminin (2μg/ml) in the presence of CCM1 media. After 3h, cells were washed
(PBS), harvested, lysed with CRIBs buffer in the presence of protease and phosphatase inhibi-
tors and incubated in the presence of GST-PAK-CRIB (Rac1) or GST-RBD-CRIB (RhoA)
beads. After washing, samples were prepared for SDS-PAGE and submitted to immunoblotting
for Rac1 (BD Bioscience) or RhoA (Santa Cruz Biotechnologies). Densitometry of the bands
was performed using ImageJ software.
FRET Imaging and Analysis
L
inv
/H
E-cad
and H
inv
/L
E-cad
OSCC cells were nucleofected with Raichu-Rac1-WT or Raichu-
Rac1-V12 plasmids for Rac1 activity and Raichu–RhoA-WT or Raichu-RhoA-Q63L plasmids
[23] for RhoA activity (0.5μg/10
6
cells) and 24h later were trypsinized and plated in plastic
dishes covered with fibronectin (2μg/ml) or poly-l-lysine (1mg/ml) + laminin (2μg/ml) in the
presence of CCM1 media. Cells were washed (PBS), fixed in formaldehyde 4% and sacarose 4%
(10 min, RT), washed (PBS) and analyzed by confocal microscopy with 2x digital zoom. Donor
probe was excited with the 458nm laser line of an Argon ion laser (Melles Griot, Albuquerque,
NM). Images were analyzed by Matlab
1
software (MathWorks, Natick, MA) using the Biosen-
sor Processing software 2.1 [24]. The mean intensity values from FRET-ratio TIFF images were
obtained on ImageJ software. Using ImageJ software, selected images were adjusted for the
same levels of brightness/contrast and a 0.5 pixel-wide Gausian filter was applied.
Migration and Adhesion Dynamics Assays
Imaging acquisition and analysis for migration assays were performed as previously described
[25]. For phase microscopy movies, images were captured at 10min intervals using a Nikon
TE300 microscope (10x 0.25 NA CFI Achro DL106 Nikon objective) with a charge coupled
device camera (Orca II, Hamamatsu Photonics) using Metamorph software (Molecular
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 4/18
Devices). For TIRF microscopy, images were taken at 3s intervals using an Olympus IX70
inverted microscope (63x 1.45 NA oil Olympus PlanAplo 660 TIRFM objective) fitted with a
Ludl modular automation controller (Ludl Electronic Products) with a charge-couple device
camera (Retiga Exi, Qimaging) and controlled by Metamorph software. GFP was excited with
the 488nm laser line of an Argon laser (Melles Griot) and a dichroic mirror (HQ485/30) and
an emission filter (HQ525/50) were used. All images and movies were analyzed using ImageJ
software and panels mounted using Adobe Photoshop
1
7 software. For analysis of migration
parameters, it was performed at least 4 independent experiments (phase contrast microscopy
movies) and the nucleus of each migratory cell was tracked using the “manual tracking”plug-
in on ImageJ. It was considered as migratory cell only cells that migrated for at least 6h. In case
of migratory cells that underwent mitosis, the tracking process was ended 1h before cytokine-
sis. To determine migration speed, it was performed the ratio between the total distance trav-
eled (distance) and the number of slices (time) that cell migrated. To analyze the cell trajectory
and persistence of migration, the X and Y coordinates obtained during the tracking of the
nucleus of the migratory cell in each slice were normalized to start at a virtual X = 0 and Y = 0
position and the variation on the position was plotted in a polar plot graph [25]. For analysis of
adhesion properties, it was used H
inv
/L
E-cad
cells expressing paxillin-GFP from at least 4 inde-
pendent experiments (TIRF microscopy) for each experimental group. Adhesion length and
area was determined by measuring, respectively, the long axis or the area of each adhesion that
assembled during the movie. The percentage of total adhesion area in each newly formed pro-
trusion was measured by the ratio of the sum of the area of all adhesions that assembled in the
protrusion by the total area of the protrusion. The adhesion assembly speed was measured
using the “kymograph”plug-in on ImageJ. For each adhesion, a line (1 pixel-wide) was drawn
in the long axis of the adhesion and the X (distance) and Y (time) coordinates originated by the
kymograph were used to measure the speed of adhesion assembly. All data were calculated
using Microsoft Excel
1
(Microsoft Corporation) and SPSS 21 software (Statistical Package for
the Social Science, IBM).
Statistical Analysis
Student t test or One-way analysis of variance (ANOVA) followed by Tukey’s post-test were
employed, using SPSS 21 software and differences were considered significant when p<0.05.
Results
Fibronectin Induces Fast Single Cell Migration of Highly Invasive
OSCCs Cells
Since the extracellular matrix composition can influence the migratory properties of various
cell types, L
inv
/H
E-cad
(Cal27) or H
inv
/L
E-cad
(SCC25) oral squamous cell carcinoma cell lines
[20] were plated on 2D- Matrigel
1
(50μl/cm
2
), laminin (2μg/ml) or fibronectin (2μg/ml)-
coated glass bottomed dishes and imaged for 24h. We tracked the migration velocity of individ-
ual as well as group of cells. Matrigel and laminin were used to mimic the laminin-rich base-
ment membrane that supports cells in an epithelial sheet, whereas fibronectin was used to
challenge the cells with the connective tissue matrix encountered when cells metastasize. On
Matrigel, both cell lines migrated collectively (S1 Movie), while on laminin, both cell types
exhibited collective as well as single cell migration (S2 and S3 Movies). While L
inv
/H
E-cad
cells
showed no changes in migration speed, H
inv
/L
E-cad
cells exhibited a ~40% increase in migration
speed on laminin when compared to Matrigel (Fig 1A). When cells were plated on fibronectin,
both cell types migrated faster than on laminin, and exhibited pronounced changes in
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 5/18
directionality. Both OSCC lines showed a ~40% increase in migration speed; but L
inv
/H
E-cad
cells migrated collectively in circles (S2 Movie), whereas H
inv
/L
E-cad
cells migrated as single
cells with persistent directionality (Fig 1B,S3 Movie).
Fig 1. Fibronectin induces faster migration speed in 2D and 3D substrates. (A) Effects of different 2D
substrates on migration speed (24h) of L
inv
/H
E-cad
(Cal27) or H
inv
/L
E-cad
(SCC25) OSCC cell lines (n = 3);
(B-E) Cell migration trajectory of L
inv
/H
E-cad
(B-C) or H
inv
/L
E-cad
(D-E) cells plated on laminin (B and D) or
fibronectin (C and E); (F) Effects of different 3D substrates on migration speed of L
inv
/H
E-cad
and H
inv
/L
E-cad
cell lines (n = 3). Results are expressed as mean ±SEM. (*)p<0.05 according to One-way analysis of
variance (ANOVA) followed by Tukey’s post-test.
doi:10.1371/journal.pone.0151338.g001
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 6/18
To complement the observations in a 2D environment, L
inv
/H
E-cad
or H
inv
/L
E-cad
OSCC
cells were plated in a 3D matrix, containing collagen (1.2mg/ml), collagen + laminin (1.2mg/
ml+10μg/ml) or collagen + fibronectin (1.2mg/ml+10μg/ml), and imaged for 24h. When com-
pared to a 3D collagen only gel, L
inv
/H
E-cad
cells showed a ~50% increase in migration speed
when plated in 3D collagen gel containing laminin or fibronectin (Fig 1F,S4 Movie) with a
slight increase in directional persistence when plated on collagen+laminin. H
inv
/L
E-cad
tumor
cells showed no changes in migration speed when plated in a collagen+laminin 3D environ-
ment, but were able to invade the collagen gel when plated in collagen+fibronectin matrices
(Fig 1F,S5 Movie). Both cells migrated poorly when plated in a 3D matrix containing only
collagen.
Since OSCC biopsies exhibit tumor islands inside the connective tissue, we developed spher-
oids from both cell lines, plated them in a collagen (1.2mg/ml) or a collagen+fibronectin
(1.2mg/ml+2μg/ml) 3D environment, and imaged for 36h. S6 movie shows that small or big
spheroids derived from L
inv
/H
E-cad
OSCCs proliferated, but showed little migratory activity.
However, spheroids of the H
inv
/L
E-cad
OSCC cells showed cells that migrated out of the spher-
oid and invaded the surrounding tissue only when plated in a collagen+fibronectin 3D
environment.
To summarize, these results in 2D and 3D matrices show that L
inv
/H
E-cad
OSCC cells
migrate more directionally when plated using conditions similar to the epithelial and blood
vessel basal lamina; whereas H
inv
/L
E-cad
tumor cells switch from a collective to a faster single
cell migration when transitioning from a laminin to a fibronectin rich connective tissue-like
environment.
OSCCs Extracellular Matrix-Derived Migration Properties Are
Associated with Changes in RhoGTPase Signaling
A differential activation of RhoGTPase signaling is a likely mechanism for the ECM-derived
differences in cell migration observed in the L
inv
/H
E-cad
and H
inv
/L
E-cad
OSCC cell lines. To
address this, we analyzed the Rac1 and RhoA activation levels by pull down and FRET assay of
cells plated on either laminin (2μg/ml) or fibronectin (2μg/ml) coated dishes. Consistent with
the increased migration speed observed for both cell types on fibronectin, fibronectin increased
Rac1 activation levels when compared to cells plated on laminin, which was accompanied by a
FRET signal mainly at the cell borders (Fig 2A); this effect was slightly, but consistently, more
pronounced in the H
inv
/L
E-cad
tumor cells. In contrast, RhoA activity was observed mainly at
the cell body and showed a decrease in the L
inv
/H
E-cad
cells plated on fibronectin, but was unal-
tered in the H
inv
/L
E-cad
cell line (Fig 2B). This decreased RhoA activity may reflect the fact that
L
inv
/H
E-cad
cells migrate collectively on fibronectin, whereas the H
inv
/L
E-cad
cells migrate as sin-
gle cells, where RhoA is necessary for formation of the contractile cell rear underlying persis-
tent directional migration [26]. These data indicate that the effects of ECM constitution on
tumor invasion process involve a differential activation of RhoGTPases that varies according to
the aggressiveness and differentiation level of the tumor cells.
Extracellular-Matrix Composition Interferes with Tumor Cell Adhesion
Properties
Since L
inv
/H
E-cad
OSCCs migrated collectively, whereas H
inv
/L
E-cad
OSCCs migrated as single
cells, we asked whether these effects correlated with the ratio of cell-cell versus cell-ECM adhe-
sions. We hypothesized that L
inv
/H
E-cad
cell line, which exhibit collective cell migration, would
exhibit increased cell-cell adhesion markers, notably cadherin, whereas highly invasive single
cells would most likely favor cell-ECM adhesions. Consistent with this hypothesis, by western
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 7/18
blotting, we observed that both cell lines expressed integrins for fibronectin, but H
inv
/L
E-cad
OSCCs presented an increase in the expression levels of α5 and β1(Fig 3B) when compared to
L
inv
/H
E-cad
OSCCs. Also, H
inv
/L
E-cad
OSCCs show a slightly increased expression of cell-ECM
adhesion related proteins, including the nascent adhesion marker paxillin, the mechanosensing
modulator vinculin and the adhesion signaling marker focal adhesion kinase (FAK). Addition-
ally, H
inv
/L
E-cad
OSCCs gained expression of N-Cadherin, a marker of the epithelial to mesen-
chymal transition (Fig 3A). These data suggest that epithelial-derived tumor cells show a
differential expression of cell-cell and cell-ECM adhesion markers according to the differentia-
tion levels.
In order to analyze the effects of ECM on cellular distribution of adhesion markers, we
observed by immunofluorescence that L
inv
/H
E-cad
OSCCs showed a localization of E-cadherin
Fig 2. RhoGTPase activation varies according to extracellular matrix composition and tumor differentiation levels. FRET analysis and pull down
assay for Rac1 (A) and RhoA (B) of L
inv
/H
E-cad
(Cal27) or H
inv
/L
E-cad
(SCC25) OSCC plated in laminin (2μg/ml) or fibronectin (2μg/ml). Raichu-Rac1-V12 and
Raichu-RhoA-Q63L represents the constitutively activated isoform. Results are expressed as mean ±SD. (*)p<0.05, n = 4.
doi:10.1371/journal.pone.0151338.g002
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 8/18
preferentially at the cell-cell contacts on both fibronectin and laminin (Fig 4A,S1 Fig) while
the ECM adhesion markers, paxillin, vinculin and FAK, all localized primarily to large, elon-
gated adhesions, as is often observed in slow migratory cells. By contrast, H
inv
/L
E-cad
OSCC
cells displayed mainly cytoplasmic E-cadherin with only weak staining at cell-cell contacts in
both ECM environments (Fig 4B). On laminin, paxillin, vinculin and FAK localized to elon-
gated adhesions similar to L
inv
/H
E-cad
OSCCs. On fibronectin, however, these adhesion mark-
ers preferentially redistributed from large adhesions to small adhesions at the cell border,
consistent with the observed increase in Rac activity and the faster migration speed. To confirm
these effects of ECM composition on the adhesion of H
inv
/L
E-cad
OSCCs, we performed live cell
imaging (TIRF microscopy) of cells expressing the nascent adhesion marker paxillin-GFP in
order to analyze adhesions properties during protrusion (Fig 4C and S7 movie). While there
was no difference in adhesion assembly speed when H
inv
/L
E-cad
OSCCs were plated on fibro-
nectin or laminin, fibronectin decreased adhesion length by ~80% (p 0.001, n = 43 adhe-
sions, Student T test), and similarly decreased both individual adhesion area by ~50%
(p 0.05, n = 221 adhesions, Student T test) as well as total adhesion area relative to protru-
sion area by 30% (p 0.01, n = 17 protrusions, Student T test). These data indicate that the
switch from laminin to fibronectin induces smaller adhesions on H
inv
/L
E-cad
OSCCs cells,
which is consistent with the phenotype of highly migratory cells. Thus, the increased persistent
migration of H
inv
/L
E-cad
OSCCs on fibronectin at least in part reflects a preference for cell-
ECM adhesion, particularly nascent signaling adhesions, rather than cell-cell adhesions.
Fig 3. Decreased cell-cell and increased cell-ECM adhesion proteins characterize invasive OSCC. Representative western blotting images of cell-cell
(E-cadherin, N-cadherin), cell-ECM (paxillin, vinculin and FAK) and integrins (α4, α5, αv, β1 and β3) from L
inv
/H
E-cad
(Cal27) or H
inv
/L
E-cad
(SCC25) OSCC
total cell lysates. Densitometry values for each protein were normalized to the loading control (β-Tubulin).
doi:10.1371/journal.pone.0151338.g003
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 9/18
Fig 4. Fibronectin induces smaller adhesion on low E-cadherin expression OSCC cell line. L
inv
/H
E-cad
(A) or H
inv
/L
E-cad
(B) invasive OSCC were plated
on laminin or fibronectin, fixed and stained for E-cadherin and actin, paxillin, vinculin and FAK. White arrows indicate the signal of E-cadherin between cells.
Scale bar = 20μm. Data regarding adhesion properties (C) were obtained using Total Internal Reflectance Fluorescent microscopy analysis of H
inv
/L
E-cad
OSCC cells expressing paxillin-GFP and plated on laminin (light gray) or fibronectin (dark gray). The data shows the assembly speed (μm/sec), adhesion
area (μm
2
), total adhesion area (as % of total protrusion area) and adhesion length (μm). Results are expressed as mean ±SEM. (*) p = 0.05; (**)p<0.01,
according to Student T—test.
doi:10.1371/journal.pone.0151338.g004
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 10 / 18
Human Oral Squamous Cell Carcinoma Biopsies Show Increased
Levels of Cell-ECM Adhesion Proteins
Since ECM-associated changes on OSCC invasiveness are likely driven by changes in adhesion,
we analyzed the adhesion proteins on biopsies from 10 patients. The sections were stained for
fibronectin, E-cadherin, paxillin, vinculin, and FAK and analyzed using confocal microscopy.
We compared regions from Tumor Adjacent Epithelia (TAE) (Fig 5, columns 1 and 2; S2 and
S3 Figs) with regions at the center of the tumor (Fig 5, columns 3 and 4; S2 and S3 Figs). Fibro-
nectin signal was observed in the connective tissue in both regions (S3 Fig). E-cadherin labeling
in TAE cells was strongest at cell-cell contacts and co-localized with actin; while the center of
the tumor cells showed a weak signal mainly within the cytoplasm, suggesting protein degrada-
tion and/or mislocalization from junctional regions. In TAE regions, paxillin and FAK stained
weakly in puncta through the cytoplasm of the epithelia basal layer and weakly co-localized
with actin; while vinculin was present mainly at the basal membrane. In the center of tumor,
cells that appeared to have detached from the tumor island showed an increase in the staining
of proteins related to cell-ECM adhesion, with paxillin showing increased labeling at the cell
border, close to the ECM, while vinculin and FAK were observed at regions of membrane
extensions of cells at the periphery of the tumor island, with some co-localization with actin.
Thus, human biopsies exhibit changes in the distribution of cell-ECM adhesion proteins, par-
ticularly at the invasive front of the tumor, that correspond with changes observed in cell-ECM
adhesion of H
inv
/L
E-cad
OSCC plated on fibronectin.
Discussion
Clinical failures in cancer therapies are due in part to the plasticity of tumor cells to a changing
microenvironment [14,15]. For example, tumor cells physically and biochemically alter extra-
cellular matrix organization [27], which appears to impact several aspects of the epithelial-to-
mesenchymal transition (EMT) and cell survival [28,29]. Furthermore, the migration of epi-
thelial-derived tumors can vary from collective to single cell migration, reflecting changes in
tumor cell-cell adhesion and the structure of the tissue that the cells are invading [6,30,31]. In
glioblastomas, for example, targeted depletion of fibronectin modifies collective cell migration,
making cancer cells sensitive to ionizing radiation [32].
We have demonstrated that 2D substrates resembling the epithelial basal membrane induce
collective cell migration in OSCC cells independently of the levels of the cell-cell adhesion pro-
tein E-cadherin. In contrast, fibronectin-enriched 2D or 3D environments induced single cell,
mesenchymal-like cell migration but specifically in the H
inv
/L
E-cad
OSCCs. Gaggioli et al
(2007) [17] demonstrated, in a 3D collagen environment, that squamous cell carcinoma cells
showed invasive behavior due to the fibroblast-mediated proteolytic ECM remodeling and
fibronectin deposition. Fibronectin also developmentally regulates migration during embryo-
genesis and determines cell fate [33]. Similarly, fibronectin is overexpressed at the invasive
zone of OSCC biopsies [18,19], where it likely contributes to the abnormal invasive behavior
of poorly differentiated cells [34].
Here we sought to determine how the ECM composition of different tumor regions affects
cell migration and the signaling mechanisms underlying these different migratory properties.
Cell migration is regulated mainly by RhoGTPases, where Rac1 stimulates actin polymerization
and nascent adhesion formation while RhoA controls cell contractility and adhesion maturation
[7,10]. During tumor invasion, the balance of RhoGTPase activation is disrupted. Rac1 activa-
tion results in a loss of cell junctions and polarity and increased cell motility [11,35]. Chen et al
2013 [36] demonstrated that mammary epithelial cells undergo EMT when plated on fibronec-
tin, through a mechanism that involves Rac1b activation, while laminin suppresses EMT.
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 11 / 18
Consistent with this observation, increased Rac1 activity drives mesenchymal-like, single cell
migration of other cancer cells undergoing EMT [6,12]. Yap, et al 2009 [37] demonstrated that
different OSCC cell lines show increased Rac1 activation when plated on fibronectin, indicating
that the microenvironment can influence the tumor invasive behavior through the modulation
Fig 5. Human oral squamous cell carcinoma biopsies show differential distribution of adhesion proteins between center of the tumor cells and
tumor-adjacent epithelia. Regions of biopsies corresponding to the epithelia adjacent to the tumor (A) and from the center of the tumor (B) were submitted
to immunostaining for E-cadherin, paxillin, vinculin or FAK (green) and actin staining (magenta). Inserts demonstrated in actin staining, were digitally
magnified (5x) to show intracellular localization. Representative images from different patients (n = 10), scale bar = 50μmor20μm.
doi:10.1371/journal.pone.0151338.g005
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 12 / 18
of cell migration-related signaling pathways. In this study, when compared to laminin, we
showed that fibronectin induced an increase in Rac1 activation and a rapid single cell migration
phenotype in H
inv
/L
E-cad
OSCCs. Interestingly, while L
inv
/H
E-cad
OSCCs also exhibited
increased Rac1 activation on fibronectin, they had lower RhoA activation. This decreased RhoA
activation may account for the decreased directionality of L
inv
/H
E-cad
OSCCs, which tended to
migrate collectively in circles, since RhoA-mediated myosin activation promotes persistent
directional migration [26]. Thus, differential RhoGTPase activity might contribute to migration
speed and persistence, as well as collective versus single cell migration on different substrates.
In addition to differential RhoGTPase expression, we observed changes in cell-ECM adhe-
sions on different substrates, with fibronectin favoring the formation of small nascent adhe-
sions in H
inv
/L
E-cad
OSCCs [38–40]. A possible explanation for the selective effect of
fibronectin in our study is the higher expression of fibronectin-related integrins observed in
H
inv
/L
E-cad
OSCCs when compared to L
inv
/H
E-cad
cells. Integrins are a family of
Fig 6. Effects of the differential composition of extracellular matrix on cell adhesion and signaling of
Oral Squamous Cell Carcinoma. OSCC with high E-cadherin levels (blue cells) shows collective and single
cell migration in the presence of laminin and collective non-directional migration in fibronectin. This switch
correlated to an increase in Rac1 and a decrease on RhoA activation and modulation of the vinculin levels in
adhesion, induced by the fibronectin-enriched environment. For OSCCs with low E-cadherin levels (orange
cells), fibronectin induced smaller adhesions and increased Rac1 signaling, which correspond to a fast single
cell migration phenotype. This model proposes that the ECM composition can trigger the tumorinvasive
behavior according to differentiation levels of OSCC cells.
doi:10.1371/journal.pone.0151338.g006
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 13 / 18
transmembrane proteins that mediate the binding of ECM proteins with intracellular proteins,
resulting in biochemical and mechanical signaling pathways that influence several steps on
tumor progression [41–44]. Consistent with our results, Chen et al 2012 [45] showed that lami-
nin induced elongated and fluxing adhesions in CHO.K1 cells, while fibronectin induced
smaller and more dynamic adhesions. Similarly, we demonstrated that both, L
inv
/H
E-cad
or
H
inv
/L
E-cad
OSCCs, when plated on laminin, showed large and elongated adhesions probably
due to the epithelial origin of the tumor. However, specifically in H
inv
/L
E-cad
tumor cells, fibro-
nectin induced smaller cell-ECM adhesions with a fast turnover, which reflected in increased
Rac1 activation [46] and faster single cell migration. Thus the ability to metastasize from a lam-
inin to a fibronectin environment might reflect a switch from cadherin-mediated cell-cell adhe-
sions to signaling integrin-ECM adhesions, which promote directional cell migration.
Several reports associate differential activation of adhesion-related proteins with a worse
patient prognosis [47–49]. Also, vulvar squamous cell carcinoma tumors silenced for the fibro-
nectin binding protein, integrin β1, show a more encapsulated and less invasive profile [50]
indicating that cell-ECM interaction is an important player during tumorigenesis. Consistent
with these findings, we demonstrated that human OSCC biopsies show decreased junctional E-
cadherin levels at the center of the tumor when compared to the epithelia adjacent to the
tumor, while the cell-ECM adhesion proteins paxillin, vinculin and FAK showed a differential
distribution in cancer cells at the border of tumor islands close to regions of contact to the
fibronectin enriched ECM. Therefore, our results (Fig 6) show that the extracellular matrix
composition is able to influence the pattern of tumor invasion and metastasis according to the
differentiation level of the tumor cells, probably through modulation of cell signaling and
changes in the balance between cell-cell and cell-ECM adhesion. These data suggest that the
invasive behavior of OSCC not only relies on intrinsic factors (i.e. mutations and abnormal
expression of proteins) but also on extrinsic factors (such as the ECM composition), which
could help to understand the failure of some tumor therapies and contribute to development of
new anti-tumorigenic approaches.
Supporting Information
S1 Fig. Differential distribution of E-cadherin on two different cell lines. L
inv
/H
E-cad
(A) or
H
inv
/L
E-cad
(B) OSCC were plated on laminin or fibronectin, fixed and stained for E-cadherin
and actin. Scale bar = 20μm.
(TIF)
S2 Fig. Distribution of cell-cell and cell-ECM adhesion molecules in OSCC human biopsies.
Original images showed in Fig 5 in the manuscript. Biopsies corresponding to the epithelia
adjacent to the tumor (A) and from the center of the tumor region (B) were submitted to actin
staining (first column) and immunostaining (second column) for E-cadherin, paxillin, vinculin
or FAK. Representative images of n = 10, digital zoom 5x, scale bar = 20μm.
(TIF)
S3 Fig. Distribution of fibronectin in OSCC human biopsies. Regions of biopsies corre-
sponding to the epithelia adjacent to the tumor (A) and from the center of the tumor (B) were
submitted to immunostaining for fibronectin (green) and actin staining (magenta). Represen-
tative images from the same patient (n = 10), scale bar = 50μm.
(TIF)
S1 Movie. Migratory properties of low and highly invasive Oral Squamous Cell Carcinoma
cell lines plated on Matrigel. Time-lapse images (right column) and cell tracking (left column)
of Oral Squamous Cell Carcinoma with H
inv
/L
E-cad
(upper line) or L
inv
/H
E-cad
(lower line)
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 14 / 18
plated for 1h on Matrigel (50μl/cm
2
) and imaged for 24h with a 10min time interval.
(AVI)
S2 Movie. Migratory properties of low invasive Oral Squamous Cell Carcinoma plated in
laminin or fibronectin. Time-lapse images (left column) and cell tracking (right column) of
Oral Squamous Cell Carcinoma with L
inv
/H
E-cad
plated for 1h on laminin (2μg/ml, upper line)
or fibronectin (2μg/ml, lower line) and imaged for 24h with a 10min time interval. This movie
corresponds to Fig 1B and 1C.
(AVI)
S3 Movie. Migratory properties of highly invasive Oral Squamous Cell Carcinoma plated
in laminin or fibronectin. Time-lapse images (left column) and cell tracking (right column) of
H
inv
/L
E-cad
plated for 1h on laminin (2μg/ml, upper line) or fibronectin (2μg/ml, lower line)
and imaged for 24h with a 10min time interval. This movie corresponds to Fig 1D and 1E.
(AVI)
S4 Movie. Migratory properties of low invasive Oral Squamous Cell Carcinoma plated in a
3D matrix. Time-lapse images (left column) and cell tracking (right column) of L
inv
/H
E-cad
OSCC were plated for 1h in a 3D matrix of collagen (1.2mg/ml, upper line), collagen+laminin
(1.2mg/ml+10μg/ml, center line) or collagen+fibronectin (1.2mg/ml+10μg/ml, lower line) and
imaged for 24h with a 10min time interval. This movie corresponds to Fig 1F.
(AVI)
S5 Movie. Migratory properties of highly invasive Oral Squamous Cell Carcinoma plated
in a 3D matrix. Time-lapse images (left column) and cell tracking (right column) of H
inv
/L
E-
cad
OSCC were plated for 1h in a 3D matrix of collagen (1.2mg/ml, upper line), collagen+-
laminin (1.2mg/ml+10μg/ml, center line) or collagen+fibronectin (1.2mg/ml+10μg/ml, lower
line) and imaged for 24h with a 10min time interval. This movie corresponds to Fig 1F.
(AVI)
S6 Movie. Migratory properties of Oral Squamous Cell Carcinoma cell lines-derived spher-
oids in a 3D extracellular matrix. Time-lapse images of spheroids obtained from Oral Squa-
mous Cell Carcinoma with L
inv
/H
E-cad
(left column) plated in a 3D matrix containing
collagen+fibronectin (1.2mg/ml+10μg/ml) or H
inv
/L
E-cad
(center and right column) plated in
a 3D extracellular matrix composed by collagen only (1.2mg/ml, center column) or collagen-
+fibronectin (1.2mg/ml+10μg/ml, right column) and imaged for 36h with a 10min time
interval.
(AVI)
S7 Movie. Extracellular matrix composition affects adhesion dynamics of highly invasive
Oral Squamous Cell Carcinoma. Adhesion dynamics of Oral Squamous Cell Carcinoma with
low E-cadherin levels transfected with paxillin-GFP, plated for 20min on laminin (2μg/ml, left
column) or fibronectin (2μg/ml, right column) and imaged using Total Internal Reflectance
Fluorescent (TIRF) microscopy for 10min with a 3s time interval. The black box represents a
digital zoom of the original movie showing the details of cell adhesion dynamics in each condi-
tion.
(AVI)
Acknowledgments
The authors thank Laura de Campos Hildebrand and Ana Luisa Homem de Carvalho for help
with the human sample biopsy collection and Karen Newell-Litwa for revision of the article.
Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 15 / 18
Author Contributions
Conceived and designed the experiments: MSF AH ML. Performed the experiments: GR LB IL
ML. Analyzed the data: GR LB IL ML. Contributed reagents/materials/analysis tools: MSF AH
ML. Wrote the paper: GR LB IL MSF AH ML.
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Extracellular Matrix Composition Affects Tumor Invasion
PLOS ONE | DOI:10.1371/journal.pone.0151338 March 15, 2016 18 / 18
