1. RESEARCH ARTICLE
Specific upregulation of RHOA and RAC1 in cancer-associated
fibroblasts found at primary tumor and lymph node metastatic
sites in breast cancer
Patricia Bortman Rozenchan1,2,3
& Fatima Solange Pasini1
& Rosimeire A. Roela1
&
Maria Lúcia Hirata Katayama1
& Fiorita Gonzáles Lopes Mundim4
&
Helena Brentani5
& Eduardo C. Lyra6
& Maria Mitzi Brentani1
Received: 2 April 2015 /Accepted: 28 June 2015
# International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract The importance of tumor–stromal cell interactions
in breast tumor progression and invasion is well established.
Here, an evaluation of differential genomic profiles of
carcinoma-associated fibroblasts (CAFs) compared to fibro-
blasts derived from tissues adjacent to fibroadenomas (NAFs)
revealed altered focal adhesion pathways. These data were
validated through confocal assays. To verify the possible role
of fibroblasts in lymph node invasion, we constructed a tissue
microarray consisting of primary breast cancer samples and
corresponding lymph node metastasis and compared the ex-
pression of adhesion markers RhoA and Rac1 in fibroblasts
located at these different locations. Two distinct tissue micro-
arrays were constructed from the stromal component of 43
primary tumors and matched lymph node samples, respective-
ly. Fibroblasts were characterized for their expression of α-
smooth muscle actin (α-SMA) and vimentin. Moreover, we
verified the level of these proteins in the stromal compartment
from normal adjacent tissue and in non-compromised lymph
nodes. Our immunohistochemistry revealed that 59 % of fi-
broblasts associated with primary tumors and 41 % of the
respective metastatic lymph nodes (p=0.271) displayed posi-
tive staining for RhoA. In line with this, 57.1 % of fibroblasts
associated with primary tumors presented Rac1-positive stain-
ing, and the frequency of co-positivity within the lymph nodes
was 42.9 % (p=0.16). Expression of RhoA and Rac1 was
absent in fibroblasts of adjacent normal tissue and in compro-
mised lymph nodes. Based on our findings that no significant
changes were observed between primary and metastatic
lymph nodes, we suggest that fibroblasts are active partici-
pants in the invasion of cancer cells to lymph nodes and sup-
port the hypothesis that metastatic tumor cells continue to
depend on their microenvironment.
Keywords Breast cancer . Tumor microenvironment .
Carcinoma-associated fibroblasts . Rho GTPases . Metastasis
Introduction
Stromal cells, including fibroblasts, endothelial cells, and im-
mune cells, play a critical role in supporting breast cancer
(BC) growth, survival, and invasion. Fibroblasts represent
the major cell type of the stromal compartment and play an
important role in coordinating interactions between stromal
and tumor cells by modulating the composition and function
of the extracellular matrix. Carcinoma-associated fibroblasts
(CAFs) present different characteristics from those of fibro-
blasts found in normal breast tissue (NAFs). CAFs form a
heterogeneous population [1], express alpha-smooth muscle
actin (α-SMA) upon activation [2], and impact BC biological
behaviors [3].
* Patricia Bortman Rozenchan
pbrozenchan@hotmail.com
1
Radiology and Oncology Department, School of Medicine of São
Paulo University, Av. Dr. Arnaldo, 455, sala 4112, São
Paulo, SP CEP 01246-903, Brazil
2
Colsan—Blood Bank Beneficent Association, São Paulo, SP, Brazil
3
Gynecological Department, School of Medicine of São Paulo Federal
University, São Paulo, SP, Brazil
4
Pathology Department, Vale do Sapucaí University, Pouso
Alegre, MG, Brazil
5
Psychiatric Department, School of Medicine of São Paulo University
(USP), Rua Ovideo Pires de Campos S/N, São Paulo, SP, Brazil
6
Brazilian Institute of Cancer Control, Av. Alcântara Machado 2576,
São Paulo, SP CEP 03102-002, Brazil
Tumor Biol.
DOI 10.1007/s13277-015-3727-1

2. The importance of interactions between tumor cells and the
surrounding stromal cells [4] has been well established. In
particular, the migratory/invasive behavior of tumor cells has
been reported to be influenced by bidirectional signals be-
tween cancer cells and tumor-associated stroma fibroblasts
and affects human breast cancer cell adhesion, migration
speed, and direction [5, 6]. During cell invasion, fibroblasts
use contractile force and proteolytic activity to reorganize col-
lagen into linear fibers to generate tracks for migration of
cancer cells [7]. Notably, family members of the Rho-small
guanosine triphosphatases (GTPases), RhoA, RAC1, and
RAC2 binding proteins, have been implicated in CAF-
mediated remodeling of the tumor microenvironment in a
manner that enhances cancer cell invasion [8]. Indeed, RhoA,
Rac1, and Rac2 induce stress fiber formation when
overexpressed in fibroblasts [9].
In the present study, we analyzed genes that were differen-
tially expressed in primary breast CAFs compared to fibro-
blasts that originated from adjacent tissue of benign breast
diseases. This analysis revealed numerous differences in
genes involved in major functional pathways, including focal
adhesion and regulation of actin cytoskeleton and tight junc-
tions. Specifically, the GTPase family members RhoA,
RAC1, and RAC2, as well as collagens and integrins, which
are known to mediate migration and invasiveness, were
shown to be differentially regulated in the context of CAFs.
To date, increased expression of these proteins, particularly
within the epithelial compartment, has been shown to be im-
portant for cellular motility, loss of adhesion, invasion, and
metastasis [10–13].
Nodal status represents one of the most powerful inde-
pendent prognostic indicators of breast carcinoma [14].
Histologically, the presence of fibroblasts in the BC micro-
environment of metastatic lymph nodes further reinforces
the role that CAFs play in tumor growth and dissemination
[15]. Le Bedis et al. [16] suggested that lymph node stroma
plays an active part in the process of lymph node metasta-
sis by creating a dynamic microenvironment that mimics
the environmental conditions present at the primary tumor
site. Previous studies by Garcia et al. [17] reported a sim-
ilar expression of metalloproteases within primary tumors
and the respective lymph nodes. Moreover, tumor–stroma
cross-talk seems to influence the metastatic lymph node
microenvironment, affecting proliferation and migration
of cancer cells [16, 18].
Because the genomic profiles of fibroblasts BC primary
and lymph node metastasis have been reported to be similar
[19], we hypothesized that similar levels of RhoA and Rac1
expression in the tumor microenvironment at the primary site
and at the lymph nodes may represent an advantage to tumor
cell behavior. In this study, we determined whether the expres-
sion of RhoA and Rac1 in stromal fibroblasts of primary tu-
mors was similar to that found in lymph node fibroblasts.
Using a tissue microarray (TMA) consisting of primary BC
samples and the corresponding lymph node metastasis, we
compared the expression level of these markers in fibroblasts
residing at the two locations, as well as in adjacent normal
tissue of primary tumors and in non-compromised lymph
nodes.
Materials and methods
Primary cell culture tissue samples
Malignant and benign breast tissue specimens were obtained
from consenting patients undergoing surgery for breast dis-
ease. Carcinoma samples were obtained from four patients
clinically staged as IIa, and benign samples were obtained
from four patients diagnosed as fibroadenoma. All tissue do-
nors were patients at Instituto Brasileiro de Controle do Cân-
cer, São Paulo, Brazil, a reference center for cancer treatment.
This study was approved by the Ethical Institutional Commit-
tee, and written explicit informed consent was obtained from
all participants. Invasive breast cancer was confirmed
histopathologically.
Primary cell culture
Fibroblasts were obtained from normal adjacent tissue sam-
ples from patients with benign breast diseases (NAF) or
with primary invasive breast cancer tumors (CAF). H&E-
stained, frozen histological sections were prepared from
each tissue sample to confirm benignity or malignancy.
After adipose tissue removal, tissue was minced in
phosphate-buffered saline (PBS), washed twice in PBS
(Na2HPO4 10 mM, NaCl 1.37 mM, KCl 27 mM, KH2PO4
2 mM, Thermo Fisher Scientific Inc., MA, USA) and in
culture medium, and then chopped into small 1- to 4-mm3
pieces under sterile conditions. A total of 15–30 fragments
were transferred to a T25 culture flask (Thermo Fisher Sci-
entific Inc.) and covered with Dulbecco’s modified Eagle’s
medium (DMEM, Thermo Fisher Scientific Inc.) supple-
mented with 20 % FBS (Thermo Fisher Scientific Inc.),
100 μg/mL ampicillin, 100 μg/mL streptomycin, and
2.5 μg/mL fungizone and maintained at 37 °C in a humid-
ified atmosphere containing 5 % CO2. Outgrowth of cells
was recorded after 10 to 20 days, and the medium was
renewed once or twice a week thereafter. After sufficient
outgrowth, the tissue fragments were removed and the cells
were passaged by mild trypsinization with trypsin 0.5 %
(Thermo Fisher Scientific Inc.).
Early passages (passage 3) of all fibroblasts were
subjected to immunocytochemical evaluation. The prolif-
eration rates and description of patients were described
before [20].
Tumor Biol.

3. cDNA microarray assembly, hybridization, and analysis
Total RNA from four NAFs and four CAFs were extracted by
TRIzol reagent (Thermo Fisher Scientific Inc.) and purified
with RNeasy minicolumns and reagents (Qiagen, Hilden, Ger-
many). RNA microarray analysis was performed with 10 μg
of biotin-labelled cRNA target prepared by a linear amplifica-
tion method from a pool of samples. The poly (A)+
RNA
(mRNA) subpopulation within the total RNA population was
primed for reverse transcription by a DNA oligonucleotide
containing the T7 RNA polymerase promoter 5′ to a d (T)24
sequence. After second-strand cDNA synthesis, the cDNA
served as the template for an in vitro transcription (IVT) reac-
tion to produce the target cRNA. This cRNA was hybridized
using CodeLinkTM
Human Whole Genome 55K Bioarray (GE
Healthcare, Buckinghamshire, UK), and the hybridization sig-
nals were normalized using the CodeLinkTM
System Software
Analysis (GE Healthcare) and subjected to a t test with 10,000
permutations. Genes with twofold differential expression
levels in the CAF versus NAF comparison were considered
differentially expressed. The gene ontology (GO) analysis was
performed using the GO Tree Machine tool (GOTM) [21],
which identifies hyper-represented categories in our gene lists
as well as the Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathways. For both GO and KEGG, we used a
hyper-geometric distribution with a p value≤0.05 [Onto-tolls].
Fluorescence microscopy
Cells were cultured on glass coverslips in 24-well plates. After
24 h, cells were fixed with 3.5 % paraformaldehyde in PBS at
room temperature, permeabilized for 5 min with 0.2 % Triton
X-100 in PBS, and blocked with 5 % bovine serum albumin
(BSA, Thermo Fisher Scientific Inc.) in PBS for 1 h. The
primary chicken polyoclonal RhoA antibody (ab23687,
Abcam Antibodies, Cambridge, UK) and mouse monoclonal
Rac1 antibody (ab33186, Abcam Antibodies) were incubated
overnight at 4 °C. After extensive washes and secondary an-
tibody staining (goat anti-mouse IgG, anti-chicken IgG, and
anti-rabbit IgG; Sigma-Aldrich, MO, USA), nuclei were
stained with 4′-6′-diamidino-2-phenylindole (DAPI, Sigma-
Aldrich). Cells were analyzed using a Zeiss 510 META con-
focal laser scanning microscope using a 488-nm argon and a
543-nm HeNe laser. Images were acquired using a Plan
NeoFluoar 40×/1 lens (Microimaging Inc., Lane Cove, New
Zealand).
Immunohistochemistry tissue samples
This study included 43 breast cancer samples that involved
axillary nodes from patients undergoing breast surgery at Hos-
pital Samuel Libânio, in Pouso Alegre, MG, Brazil from 1997
to 2005. This study was approved by the Institutional Ethics
Committee of Hospital Samuel Libânio (no. 1119/09). The
median age of patients was 58 years (range 38–88 years). All
patients were diagnosed with invasive ductal carcinomas at
clinical stages II and III in 37.2 or 62.8 % of cases, respectively
(two unknown). Statuses of the estrogen receptor (ER), pro-
gesterone receptor (PR), HER-2, and histological grade (HG)
are listed in Table 1. HER-2 was assessed by the HercepTestTM
(Dako, Glostrup, Denmark A/S) system. Membrane-based
staining cases with scores 0–1 or chromogenic in situ hybrid-
ization (CISH) negative were categorized as negative, while
cases with score ≥3 were considered as positive. The cases
with an immunohistochemistry score of 2 were further con-
firmed by CISH analysis (Ventana Medical Systems, Inc., a
member of the Roche Group, Tucson, AZ) [22].
Additionally, we included 10 female BC cases (aged be-
tween 39–84 years) in spite of analyzing the stromal compart-
ment of adjacent normal tissue of primary tumors and not
compromised lymph nodes. All samples were subjected to a
pathology analysis, where tumor margins were considered to
assure consistency within the tissues. The Pathology
Reporting of Breast Disease was used as guidelines [23].
Construction of TMA
Two distinct TMAs were constructed from the stromal com-
ponent of primary tumors and lymph node samples,
Table 1 Clinical and pathological parameters of 43 breast cancer
patients
Characteristic No. of patients (%)
Age median (range) 58 (38–88)
Clinical stage
I 1 (2.3)
II 16 (37.2)
III 26 (60.5)
Histologic grade
Grade I 9 (20.9)
Grade II 15 (34.9)
Grade III 19 (44.2)
ER—primary tumor
Negative 18 (41.5)
Positive 24 (55.8)
Missing 1 (2.3)
PR—primary tumor
Negative 17 (39.5)
Positive 21 (48.8)
Missing 5 (11.6)
HER-2
Negative 33 (76.7)
Positive 9 (20.9)
Missing 1 (2.3)
Tumor Biol.

4. respectively. Representative areas of each component were
surrounded by a marking pen at the donor blocks and collect-
ed using the Manual Tissue Arrayer I (Beecher Instruments
Inc., Sun Prairie, USA). Samples were orderly arranged in a
grid, and the first core was represented by a fragment or nor-
mal liver used as a reference in both TMAs. The first TMA
was built with samples of stromal component of the tumors in
order to enable the assessment of fibroblast cells within the
desmoplastic contingent of the carcinomas.
The second TMA consisted of 43 majorly compromised
lymph node samples from cases with axillary lymph node
metastasis. Because the width of the metastatic element at
the lymph nodes was frequently limited to 0.5 to 1.0 cm, it
was seldom possible to separate distinct areas of stromal and
epithelial elements. The chosen areas at the donor block in-
volved stromal and epithelial elements, which were further
evaluated separately.
From each of the two TMAs, 3-μm-thick slices were ob-
tained and collected on slides with special adhesives
(Instrumedics Inc., NJ, USA). Two sets of triplets, separated
by a gap of 40 cuts, were submitted for immunohistochemical
analyses in order to represent two levels of the same sample.
Immunohistochemistry
The immunohistochemical reactions were performed using
the complex streptavidin-biotin peroxidase (StreptABC, Dako
Corporation, Glostrup, Denmark). After deparaffinization of
tissue sections, antigen retrieval was performed using a pres-
sure cooker in citrate buffer pH 6.0, followed by blocking
endogenous peroxidase with hydrogen peroxide solution
(3 %). The sections were incubated with primary antibodies,
Rac1 (1:1,000; ab33186, Abcam) and RhoA (1:175; ab23687,
Abcam), vimentin (1:2,000; clone v9, Dako Corporation), and
α-SMA (clone: HHF-35; Cell Marque, CA, USA). After in-
cubation with primary antibody and primary blocking, a poly-
mer–peroxidase (Novolink, Leica, Wetzlar, Germany) ampli-
fication step was performed. Antigen detection was carried
out in a solution containing 3,3-diaminobenzidine (Sigma-
Aldrich) and 6 % H2O2. Counterstaining was performed with
Harris hematoxylin (Merck, NJ, USA). Carcinoma ductal in-
vasive of breast was used as positive control for RhoA, and
large intestine adenocarcimona was the positive control used
for Rac1. Negative controls were performed removing first
antibodies.
Evaluation of immunohistochemical essays
All reactions were assessed by two independent blinded ob-
servers. Disparities between the two pathologists were
reevaluated by consensus. Results from the epithelial compo-
nent and stromal component were reported separately either to
carcinomas or to lymph node samples.
The presence of RhoA and Rac1 staining was assessed in
the stromal population and separately in primary tumors or in
lymph node metastases. In relation to the immunohistochem-
ical result evaluation, RhoA was scored according to the
Allred system [24]. Samples with a score above 4 were con-
sidered positive. For Rac1 evaluation, the percentage of pos-
itive cells was assessed and classified into three groups: (1)
0 %, (2) 1−33 %, and (3) ≥34 %. Samples with greater than
10 % of cells that were positive for α-SMA and vimentin were
classified as positive. For all cases, cells presented in 10 fields
at a magnification of ×400 were counted.
Statistical methods
Correlations between categorical antigen expression and other
clinicopathological parameters were studied with the Fisher’s
exact test or chi-square test, where appropriate. Spearman’s
rank correlation coefficient was calculated to assess the rela-
tionships involving categorical antigen expression. All statis-
tical tests were two-sided, with significance defined as
p<0.05. Analyses were performed using the software SPSS
version 10.0 for Windows (SPSS Inc., IL, USA).
Results
First, we evaluated the gene expression profile of stromal fi-
broblasts derived from four benign (NAF) and four malignant
(CAF) breast tissues using the CodeLinkTM
Human Whole
Genome 55K Bioarray. Using a twofold cutoff, we found 1,
111 genes differentially expressed in CAFs compared to
NAFs. To further examine the biological functions of these
genes, an analysis of the KEGG database revealed differential
expression of several pathways, including Boxidative phos-
phorylation,^ “focal adhesion,” BMAPK signaling pathway,^
Bleukocyte transendothelial migration,^ Bregulation of actin
cytoskeleton,^ and Btight junctions^ (Fig. 1). Interestingly,
all of these pathways are involved in important processes like
cell/cell communication, migration, and invasiveness. Focal
adhesion was ranked the second top significant pathway, and
in microarray analysis, we found that both RhoA and Rac1
were upregulated in CAFs compared to NAFs.
Using confocal assays, we confirmed that protein levels of
RhoA and Rac1 were elevated in carcinoma-associated fibro-
blasts (Fig. 2). Strong RhoA staining was localized to the
cytoplasm, while Rac1 staining was associated with the plas-
ma membrane.
To further examine correlations between altered genes
found in the microarray data with patterns of protein expres-
sion, we analyzed the expression of RhoA and Rac1 in the
stromal component of 43 samples of primary breast carcinoma
and metastatic ipsilateral axillary lymph nodes by immunohis-
tochemistry. In a few metastatic lesions cases, it was not
Tumor Biol.

5. Fig. 1 Distribution of
differentially expressed genes in
CAFs. Representative pathways
were identified for genes
differentially expressed (black
bars) between CAFs and NAFs in
our cDNA microarray platform
(gray bars). The gene ontology
(GO) analysis was performed
using the GO Tree Machine tool
(GOTM), which identifies hyper-
represented categories in our gene
lists as well as the Kyoto
Encyclopedia of Genes and
Genomes (KEGG) pathways. For
both GO and KEGG, we used a
hyper-geometric distribution with
a p value≤0.05 [Onto-tolls]
Fig. 2 CAF-specific staining of
RhoA and Rac1. CAF (a, c) and
NAF (b, d) confocal
photomicrographs of typical
fields are shown. Merged images
of a cytoplasmatic staining of
RhoA (a, b) and cytoplasmatic
membrane staining of Rac1 (c, d)
validated the gene expression
analysis; Rac1 and RhoA were
upregulated in CAFs
Tumor Biol.

6. possible to count the preferred number of cells. These proteins
were also analyzed in fibroblasts of adjacent normal tissue of
primary tumors and in non-compromised lymph nodes of 10
breast cancer cases.
To distinguish fibroblasts (CAFs) from tumoral cells, we
evaluated two fibroblast cell markers: vimentin and α-SMA.
Vimentin staining was observed in 37/43 (86.0 %) of fibro-
blasts of primary tumors and in 35/41 (85.4 %) of lymph nodal
fibroblasts. α-SMA staining was observed in 51.2 % of pri-
mary lesions and in 41 % of nodal metastatic lesions (Fig. 3).
Positive RhoA staining in stromal cells was seen in 36/43
cases of primary tumors (59 %) and in 25/35 (41 %) of respec-
tive metastatic lymph nodes (p=0.271). Similarly, Rac1 stain-
ing in stromal cells was observed in 28/39 cases of primary
tumors (57.1 %), whereas the frequency of Rac1 staining in
stromal cells found in the lymph nodes was 21/38 (42.9 %)
(Table 2). In summary, the status of RhoA and Rac1 was
similar between primaries and lymph nodes (Fig. 4), and all
markers were detected in the cytoplasm. Importantly, RhoA or
Rac1 expression was not detected in fibroblasts of adjacent
normal tissue or in non-compromised lymph nodes (Fig. 5),
suggesting that the presence of RhoA or Rac1 in CAFs may
help facilitate tumorigenesis.
Discussion
A large amount of data have shown that the microenvironment
is important to breast carcinoma epithelial cells [25–29]. Ad-
ditionally, we have shown that reciprocal changes in gene
expression profiles of important cellular function pathways
occur when breast epithelial cells were co-cultured with
primary fibroblasts, reinforcing the idea that interactions be-
tween these two cell types impact cell signaling and behavior
[20]. In line with these works, we compared gene expression
data generated in CAFs and NAFs to show that a large number
of genes present in the focal adhesion pathways, including the
GTPAses RhoA and Rac1, were differentially expressed in
CAFs.
Cell motility and invasiveness require cytoskeleton reorga-
nization, which involves formation of the filopodia/
lamellipodia and changes in focal adhesion complexes. Rho
proteins are involved in stress fiber formation and focal adhe-
sion, while Rac proteins stimulate lamellipodia and
membrane-ruffle formation [30–33]. Increasing evidence sug-
gests that the RhoA and Rac1 proteins play an important role
in cell migration, loss of adhesion, invasion, and metastasis in
tumors [10, 34–37]. Building on these studies, we used im-
munohistochemistry to investigate whether the RhoA and
Rac1 expression patterns were similar in fibroblasts of prima-
ry BC tumors and matched lymph node metastases.
Fig. 3 CAF-specific α-SMA and
vimentin staining at primary at
metastatic tumor sites. α-SMA (a,
b) and vimentin (c, d) staining in
CAFs in the primary tumors (a, c)
and in their counterpart lymph
node metastasis (b, d). Original
magnification ×400
Table 2 Correlation among the proportion of biological marker
expression in stromal tissue between the primary tumors and lymph
node metastasis of 43 breast cancer patients
Variable Primary tumor Lymph node p
RhoA
Negative 7 (41.2) 10 (58.8) 0.27
Positive 36 (59.0) 25 (41.0)
Rac1
Negative 11 (39.3) 17 (60.7) 0.16
Positive 28 (57.1) 21 (42.9)
Stroma tissue: only fibroblast cells were analyzed; Values of p (two-sided)
less than 0.05 were considered significant
Tumor Biol.

7. Early studies noted that RhoA overexpression result-
ed in mouse fibroblast transformation [38] and stress
fiber formation [39]. It was previously shown that high-
ly metastatic mesenchymal sarcoma cells primarily use
an ameboid mode of cell invasion that depends on the
activity of the Rho family of GTPases [40]. Some au-
thors have described the role of Rho GTPases in fibro-
blasts [41–43]. Verghese et al. [41] reported that cyto-
skeletal regulation by Rho GTPases in breast fibroblasts
enhanced migration and invasion in consequence of mir-
26b dysregulation.
In agreement with Halon et al. [10], we show that a high
proportion of cells in the BC tumor–stromal compartment
were positive for RhoA and Rac1. In addition, we have shown
that activated fibroblasts, as identified by α-SMA, were pres-
ent in the majority of metastatic lymph node cases, whereas
non-involved lymph nodes were devoid of myofibroblasts.
The epithelial-to-mesenchymal transition (EMT) repre-
sents one possible mechanism by which a cancer cell metas-
tasizes and may facilitate the re-localization of fibroblasts to
the lymph node. Past work has emphasized RhoA as a medi-
ator of this process via integrin β1/TGF β activation [44].
Fig. 4 RhoA and Rac1 staining
in primaries and in lymph node
metastasis. Representative cases
depicting RhoA and Rac1 (a, c) in
CAFs in the primary tumors and
in the respective lymph node
metastasis (b, d). Original
magnification ×400
Fig. 5 Lack of RhoA and Rac1
staining in fibroblasts of adjacent
normal tissue and in non-
compromised lymph nodes.
Negative expression of RhoA and
Rac1 in fibroblasts of adjacent
normal tissue (a, c) and non-
compromised lymph nodes (b, d).
Original magnification ×400
Tumor Biol.

8. Moreover, EMT is a recognized source of CAFs, as it pro-
duces myofibroblast cells with enhanced migratory capacity,
invasiveness, and increased expression of ECM proteins [45].
In line with these data, we found that 41 % of fibroblasts of
lymph nodes origin were positive for RhoA and α-SMA.
CAFs may also arrive at lymph nodes in co-migration with
tumor cells, referred to as the collective pathway of invasion
[46]. Reorganization of collagen fibers by mammary fibro-
blasts can create avenues for invasion [5], and the Rho-
family of GTP-binding proteins may regulate fibroblast-
mediated collagen reorganization [8]. Therefore, RhoA and
Rac1 may be involved in both pathways.
Our study brings some clarification regarding the role of
the microenvironment in lymph node metastasis in BC. How-
ever, we acknowledge that our study is limited by the number
of samples used in immunohistochemical assays, as well as by
the presence of non-distinct histological types, as different
subtypes represent different fibroblasts populations [47].
In conclusion, we demonstrate positive expression of
RhoA and Rac1 in CAFs in paired primary breast cancer
and in the respective lymph node metastasis, suggesting that
a similar microenvironment may be present at both sites. Im-
portantly, as we were unable to detect positive expression of
these proteins in fibroblasts of normal adjacent tissue or in
non-committed lymph nodes, our findings may highlight the
stroma as an active participant in the metastatic process and
suggest that metastatic tumor cells may continue to be depen-
dent on their supportive microenvironment.
Acknowledgments The authors are grateful to Ana Lúcia Garippo for
her technical assistance in confocal microscopy. This research was sup-
ported by Fundação de Amparo à Pesquisa no Estado de São Paulo
(FAPESP) 01/13513-1, 05/51593-5, 04/04607-8, 05/60333-7, 2014/
03090-3 and 09/10088-7 and Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq).
Conflicts of interest None
Ethical approval All procedures performed in studies involving hu-
man participants were in accordance with the ethical standards of the
institutional and/or national research committee and with the 1964 Hel-
sinki declaration and its later amendments or comparable ethical
standards.
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