Exosomes in Human Breast Milk Promote EMT · Biology of Human Tumors Exosomes in Human Breast Milk Promote EMT Wenyi Qin1,2,Yoshikazu Tsukasaki1, Santanu Dasgupta1, Nitai Mukhopadhyay3,

Biology of Human Tumors

Exosomes in Human Breast Milk Promote EMTWenyi Qin1,2, Yoshikazu Tsukasaki1, Santanu Dasgupta1, Nitai Mukhopadhyay3,Mitsuo Ikebe1, and Edward R. Sauter1,2

Abstract

Purpose: Pregnancy increases breast cancer risk for all womenfor at least 5 years after parturition. During weaning and involu-tion, the breast microenvironment becomes tumor promotional.Exosomes provide cell–cell communication during physiologicprocesses such as lactation, but also in breast cancer. We deter-mined whether molecules in milk exosomes from healthy lactat-ing women modulate the development and progression of breastcancer.

Experimental Design: Thirteen nursing women providedthree (transitional, mature, and wean) milk samples. Exo-somes were extracted and MCF7 and MCF10A breast cellslabeled. The expression of six proteins linked to breast cancerwas measured. On the basis of the findings, TGFb2 concen-tration in exosome samples was measured, breast cells incu-bated with the exosomes and effect (epithelial–mesenchymaltransition, EMT) on EMT-related proteins [E-cadherin,

a-smooth muscle actin (a-SMA), filamentous (F)-actin andvimentin] measured.

Results:Humanmilk exosomes entered benign andmalignantbreast cells. The greatest change in wean milk protein was inTGFb2 (P ¼ 0.01). Exosomes with a high (but not low) level ofTGFb2 led to EMT in both cancer and benign cells, based on (i)change in cell morphology, actin cytoskeleton, and loss of cell–cell junction structure and (ii) increaseda-SMA and vimentin anddecreased E-cadherin.

Conclusions: TGFb2 is significantly upregulated in breast milkexosomes duringweaning/early involution. Breastmilk exosomescontaining high levels of TGFb2 induce changes in both benignand malignant breast epithelial cells, consistent with the devel-opment and progression of breast cancer, suggesting a role forhigh TGFb2-expressing breast milk exosomes in influencingbreast cancer risk. Clin Cancer Res; 22(17); 4517–24. �2016 AACR.

IntroductionPregnancy increases breast cancer risk(at least for the short term)

Pregnancy-associated breast cancers (PABCs) diagnosed afterchildbirth are generally aggressive, with a poor prognosis. More-over, pregnancy has a lasting influence on breast cancer risk,beyond the time of PABCs, which is variably defined as lastinganywhere from 1 to up to 10 years after childbirth. Epidemiologicdata suggest that pregnancy increases a woman's risk of develop-ing breast cancer compared with nulliparous women over theshort term, but that the length in years of this increased riskdepends on the age at which the pregnancy occurs. Our under-standing of the human biology of PABCs is very limited. Ourultimate goal is to identify biologic markers (biomarkers) thatwill help predict which parous women are most likely to developbreast cancer.

Immediately following parturition, there is an increased risk ofbreast cancer observed for all age groups (1). This is not surprising,as the growth factors required to allow breast glandular prolifer-

ation, as well as the extensive remodeling required during invo-lution, may stimulate the growth of already present neoplasticbreast epithelial cell(s). The stimulation of extant neoplastic cellsversus their inhibition is a key concept in whether pregnancy andlactation decrease or increase breast cancer risk. Over the longterm, parity is protective for women whose first full-term preg-nancy (FFTP) was completed at a young age (variously defined as< 20 up to age 26), and increased in parous women whose FFTPoccurred after 35 years of age (2). The time of weaning, a period ofbreast involution and remodeling, appears critical to future breastcancer risk (3).

There is good evidence that during involution, which isinitiated by weaning, the breast microenvironment becomestumor promotional (3). A variety of proteins are important tothe involution process. Milk fat globule EGF factor 8 (MFG-E8)or lactadherin, which is secreted from exosomes (discussedbelow) fulfills a tethering function between apoptotic cells.MFG-E8 is involved in the recognition and clearance of apo-ptotic mammary epithelial cells during mammary involution(4). Matrix metalloproteinase (MMP)-dependent reorganiza-tion of the extracellular matrix during breast involution releasesgrowth factors, supporting breast metastasis (5). TGFb has bothpositive and negative effects on tumorigenesis (6). In mousemodels, TGFb is induced during mammary gland lactation andinvolution (7), and it mediates proapoptotic effects duringinvolution (1). On the other hand, TGFb promotes tumorgrowth through activation of the stroma and it stimulatesproliferation of cells in the healing wound (8). TGFb is linkedto breast cancer in preclinical models (7) and to prognosis inhuman breast cancer (9). TGFb-containing exosomes frommesothelioma cells and breast cancer cells converted fibroblaststo a myofibroblast phenotype (10).

1DepartmentofCellular&MolecularBiology,UniversityofTexasHealthScience Center, Tyler, Texas. 2Department of Surgery, University ofTexasHealth ScienceCenter,Tyler,Texas. 3Department ofBiostatistics,Virginia Commonwealth University, Richmond, Virginia.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Edward R. Sauter, University of Texas Health ScienceCenter, 11937 US Hwy 271, Tyler, TX 75707. Phone: 903-877-8855; Fax: 903-877-8655; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-16-0135

�2016 American Association for Cancer Research.

ClinicalCancerResearch

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Milk exosomesExosomes are cellular organelles that are shed into the sur-

rounding extracellular environment (11). They contain mem-brane and cytoplasmic components of the cell. While someexosomes are quickly degraded, others are not and can travel bydiffusion in body fluids such as breast milk. Exosomes are knownto contain a wide variety of proteins, lipids, and RNA (12).Exosomes provide cell–cell communication during physiologicprocesses such as lactation, but also in breast cancer (12). Exo-somes have been shown to be critical for directional migration ofcancer cells (13). While exosome isolation and analysis fromhuman blood has been widely reported, there are few reports onhuman breast milk exosomes (one focused on how to isolate theexosomes [14] and a second on exosome populations related toallergic sensitization [15]).

Epithelial–mesenchymal transitionDuring epithelial–mesenchymal transition (EMT), an epi-

thelial cell loses polarity and cell–cell contacts, acquiringmigratory and invasive properties (16). EMT occurs both innormal developmental processes including mesoderm forma-tion and neural tube formation, in wound healing, and incancer progression. TGFb is known to induce EMT in cancer,can promote tumor invasion and is associated with breastcancer bone metastasis (17). The role of TGFb in cancer devel-opment is more complex, as TGFb can also lead to cellularapoptosis (6).

Epithelial cell–cell junctions (subapical tight junctions,adherens junctions and desmosomes at lateral surfaces, andscattered gap junctions at lateral surfaces) maintain cell–cellcontact (18). EMT requires restructuring of these junctionsand of the actin cytoskeleton. During the destabilization ofadherens junctions, epithelial (E)-cadherin is cleaved andthen degraded (19). There is also upregulation of a-smoothmuscle actin (SMA) and reorganization of filamentous (F)-actin (20). This reorganization leads to increased migratoryand invasive capabilities (20). Proteins such as vimentin,which promote cell migration and invasion, are also upregu-lated (20).

SummaryDuring breast involution, the tumor promotional microen-

vironment may drive epithelial cells in the breast toward cancerdevelopment or progression. We investigated whether breast

milk exosomes contained proteins involved in breast involu-tion, which of the detected proteins was increased at the time ofinvolution, whether these protein(s) change benign cellstoward a cancer phenotype, and/or make cancer cells moreaggressive. We chose two immortalized breast cell lines forstudy, one derived from a patient with cancer and the secondfrom a patient with benign breast disease. Both have epithelialcharacteristics. MCF7 cells are derived from pleural metastasesof a lobular breast carcinoma. The cells require estrogen sup-plementation or genetic manipulation for tumor formation inimmunodeficient mice (21). MCF10A cells are derived from awoman with benign fibrocystic changes. The cells are immortal,but nontumorigenic unless genetically modified (22). Weobserved that breast milk exosomes from healthy lactatingwomen containing high levels of TGFb2 induced changes inbenign epithelial cells consistent with transformation towardneoplasia, and changes in breast cancer cells toward a moreaggressive, invasive phenotype.

Materials and MethodsPreparation of milk samples

A total of 38matchedmilk samples (onewean samplemissing)were collected from 13 lactating women (transitional: within 10days of lactation start; mature milk: collected 2 months afterlactation start, and wean (involution)milk: collected once breast-feedings were significantly curtailed at the end of nursing) afterInstitutional Review Board informed consent was obtained. Thewomen ranged in age from 25 to 37 years. The samples wereseparated by centrifugation into three components: fat, superna-tant, and cell pellet, snap frozen, labeled, and placed in a �80�Cfreezer until analysis.

Isolation and resuspension of exosomesThe ExoQuick eExosome Isolation Kit (System Biosciences)

was used as per the manufacturer's instructions. A total of 200 mLof milk supernatant, PBS, and exosome isolation reagent added,incubated at room temperature, centrifuged, and the excesssupernatant removed. The samples were centrifuged a secondtime to remove additional supernatant. A total of 500 mL of PBSwas then added to each pellet, and the tube vortexed overnight at4�C to fully dissolve the pellet. After dissolution, the sample wascentrifuged, the supernatant aliquoted, and the remaining pelletdiscarded. The remaining specimen contained exosomes foranalysis.

Analysis of total milk protein and milk exosome proteinA Bicinchoninic Acid Kit (Life Technologies) was used to

measure total milk protein and milk exosome protein. Afteradding standard and sample to each well, reagent was added,the plate mixed on a plate shaker, the plate incubated andabsorbance read at 562 nm on a plate reader.

Exosome labeling; coculture of breast milk exosomes andbreast cells

Exosomes isolated from milk supernatant were labeled usingan Exo-Green Exosome Labeling Kit as per the manufacturer'sinstructions (System Biosciences). MCF7 and MCF10A cells weseparately cocultured with breast milk exosomes containing high(1.5 ng/mL) or low (0.06 ng/mL) levels of TGFb2 for 48 hours.Cells were cultured in exosome-depleted specific FBS medium

Translational Relevance

Pregnancy-associated breast cancer is an aggressive form ofthe disease that is difficult to both diagnose and treat for whichthere is no effective screening tool. Relatively little is knownregarding the molecular drivers of the disease, especially inhumans. We report that TGFb2 levels in milk exosomessignificantly increase during breast involution, a time whenthe breast microenvironment is tumor promotional. We fur-ther demonstrate that milk samples with high (but not low)levels of TGFb2 appear to drive both breast cancer and benignbreast cells toward an aggressive phenotype, more prone totumor invasion and metastasis.

Qin et al.

Clin Cancer Res; 22(17) September 1, 2016 Clinical Cancer Research4518




(System Biosciences) as recommended. The cells were imagedusing a confocal microscope to confirm the presence of exosomeswithin the cells.

ELISAThe expression of 6 proteins (MMP2, MMP3, MMP9, TGFb1,

TGFb2, and MFG-E8) in breast milk exosomes was assessed induplicate by ELISA. A TGFb2 ELISA Kit (R&D Systems) was used,following the manufacturer's instructions. An MMP9 ELISA Kitwas purchased from Abcam.MMP2, MMP3, TGFb1, andMFG-E8ELISA kits were purchased from R&D Systems. The milk exosomepellet was resuspended in buffer, an assay diluent added, thenstandard, control, or activated sample added to each well. Afterincubation, the cells were washed, conjugate added, followed bywashing and the addition of substrate. A second incubation for 20minutes was followed by adding stop solution. Absorbance wasassessed with a plate reader at 450 nm.

EMTMCF7 and MCF10A cells were obtained from the ATCC. The

MCF7 line was derived from a woman with breast cancer,the MCF10A line from a woman with benign breast disease.The cells were washed twice with FBS, cell number and viabilityassessed using trypan blue, and the cells resuspended to a finalconcentration of 105 cells/mL in FBS-containing culture media.1.5 � 105 cells were suspended into the wells of a 6-well tissueculture plate, incubated for 72h-MCF7 or 96h-MCF10A (toobtain similar plate confluence) then milk exosomes contain-ing a high (1.5 ng/mL) or low (0.06 ng/mL) concentration ofTGFb2 added, with or without 1.5 mg TGFb2-blocking antibody(R&D Systems). Cell morphology was then visualized byinverted light microscopy.

Immufluorescence was performed onMCF10A cells before andafter treatment. Both negative (PBS-treated cells) and positive(pharmacologic treatment of cellswith 10ng/mLTGFb2) controlswere included for each protein evaluated. Cells were treated with200mgbreastmilk exosomes containing low(0.06ng/mL)or high(1.5 ng/mL) levels of TGFb2. The following EMT-related proteinswere evaluated: E-cadherin, a-SMA, F-actin, and vimentin. Cellscultured on glass coverslips were fixed in 4% formaldehyde,washed twice with PBS, permeabilized with 0.1% Triton X-100,washed again and blocked with 1% BSA. Cells were then incu-bated at 4�C overnight with the indicated primary antibodies (E-cadherin: Cell Signaling Technology; a-SMA: R&D Systems;vimentin: Sigma) and then incubated for 1 hour with fluorescentsecondary antibody (Invitrogen), Alexa 647 phalloidin for F-actin(Life Technologies) and 40,6-diamidino-2-phenylindole (DAPI)for nuclear staining (Sigma). Fluorescence images were viewedwith a Leica TCS SP8 laser-scanning confocal microscope (LeicaMicrosystems).

Quantitative real-time (RT) PCR analysis was performed induplicate to measure the expression of E-cadherin after treatmentofMCF10A cellswithnegative andpositive controls, aswell as lowor high levels of TGFb2. Briefly, total RNA was extracted fromMCF10A cells with an RNeasy PlusMicro Kit (Qiagen). Total RNA(800 ng) was reverse transcribed to cDNA with a QuantiTectReverse Transcription Kit (Qiagen). RT-PCR reactions were per-formed in duplicate using an IQ SYBR Green Supermix (Bio-Rad)according to the manufacturer's instructions using a MyiQ RT-PCR detection system (Bio-Rad). Relative changes in gene expres-sion were calculated using the 2�DDCt formula. GAPDH mRNAlevels served as the internal control. The E-cadherin and GAPDHprimers were purchased from Qiagen.

Statistical analysisExpression levels of the six proteins (Table 1)were summarized

using their mean and median level at each of the time points.Post-baseline levels were compared with baseline using a paired ttest for statistical significance. As the sample sizewas small, no testfor normality was performed. All analyses were performed usingstatistical software R version 3.0.2.

ResultsMilk exosomes from healthy lactating women enter breastcancer and benign breast cells

We used confocal microscopy to confirm that, when cocul-tured, human milk exosomes get inside of both MCF7 andMCF10A breast cells (Fig. 1). Confocal imaging demonstratedGFP binding to CD63, a protein commonly used to identifyexosomes. Proliferation was measured in MCF7 cells and notedto be significantly increased (P < 0.01) after treatment with milkexosomes containing a high level of TGFb2 versus control.

TGFb2 is significantly upregulated in wean milk exosomesWe were able to detect each of the six proteins in the exosomes

of all milk samples. For each protein, we compared expression intransitional milk to milk collected later (in mature milk or weanmilk). TGFb2 expression in the 38 samples ranged from 0.55 to53.2 ng/mL, with a mean of 9.4 ng/mL. We considered highexpression to be above the mean, and low expression below themean. In all cases,median andmeanprotein expression decreasedin mature milk. This decrease was statistically significant forTGFb1, MMP3, and MFG-E8 (Table 1; Fig. 2). Protein expressionin wean milk significantly changed in three of the proteins,increasing for TGFb2 (P ¼ 0.01) and decreasing for MMP2 andMMP3. The median (mean) increase in TGFb2 from transitionaltoweanmilkwas 2.2-fold (4-fold), and frommature toweanmilk5.9-fold (6.4-fold). There was no correlation (P ¼ 0.46) betweennursing length and TGFb2 expression.

Table 1. Protein expression in breast milk exosomes

Median (Mean) milk concentration (pg/mg) P PProtein Transitional Whole Wean Trans. vs. Whole Trans. vs. Whole

TGFb1 80.2 (76.4) 47.6 (48.8) 83.0 (86.0) 0.01 0.54TGFb2 503.5 (474.2) 187.0 (295.8) 1,096.0 (1,883.0) 0.17 0.01MMP2 0.48 (0.49) 0.34 (0.40) 0.27 (0.29) 0.16 <0.01MMP3 0.31 (0.33) 0.12 (0.13) 0.14 (0.18) <0.01 0.05MMP9 65.0 (167.7) 28.3 (143.5) 44.4 (92.3) 0.77 0.24MFG-E8 1,288.0 (1,537.0) 1,114.0 (1,028.0) 1,001.0 (1,147.0) 0.05 0.15

NOTE: Comparisons with a P value < or ¼ 0.05 are in bold.Abbreviations: MMP, matrix metalloproteinase; MFG, milk fat globulin.

TGFb2 in Breast Milk Exosomes

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Milk exosomes promote breast cancer through EMTWe determined the dose-dependent role of TGFb2 containing

milk exosomes on EMT. Milk exosomes containing high or lowconcentrations of TGFb2 or PBS control were added toMCF7 (Fig.3) or MCF10A (Fig. 4) cells. Exosomes with high TGFb2 (but not

low TGFb2) demonstrated morphologic changes consistent withEMT, including disruption of cell–cell junctions with cellularextensions (, Fig. 3A and B, right). A TGFb2-blocking antibodyreversed theEMT (Fig. 3B, left). Pharmacologic TGFb2 (10ng/mL)also induced EMT (Fig. 4).

Figure 2.Expression of TGFb1, TGFb2, MMP2,MMP3, MMP9, and MFG-E8 in breastmilk exosomes. Expression wasmeasured in matched transitional,mature and wean milk samples fromnursing mothers. The box plots spanthe first to the third quartile, with thebold line in the middle representingthe median. The extended linesbeyond the box show the 95%confidence region and points beyondthat the outlier results. Asterisksindicate a significant difference(P < 0.05) between expression inwhole or wean milk versustransitional milk.

A B C

D E F

Figure 1.Confocal image of MCF7 (A–C) andMCF10a (D–F) cells cocultured withmilk exosomes demonstrating GFPbinding to CD63, a protein used toidentify exosomes. A and D, withoutGFP; B and E, GFP with CD63 staining;C and F, merged image. Arrowindicates internalized exosome. Scalebar, 25 mm.

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Effect of TGFb2-containing milk exosomes on EMT-relatedproteins

The actin cytoskeletal structure dramatically changes duringEMT, which is correlated with a migration phenotype of thetransformed cells. During this transition, the cells lose cell–celljunctions. MCF10A cells show a nonmigratory phenotype thatis accompanied with the expression of E-cadherin, a key adhe-rens junction component, at the cell–cell interface and lowexpression of a-SMA and vimentin (Neg controls, Fig. 5A andB). TGFb2 stimulation markedly changed the actin cytoskeletalstructure showing stress fibers and cell–cell junction structureswith the loss of E-cadherin (Pos control, Fig. 5A and B). Duringthis transition, there is a notable increase in a-SMA andvimentin expression. A similar actin cytoskeletal change and

loss of E-cadherin expression were evident after high (TGFbH)but not low (TGFbL) TGFb2 exosome treatment.

We quantified the average change in E-cadherin after eachtreatment. Compared with control, there was a > 60% decreasein expression in the positive control and in the TGFb2H-treatedgroup,whereas thedecrease after treatmentwith TGFb2Lwas 21%(Supplementary Fig. S1).

DiscussionPregnancy is known to influence future breast cancer risk, and

PABC generally portends a poor prognosis. Current assessment ofhow pregnancy influences future breast cancer risk is focused onclinical factors during the period between menarch and FFTP,

High TGFβ2 milk exosome

Control TGFβ2

Low TGFβ2 milk exosome

Figure 4.MCF10A cells were coincubated for 96hours with PBS (control), 10 ng/mLTGFb2 or milk exosomes with low orhigh TGFb2 levels. Magnification200�.

PBS High TGF-β2Highighh TTTTTTTGFGFGFGFGFGFGGGF----βββββββββββββ2222222222222222TGFβ2L

High TGFβ2+NAb Low TGFβ2+NAb

TGFβ2H

Figure 3.EMT and filopodia formation in MCF7cells after 72-hour coincubation withmilk exosomes expressing high (H)but not low (L) TGFb2 or PBS (control;top row). Reversal of EMT andfilopodia formation followingtreatment with a TGFb2 neutralizingantibody (Nab; bottom, left twopanels). Magnification 200� (bottom,right panel). Close-up of filopodiaformation (arrows). Magnification200�.






NegA

B

control Pos control TGFβ2L TGFβ2H

E-cadherin

α-SMA

F-Actin

Merge

Neg control Pos control TGFβ2L TGFβ2H

Vimentin

α-SMA

F-Actin

Merge

Figure 5.EMT of MCF10A cells after the addition of milkexosomes. MCF10A cells were stimulated withPBS (Neg control), pharmacologic TGFb2 (Poscontrol), or milk exosomes containing low (L)or high (H) levels of TGFb2. During EMT,E-cadherin demonstrates decreasedexpression, a-SMA and vimentin wereupregulated and F-actin indicatedmore stressfibers and an asymmetric cell structure. Mergefigures demonstrate nuclear DAPI stainingwith membranous E-cadherin (A) or vimentin(B). Scale bar, 25 mm.


Qin et al.




including length in years, diet, adiposity, physical activity, andalcohol intake (23). While these factors provide insight into risk,they are insufficiently informative at the individual patient risklevel to clearly guide a pregnant or lactating woman's cancerprevention efforts.

In preclinical studies, Schedin and colleagues reported thattumors developing in an involuting mammary gland werelarger, greater in number and had a higher proliferation indexthan tumors that developed in a nulliparous mammary gland(24, 25). Our prior work demonstrated that of 16 proteinslinked to breast cancer which we analyzed in lactating women,the greatest changes, regardless of age at FFTP, were withkallikreins (KLK) 6, 8, and TGFb2 (26). Among KLKs and TGFbisoforms, the greatest increase in wean milk supernatant col-lected at the time of breast involution was in TGFb2. Amongwomen whose breast cancer was detected while they werenursing, we found that TGFb2 expression was significantlyhigher in breast milk collected from the cancer containingcompared with the matched clinically normal breast (27).

In the current study, we demonstrate that milk exosomes fromhealthy lactating women can be reliably isolated, that proteinsinvolved in breast involution can be measured in exosomes, andthat the exosomes enter human breast cancer and benign breastcells. The decrease in exosomal protein expression in mature(compared with transitional) milk is consistent with our priorreports in breast milk supernatant, even after controlling for totalmilk protein (28). Among the six proteins analyzed in the currentstudy, only one, TGFb2, significantly increased inwean comparedwith transitional milk. Given our observations on TGFb2 expres-sion, that the protein increases both in milk supernatant and inmilk exosomes at the time of breast involution, as well as the factthat expression is significantly higher in the milk of cancer con-taining than matched clinically normal breasts, we elected tofocus our mechanistic studies on this protein.

We observed that TGFb2-containing exosomes increase theproliferation of breast cancer cells as well as dose-dependent EMTin both breast cancer cells and cells derived from a woman withbenign disease, with a marked change in cytoskeletal reorganiza-tion. Exogenously added chemical sources of TGFb2 have previ-ously been shown to induce EMT, and we confirmed this changein MCF10A breast cells; however, little is known regarding ifTGFb2 from human body fluids, and specifically from breastmilk exosomes, drives EMT (29).

The ability of TGFb isoforms to induce EMT appears cell line–dependent. While some cell lines such as MCF10A undergo EMTwhen TGFb is used alone (30), other cell lines such as MCF7

require additional reagents (30, 31). Consistent with thesereports, we observed that pharmacologic TGFb2 induced EMT inMCF10A cells (Fig. 3), whereas it did not in MCF7 cells (data notshown). Moreover, TGFb2 induced changes in EMT-related pro-teins in MCF10A-treated cells, whereas it did not in MCF7 cells(data not shown).

Both pharmacologic TGFb2 and high-dose TGFb2 decreased E-cadherin (Fig. 5A) and increased a-SMA, actin stress fibers andvimentin (Fig. 5B), the latter three of which are increased withEMT. The dose of TGFb2 in milk exosomes (1.5 ng/mL) was lessthan pharmacologic TGFb2 (10 ng/mL). There is evidence of adose–reponse, perhaps best observed comparing pharmacologictreatment to high-dose TGFb2 milk exosome treatment for cellmorphology (Fig. 4) and F-actin structure (Fig. 5A and B). This isconsistent with the observation that the induction of EMT, andchange in protein expressionwith EMT, is both cell line- and dose-dependent.

Our findings suggest that breast milk exosomes containing highlevels of TGFb2may promote EMT.Our ultimate goal is to identifyone or more biomarkers of breast cancer risk in parous women bynoninvasively analyzing a readily available body fluid, breastmilk.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Dasgupta, M. Ikebe, E.R. SauterDevelopment of methodology: W. Qin, S. Dasgupta, N. Mukhopadhyay, E.R.SauterAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. Tsukasaki, S. Dasgupta, M. Ikebe, E.R. SauterAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Tsukasaki, S. Dasgupta, N. MukhopadhyayWriting, review, and/or revision of the manuscript: S. Dasgupta, N. Mukho-padhyay, M. Ikebe, E.R. SauterAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): W. Qin, S. DasguptaStudy supervision: S. Dasgupta, M. Ikebe, E.R. Sauter

Grant SupportThis projectwas fundedbyAvonFoundation forWomen (grant no. 02-2012-

090).The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received January 15, 2016; revisedMarch 10, 2016; acceptedMarch 27, 2016;published OnlineFirst April 8, 2016.

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2016;22:4517-4524. Published OnlineFirst April 8, 2016.Clin Cancer Res Wenyi Qin, Yoshikazu Tsukasaki, Santanu Dasgupta, et al. Exosomes in Human Breast Milk Promote EMT

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Exosomes in Human Breast Milk Promote EMT · Biology of Human Tumors Exosomes in Human Breast Milk Promote EMT Wenyi Qin1,2,Yoshikazu Tsukasaki1, Santanu Dasgupta1, Nitai Mukhopadhyay3,