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Archives of Medical Research 44 (2013) 208e214
ORIGINAL ARTICLE
Elevated Concentration of Microvesicles Isolated from PeripheralBlood in Breast Cancer Patients
Octavio Galindo-Hernandez,a,* Socrates Villegas-Comonfort,a,* Fernando Candanedo,a
Mar�ıa-Cristina Gonz�alez-V�azquez,a Sonia Chavez-Oca~na,b Xicotencatl Jimenez-Villanueva,c
Monica Sierra-Martinez,b and Eduardo Perez Salazara
aDepartamento de Biologia Celular, CINVESTAV-IPN, Mexico D.F., MexicobLaboratorio de Genetica y Diagnostico Molecular, Unidad de Investigacion, cServicio de Oncologia, Hospital Juarez de Mexico, Mexico D.F., Mexico
Received for publication August 27, 2012; accepted February 28, 2013 (ARCMED-D-13-00254).
*These authors con
Address reprint re
Biologia Celular, Cin
Mexico; Phone: (þ5
E-mail: [email protected]
0188-4409/$ - see frohttp://dx.doi.org/10
Background and Aims. Breast cancer is the most common cancer and the main cause ofcancer deaths in women worldwide. Microvesicles (MVs) are fragments of the plasmamembrane secreted from cytoplasmic membrane compartments by normal and malignantcells. An increase in MV number has been found in peripheral blood of patients withseveral diseases including cancer. We hypothesized that MV number and the relativeamount of focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR)proteins in plasma fractions enriched in MVs and deprived of platelet-derived MVs arerelated to the presence of breast cancer.
Methods. Plasma fractions enriched in MVs and deprived of platelet-derived MVs wereobtained by differential centrifugation of blood samples. MV number was evaluated byBD TruCOUNT Tubes (BD Biosciences). FAK and EGFR proteins were analyzed byWestern blot.
Results. MV number in plasma fractions enriched with MVs and deprived of platelet-derived MVs is higher in breast cancer patients with stages IeIV as well as withT2eT4 tumors, in comparison to control group. In addition, plasma fractions enrichedin MVs present FAK and EGFR proteins and their amount is increased in some stagesof breast cancer in comparison to control group.
Conclusions. Our findings strongly suggest that MV number and the amount of FAK andEGFR in plasma fractions enriched in MVs are associated with some stages of breastcancer. � 2013 IMSS. Published by Elsevier Inc.
Key Words: Breast cancer, Microvesicles, EGFR, FAK.
Introduction
Breast cancer is the most common diagnosed cancer andthe main cause of cancer death in women worldwide,accounting for 23% (1.38 million) of the total new cancercases and 14% (458, 400) of the total cancer deaths in2008. Moreover, incidence of breast cancer is highest inWesternized high income countries but has been increasingin low- and middle-income countries including Mexico.
tributed equally to this work.
quests to: Eduardo Perez Salazar, Departamento de
vestav-IPN, Av. IPN #2508, 07360 M�exico, D.F.,
2) (55) 5747-3991; FAX: (þ52) (55) 5747-3393;
investav.mx
nt matter. Copyright � 2013 IMSS. Published by Elsevier.1016/j.arcmed.2013.03.002
The increase in breast cancer may be the result of familyhistory, hormone replacement therapy, body and abdominalmass, alcohol consumption and reproductive patternsincluding early menarche, late menopause, null parity andlate age at first delivery (1e3).
Microvesicles (MVs) are fragments of the plasmamembrane secreted from cytoplasmic membrane compart-ments by normal and malignant cells and their functionare dependent on cargo that they carry and the cell typewhich they originate (4). Exogenous stimuli includingepinephrine, adenosine diphosphate, collagen, calciumionophore (A23187) and amphiphiles induce MVs releasein several cell types (5e7). Moreover, detachment of breasttumor cells induces secretion of exosomes, as well as MVs
Inc.
209Microvesicles in Breast Cancer Patients
can also be shed from blood cells during their isolation(8,9). The MVs are small in size, expose phosphatidylserineon the outer leaflet of their membrane, present antigens re-flecting their cell origin, and are divided by size and origininto two groups: exosomes and microparticles. Exosomesare the smaller vesicles (30e100 nm), more homogeneousin size and released by endosomal compartments, whereasmicroparticles are larger vesicles (0.1e1 mM) and releasedfrom surface membranes during membrane blebbingthrough a calcium-dependent mode (10,11).
An increased number ofMVs has been found in peripheralblood of patients with several diseases including cardiovas-cular disorders, infections, autoimmune diseases and cancer(12e14). Particularly, tumor-derived MVs (TMVs) presentbioactive molecules including microRNAs, mRNAs andproteins. It has been suggested that these TMVs facilitateextracellular matrix invasion and immune response evasionas well as demonstrating uptake by normal cells (14e17).
In the present study our findings show that MV numberin plasma fractions enriched in MVs and deprived ofplatelet-derived MVs is higher in breast cancer patients instages IeIV as well as with T2eT4 tumors in comparisonto control group. In addition, we demonstrate that plasmafractions enriched in MVs present FAK and EGFR proteinsand their amount is increased in certain stages of breastcancer in comparison to the control group.
Materials and Methods
Materials
Focal adhesion kinase (FAK) antibody (Ab) C-20,epidermal growth factor receptor (EGFR) Ab 1005, majorhistocompatibility complex class I (MHC-I) Ab BRA23/9and CD9 Ab C-4 were obtained from Santa Cruz Biotech-nology (Santa Cruz, CA). Flotillin-2 Ab and TruCOUNTtubes were obtained from BD Biosciences (San Jose, CA).
Patients
Fifty female patients (median age 55.3 years, range 41e84years) with biopsy-proven breast cancer at different clinicalstages and without receiving therapy were studied. Thecontrol group consisted of 31 healthy females (medianage 42.7 years, range 16e86 years). All study participantssigned informed consent, and the ethics committee ofHospital Juarez de Mexico approved the protocol.
Blood Samples and Isolation of Plasma FractionsEnriched in MVs and Deprived of Platelet-derived MVs
Peripheral blood was drawn into polypropylene tubes con-taining sodium citrate (Vacutainer System, BD Biosci-ences). Plasma fractions enriched in MVs and deprived ofplatelet-derived MVs were obtained as described previously
with some modifications (18). Briefly, whole blood sampleswere centrifuged for 15 min at 1500 g, and plasmas werecollected and centrifuged for an additional 30 min at3000 g. Next, plasma fractions were carefully aspiratedfrom the pellet and centrifuged at 15,000 g for 30 min,and these fractions were collected, aliquoted and keptfrozen at �80�C until analysis.
MV aliquots were thawed on ice and centrifuged at17,570 g for 30 min. Pellets were reconstituted in 8 mlphosphate buffered saline (PBS)/10.9 mM sodium citratesolution and centrifuged at 100,000 g for 2 h. The newpellets were reconstituted in 75 ml PBS/sodium citrate solu-tion. These plasma fractions enriched in MVs and deprivedof platelet-derived MVs were used to determine the MVnumber and Western blot assays.
Determination of Absolute Number of MVs
Determination of absolute counts of MVs was performedby flow cytometry with the use of BD TruCOUNT Tubes(BD Biosciences) as described previously (19,20). Absolutenumbers of MVs were calculated according to the formula:(number of events in region containing MVs/number ofevents in absolute count bead region) � (number of beadsper test/test volume) 5 Absolute count of MVs.
Absolute Count of MVs
Western blotting. Equal volumes of plasma fractions en-riched in MVs were separated by SDS-PAGE using 10%separated gels followed by transfer to nitrocellulosemembranes. After transfer, membranes were blocked using5% nonfat dried milk in PBS, pH 7.2/0.1% Tween 20 (washbuffer) and incubated overnight at 4�C with primary Ab.The membranes were washed three times with wash bufferand incubated with secondary Ab (horseradish peroxidase-conjugated) (1:5000) for 2 h at 22�C. After washing threetimes with wash buffer, the immunoreactive bands werevisualized using ECL detection reagent. Autoradiogramswere scanned and the labeled bands were quantified usingthe Image J software (NIH, Bethesda, MD).
Statistical Analysis
Statistical analysis was performed by nonparametricMann-Whitney test in all experiments using the GraphPadPrism 5 software. Differences were considered significantat p !0.05.
Results
Number of MVs in Plasma of Breast Cancer Patients
Plasma fractions enriched in MVs and deprived of platelet-derived MVs were obtained by using differential centrifu-gation of blood samples. To substantiate that those fractions
210 Galindo-Hernandez et al./ Archives of Medical Research 44 (2013) 208e214
were enriched in MVs, we determined whether the MVfractions were enriched in CD9, flotillin-2 and MHC-Ibecause these proteins are molecular markers associatedwith the different classes of MVs (4,14,21). Plasma frac-tions enriched in MVs were analyzed by Western blottingusing anti-CD9, anti-flotillin-2 and anti-MHC-I Ab. Asillustrated in Figure 1A, MV fractions showed the presenceof CD9, flotillin-2 and MCH-I, whereas these proteins wereundetectable in total plasma and plasma deprived of MVs.
We determined MV number in plasma fractions en-riched in MVs. A set of 50 blood samples from womenwith diagnosis of breast cancer and a control group of31 blood samples of healthy women were analyzed. Theclinical characteristics of women with breast cancer arepresented in Table 1. Our findings showed that MV
Figure 1. Number of microvesicles (MVs) in plasma of women with breast
cancer Plasma fractions enriched in MVs and deprived of platelet-derived
MV were isolated of blood samples of healthy women (control group) and
women with different stages of breast cancer (BC). (A) Total plasma (TP),
plasma fractions enriched in MVs (MVsF) and plasma deprived of MVs
(PDMVs) were analyzed by Western blotting with anti-CD9, anti-flotil-
lin-2 and anti-MHC-I. The autoradiogram shown is representative of at
least three independent experiments. (B) The graph represents the mean
� SD of MV number of control group and BC patients. (C) The graph
represents the mean � SD of MV number of control group and women
with different stages of BC. (D) The graph represents the mean � SD of
MV number of patients with different stages of BC. Asterisks denote
comparisons made to control group (B and C) or in situ (D) values. *p
!0.05, ***p !0.0001 using one-way ANOVA. Number of analyzed
samples is indicated.
number in MV fractions of breast cancer patients wassignificantly elevated when compared with control group(Figure 1B).
We then compared the MV number between controlgroup and patients with different stages of breast cancer,and our findings did not show a significant differencebetween MV number of control group and patients within situ breast cancer, whereas significant differences werefound between control group and patients with stages IeIVbreast cancer (Figure 1C). Moreover, comparison of MVnumber between patients with in situ breast cancer andpatients with stages IeIV breast cancer showed onlya significant difference between patients with in situ andstage III breast cancer (Figure 1D).
Number of MVs in Plasma of Women with Breast Cancerand Different Tumor Sizes
We studied the relationship between the MV number of thecontrol group and the different tumor sizes of patients(T0eT4). Our results revealed significant differencesbetween MV number of control group and patients withT2eT4 tumors, whereas we did not find any significantdifference between the MV number of control group andpatients with T0eT1 tumors (Figure 2A). In addition,comparison between the MV number of patients with T0and T1eT4 tumors showed only a significant differencebetween the MV number of patients with T0 and T3tumors. However, comparison of MV number of patientswith T3 tumor with patients with T1, T2 and T4 tumorsshowed significant differences between patients with T3and T1eT2 tumors (Figure 2B).
MVs in Plasma of Breast Cancer Patients Contain FAKand EGFR
We determined the presence of FAK and EGFR proteins inthe plasma fractions enriched in MVs of breast cancerpatients and control group. MV fractions were analyzedby Western blotting with anti-FAK and anti-EGFR Ab.As shown in Figure 3A, FAK protein was undetectable inthe MV fractions of the control group; however, FAK wasdetected in the MV fractions of breast cancer in situ, andstages I, II and III. Comparison between FAK levels ofthe control group and patients with breast cancer in situand stages I, II and III showed a significant differencebetween control group and all breast cancer stages studied.
The EGFR protein was undetectable in the plasma frac-tions enriched in MVs of the control group as well as inpatients with breast cancer stages II and III, but it wasdetected in patients with breast cancer in situ and stage I.The comparison between EGFR levels of control groupand patients with breast cancer in situ and stage I showedsignificant differences between the FAK levels of controlgroup and patients with breast cancer in situ and stage I(Figure 3B).
Table 1. Clinical and pathological information of patients
ID TNM Stage Tumor size (cm) ER PR Her2/neu Ki67 Histology Age (years)
Situ 6 In situ NA (�) (�) (�) NA ISCD 57
Situ 7 In situ NA NA NA NA NA ISCD NA
Situ 11 In situ NA NA NA NA NA ISCD NA
G-I 7 TxNxMx I 2 NA NA NA NA ISCD 52
G-I 27 T3NoMx I O5 NA NA NA NA IDC 84
G-I 97 TxNxMx I 2 NA NA NA NA ISCD 52
G-I 19 T2N1Mo IB O2 (þ) (þ) (�) (þ) ISCD 64
G-I 12 T1bNoMo I 0.7 (þ) (þ) (�) (þ) IDC NA
G-I 30 T2No Mo I NA NA NA NA NA IDC NA
GI 9 TxNxMx I NA NA NA NA NA IDC 74
G-I 4 T2NoMx I O2 (þ) (þ) (þ) NA ISCD 69
G-I 41 T2NoMx I O1.5 (þ) (þ) (þ) NA ISCD 55
G-II 1 T2NoMo IIA 4 (þ) (þ) (�) (þ) IDC NA
G-II 14 T2N1Mo IIB 5 NA NA NA NA IDC 46
G-II 5 T4N1Mx IIB 2 (þ) (þ) (�) NA ISCD 42
G-II 26 T2NoMx II A 3.5 (þ) (þ) (�) (þ) IDC 64
GII 32 T2NoMo IIA 4 (þ) (þ) (þ) NA IDC 58
GII 21 T2NoMo IIA O2 (þ) (þ) (þ) NA ISCD 41
G-II 15 T2N1Mx IIB O2 NA NA NA NA NA NA
G-II 3 T1NoMo IIA 2 (þ) (þ) (�) (þ) ISCD NA
G-II 4 T2N2Mx IIA O2 NA NA NA NA ILC 46
G-II 5 T3NoMo IIB 8 (þ) (�) (�) (þ) IDC 56
G-II 28 T3N1Mx II 5 (þ) (�) (�) NA ISCD NA
G-II 25 T2NoMo IIA 1.2 (�) (�) (�) NA IDC NA
G-II 1 T2NoMo IIA 4 (þ) (þ) (-) (þ) IDC NA
G-II 29 T3NoMo IIB 7.5 NA NA NA NA IDC NA
G-II 32 T2N1Mo II NA NA NA NA NA IDC NA
G-II 35 T2N1Mo II NA NA NA NA NA MC NA
G-II 36 T2NoMo IIA NA NA NA NA NA IDC NA
G-II 37 T3NoMo IIB NA NA NA NA NA IDC NA
G-II 26 T2NoMo IIA 2.5 (�) (�) (�) NA ISCD NA
G-II 71 T2NoMo IIA 2.5 (�) (�) (�) NA IDC NA
G-II 81 T2NoMo IIA 2 (�) (þ) (�) (þ) ISCD NA
G-II 9 T2NoMo IIA O2 NA NA NA NA ISCD 48
G-II 24 T2NoMo IIA O2 NA NA NA NA IDC NA
G-III 44 T3NoMo IIIC 7 (þ) (�) (�) (þ) IDC 49
G-III 18 T3NoMo IIIA 7 (�) (�) (�) (þ) IDC NA
G-III 34 T2N1Mo III NA NA NA NA NA IDC NA
G-III 31 T3N1Mx III NA NA NA NA NA IMC NA
G-III 6 T3N1Mx III 10 (þ) (þ) (þ) NA ISCD NA
G-III 7 T4BN2Mx III 7 (�) (�) (þ) NA IDC NA
G-III 28 T4BN1Mo III 7 (þ) (þ) (þ) NA ISCD NA
G-III 61 T3N2Mo IIIA NA (þ) (þ) (þ) (þ) ISCD NA
G-III 64 T4BN2Mx IIIB 8.5 (þ) (�) (�) (þ) ISCD NA
G-III 65 T3N1Mx III NA (þ) (�) (�) (þ) MC NA
G-III 78 T4BN2Mx IIIB NA (þ) (�) (�) (þ) ISCD NA
G-III 34 T4bN2Mx IIIB 8.5 (þ) (þ) (�) NA IDC 55
G-IV 68 T4dN1Mx IV 4 (�) (�) (þ) (þ) ISCD 44
G-IV 22 T4N3M1 IV 2 (þ) (þ) (þ) NA IDC 45
G-IV 18 T3N1M1 IV 7 (þ) (�) (þ) NA ISCD 60
IDC, infiltrating ductal carcinoma; ISDC, in situ ductal carcinoma; ILC, infiltrating lobular carcinoma; IMC, infiltrating mucinous carcinoma; MC, mixed
carcinoma; NA, not available.
211Microvesicles in Breast Cancer Patients
Discussion
This study was designed to determine in plasma fractionsenriched in MVs, which were deprived of platelet-derivedMVs, the number ofMVs andwhether these fractions presentFAK and EGFR proteins in breast cancer patients anda control group of healthy women. MV fractions were
deprived of platelet-derived MVs because the vast majorityof MVs present in plasma are of platelet origin (O80%)(22). Our findings demonstrate that number of MVs is higherin women with breast cancer in comparison to healthywomen. Comparisons between control group and all stagesof breast cancer demonstrate significant differences in the
Figure 2. Number of microvesicles in plasma of women with BC and
different tumor sizes. MV fractions were isolated of blood samples of
control group and women with BC and different tumor sizes. (A,B) The
graph represents the mean � SD of MV number of control group and
patients with BC and different tumor sizes. Asterisks denote comparisons
made to control group (A) or T0 tumor (B) values. ***p !0.0001 using
one-way ANOVA. Number of analyzed samples is indicated. Figure 3. FAK and EGFR are present in the plasma fractions enriched in
MVs. MV fractions were isolated of blood samples of control group and
women with different stages of breast cancer. (A,B) MV fractions were
analyzed by Western blotting with anti-FAK and anti-EGFR. Graphs repre-
sent the mean � SD of analyzed samples and are expressed as the fold of
EGFR or FAK above control group. Autoradiograms shown are represen-
tative of at least three independent experiments. Asterisks denote compar-
isons made to control. *p !0.05, **p !0.001, ***p !0.0001. Number of
analyzed samples is indicated.
212 Galindo-Hernandez et al./ Archives of Medical Research 44 (2013) 208e214
MV number of control group and breast cancer stages IeIV.In contrast, a comparison between breast cancer stages IeIVdoes not show any significant difference in the MV number.However, a significant difference of MV number betweenbreast cancer in situ stage and stage III is found when breastcancer in situ is compared with breast cancer stages IeIV. Inaddition, comparison of control group with the differenttumor sizes (T0eT4) in breast cancer patients show signifi-cant differences in the MV number of the control groupand T2eT4 tumors, whereas comparison between T0 andT1eT4 tumors shows a significant difference of MV numberof T0 and T3 tumors. To further substantiate these findings,a larger number of breast cancer patients remain to beanalyzed, particularly in situ and breast cancer stage IVbecause we analyzed a small number of patient samples inthis study. With these results, we propose that the numberof MVs in plasma fractions enriched in MVs and deprivedof platelet-derived MVs is related with breast cancer stagesIeIV but is not related with in situ breast cancer in Mexicanwomen. In agreement with our proposal, it has been demon-strated that in comparison to control group, the number ofMVs in gastric cancer patients is significantly elevated inall stages (18). Moreover, another study demonstrated thatplasma number of microparticles is significantly higher in
gastric cancer patients in comparison to healthy controlsand is highest in patients with stage IV than those in patientswith stages IeIII (23).
The amount of MVs released by tumor cells has beenshown to correlate with their invasiveness both in vitroand in vivo as well as the fact that MV-mediated cargotransfer to adjacent or remote cells mediates many stagesof tumor progression including angiogenesis, escape fromimmune surveillance, extracellular matrix (ECM) degrada-tion and metastasis (4,24). MVs from tumor cells facilitatethe transfer of soluble proteins including nucleic acids,functional transmembrane proteins, chemokine receptortissue factor and receptor tyrosine kinases such as EGFRand HER-2 (14,25). We propose that in breast cancer stagesIeIV the cancer cells secrete a large amount of MVs andplay an important role in tumor progression because theyare transferred to other cancer cells with their cargo.
213Microvesicles in Breast Cancer Patients
FAK is a nonreceptor tyrosine kinase that localizes tofocal adhesions, the multimolecular structures of contactbetween cytoskeleton and ECM (26), and its activation ismediated by diverse signaling molecules including bioac-tive lipids, polypeptide growth factors, activated variantsof Src, ECM proteins and fatty acids (27,28). Moreover,FAK has been implicated in regulation of spreading, differ-entiation, proliferation, apoptosis, invasion survival, migra-tion and angiogenesis (28e30). Because the FAK gene isamplified in breast cancer and its protein is overexpressedand that it correlates with increased invasion and metastasis(31e33), we studied the presence of FAK in the plasmafractions enriched in MVs. Our findings demonstrate lowlevels of FAK in the MV fractions of the control group;however, FAK levels are significantly higher in the MVfractions of patients with breast cancer, where MV fractionsof stage III breast cancer present the highest level of FAKprotein. We propose that MVs released in breast cancerpatients mediate intercellular communication by the trans-fer of kinases including FAK, and that this kinase playsan important role in cell migration/invasion. Therefore,plasma fractions enriched in MVs of stage III breast cancerpresent the highest amount of FAK because these cells areable to migrate and produce invasion.
The epidermal growth factor (EGF)/ErbB receptorfamily has four members EGFR/ErbB1/HER1, ErbB2/Neu/HER2, ErB3/HER3 and ErbB4/HER4. EGFR controlsa wide variety of biological responses including prolifera-tion, differentiation, migration and apoptosis. It is ex-pressed in several cell types including epithelial andmesenchymal cells and its overexpression or dysregulationhas been described in human malignancies (34e36). EGFRmutations are rare in breast cancer but are highly expressed,with or without gene amplification, in basal breast cancer,which is a subset of triple negative breast cancer (TNBC).TNBC is characterized by the absence of estrogen receptor,progesterone receptor and HER-2 expression and representsa subset of breast cancer with a particularly aggressivephenotype and poor clinical outcomes with distinct patternof metastasis where EGFR expression status correlatesnegatively with patient survival (37e39). We determinedthe presence of EGFR in the plasma fractions enriched inMVs. Our findings show the presence of EGFR in theMV fractions of in situ and stage I breast cancer, whereasMV fractions of control group as well as breast cancerstages II and III present low levels of this receptor. Wepropose that cells expressing high levels of EGFR are ableto transfer this receptor to cells with low levels of EGFRexpression, and that this receptor is only transferred in earlystages of breast cancer (in situ and stage I). In line with thisproposal, it has been demonstrated that EGFR is a cargo ofMVs and that glioma cells express a truncated and onco-genic form of EGFR (EGFRvIII), which is transferredbetween the glioma cells by using MVs (40e42).
Because a small number of patient samples werestudied, we conclude that these findings strongly suggestthat MVs number in plasma fractions enriched in MVsand deprived of platelet-derived MVs is associated withbreast cancer stages IeIV and with the T2eT4 tumors. Inaddition, plasma fractions enriched in MVs present FAKand EGFR proteins, where FAK present in the MV fractionsis associated with breast cancer in situ and stages IeIII;whereas EGFR is associated with in situ breast cancerand stage I.
AcknowledgmentsWe are grateful to Luis Castro-Sanchez and Nora Ruiz for theirtechnical assistance. This work was supported by a grant fromCONACYT (83802) and UC-Mexus. O. G-H and S. V-C aresupported by a CONACYT predoctoral training grant. C. G-V issupported by a ICytDF postdoctoral grant.
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