European Journal of Cancer (2014) 50, 1025– 1034

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Regulation of exosome release from mammary epithelial and breast cancer cells – A new regulatory pathway Andrew Riches a,⇑, Elaine Campbell a, Eva Borger b, Simon Powis a a b

School of Medicine, Medical & Biological Sciences Building, University of St Andrews, KY16 9TF Scotland, UK School of Biology, Medical & Biological Sciences Building, University of St Andrews, KY16 9TF Scotland, UK

Available online 22 January 2014

KEYWORDS Exosomes Mammary epithelial cells Breast cancer Regulation

Abstract Purpose: Exosomes are small 50–100 nm sized extracellular vesicles released from normal and tumour cells and are a source of a new intercellular communication pathway. Tumour exosomes promote tumour growth and progression. What regulates the release and homoeostatic levels of exosomes, in cancer, in body fluids remains undefined. Methods: We utilised a human mammary epithelial cell line (HMEC B42) and a breast cancer cell line derived from it (B42 clone 16) to investigate exosome production and regulation. Exosome numbers were quantified using a Nanosight LM10 and measured in culture supernatants in the absence and presence of exosomes in the medium. Concentrated suspensions of exosomes from the normal mammary epithelial cells, the breast cancer cells and bladder cancer cells were used. The interaction of exosomes with tumour cells was also investigated using fluorescently labelled exosomes. Results: Exosome release from normal human mammary epithelial cells and breast cancer cells is regulated by the presence of exosomes, derived from their own cells, in the extracellular environment of the cells. Exosomes from normal mammary epithelial cells also inhibit exosome secretion by breast cancer cells, which occurs in a tissue specific manner. Labelled exosomes from mammary epithelial cells are internalised into the tumour cells implicating a dynamic equilibrium and suggesting a mechanism for feedback control. Conclusions: These data suggest a previously unknown novel feedback regulatory mechanism for controlling exosome release, which may highlight a new therapeutic approach to controlling the deleterious effects of tumour exosomes. This regulatory mechanism is likely to be generic to other tumours. Ó 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author: Address: School of Medicine, Medical & Biological Sciences Building, University of St. Andrews, KY16 9TF Fife, Scotland, UK. Tel.: +44 (0)1334 463603. E-mail address: [email protected] (A. Riches).

0959-8049/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejca.2013.12.019

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1. Introduction Exosomes are small 50–100 nm sized extracellular vesicles released from normal and tumour cells and represent a new intercellular communication pathway [1–9]. Importantly, what regulates the release and homoeostatic levels of exosomes in body fluids remains undefined for both normal and tumour cells. Exosomes are released by tumour cells and patients with cancer have been shown to have increased numbers in their blood [10,11]. They modulate the immune response [12], induce angiogenesis [13], transfer genetic information [14], modulate niche formation in metastasis [15] and growth of tumour cells [16,17], thus promoting tumour growth and progression. Exosomes can also deliver mRNA and microRNA to other cells [18]. They are also potential tumour biomarkers [17,19,20]. Tumour associated fibroblasts release exosomes which interact with tumour cells to promote metastasis via Wnt-planar cell polarity signalling [21]. Cancer exosomes can also transmit information which induces fibroblasts to differentiate into myofibroblasts [22]. The secretion and composition of exosomes derived from myeloma, lymphoblastoid and breast cancer cells is regulated by heparanase [23]. Several groups have demonstrated that labelled tumour exosomes can be taken up by tumour cells [24–27]. The significance of the interaction of exosomes with tumour cells and normal cells has not been fully elucidated. In particular it is not known what regulatory mechanisms exist that influence exosome release in normal or tumour tissues. To investigate this putative regulatory pathway, we have utilised a normal mammary epithelial cell line and a breast cancer cell line derived from it. The normal and tumour cell lines were derived from the same patient. These tumour cells exhibit gene alterations similar to those observed in a panel of sporadic breast cancers [28].

2. Materials and methods 2.1. Cell culture The human mammary epithelial cell line (HMEC B42) was established as a primary cell culture (Tayside Committee on Medical Research Ethics LREC 168/02). The cells were maintained in HuMEC cell culture medium (Gibco) with the supplied supplements. The B42 clone 16 cell line was derived from the B42 line following exposure to gamma irradiation and a cloned line selected [28]. The cells were maintained in Mammary Epithelial Basal Medium with the supplied supplements (Lonza). The MGH-U1 cell line [29] was maintained in DMEM:F12 (1:1) (Gibco) with added foetal calf serum

(7%), penicillin 100 lg/ml, L-glutamine 2 mM and streptomycin 100 U/ml (Sigma). 2.2. Preparation of exosomes Exosomes were prepared using well established methods [30]. As the cell culture media contains bovine pituitary extract which contains large numbers of microvesicles, all the experiments in which exosomes are counted or collected were undertaken using culture medium that had been firstly spun at 300g for 10 min followed by an overnight spin at 100,000g at 4 °C on a Beckman Coulter Optima L-100XP ultracentrifuge. The resultant supernatant was checked to enumerate any background microvesicle count using the Nanosight LM10. Concentrated samples of exosomes were prepared by incubating the relevant cell population for two consecutive 12 h periods with microvesicle free culture medium. Following collection of the supernatant, the exosomes were concentrated by differential centrifugation; 300g for 10 min to remove any cells and debris, 10,000g for 30 min to remove large particles and ultracentrifugation at 100,000g for 2 h to pellet the exosomes. The exosomes were then suspended in a small volume of microvesicle free medium and the exosome concentration measured using the Nanosight. Samples were stored at 20 °C before use. To measure exosome production by the cells, cells were plated in 25 cm2 Nunc tissue culture flasks and grown to sub-confluence. Three separate flasks were used per treatment and at least three independent replicate experiments were undertaken. The medium was removed and the cells rinsed twice in Dulbeccos Phosphate Buffered Saline (DPBS) to remove any microvesicles. Microvesicle free medium (3 ml) was then added to the cells. Sampling of the medium at this stage demonstrated that no microvesicles could be detected. Concentrated suspensions of exosomes from the HMEC B42, B42 clone 16 and MGH cell lines were added in volumes of less than 200 ll per 3 ml of exosome depleted medium. Supernatants were collected from the cells at appropriate times and the supernatant spun at 300g for 10 min followed by centrifugation in Eppendorf tubes at 20,000g for 20 min. The resultant supernatant was used to measure exosome concentrations. Following removal of the culture medium, 3 ml of TrypLEe Express (Life Technologies) was added per flask and the cells resuspended once detached. The cellularity was determined by counting on a Coulter Counter Model ZB. 2.3. Analysis of exosome concentrations The supernatants were introduced into the sample chamber of a Nanosight LM10 analysis unit with a

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635 nm laser [31] (Nanosight, Salisbury, United Kingdom (UK)). Samples were analysed based on our previous studies of exosomes [32]. All samples were diluted to give counts in the linear range of the instrument (i.e. 3  108 to 109 per ml) [31]. The particles in the laser beam undergo Brownian motion and videos of these particle movements are recorded. The Nanosight Tracking Analysis (NTA) 2.3 software then analyses the video and determines the particle concentration and the size distribution of the particles. Three videos of 30 s duration were recorded for each sample at appropriate dilutions with a shutter speed setting of 1500 (exposure time 30 ms) and camera gain of 560. The detection threshold was set at 6 and at least 1000 tracks were analysed for each video. 2.4. Western blotting

Fig. 1. Time course of exosome production. Exosome production by human mammary epithelial cell (HMEC) B42 cells ( ) and B42 clone 16 cells (d) with time after changing to exosome depleted medium. The number of exosomes produced in vitro was quantified using NTA. Three separate flasks were used per time and the exosome numbers quantified from three video recordings. Error bars are plotted as s.e.m.

Concentrated samples of exosomes were suspended in reducing buffer and the proteins analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane (BA85, Whatman, UK) and incubated overnight with specific antibodies at 4 °C. Anti-Alix (sc53538) and Tsg 101 (sc7964) mouse monoclonal antibodies were purchased from Santa Cruz (Framlington, UK) [32].

Fig. 2. Time course of exosome production by human mammary epithelial cells and breast cancer cells. The number of exosomes produced in vitro was quantified using NTA. Three separate flasks were used per treatment and the exosome numbers quantified from three video recordings. The results of replicate experiments are presented in Table 1. Error bars are plotted as s.e.m. (A) Number of exosomes produced per flask in a 24 h collection period compared to the number produced per flask in two consecutive 12 h collection periods (A + B) by human mammary epithelial cell (HMEC) B42 mammary epithelial cells. (B) Number of exosomes produced in a 24 h collection period per flask compared to the number produced per flask in two consecutive 12 h collection periods (A + B) by B42 clone 16 breast cancer cells. There was a marked difference between the number of exosomes produced per flask in two consecutive 12 h periods (A + B) and the number produced in a 24 h collection period, for the HMEC B42 cell line P = 0.005, n = 3, and for the B42 clone 16 breast cancer cell line P = 0.0005, n = 3, using the two tailed unpaired t test. There was no difference in the number of cells per flask at the end of the 24 h period and two consecutive periods of 12 h (HMEC B42 cell line, P = 0.56, n = 3; B42 clone 16 cell line P = 0.78, n = 3).

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2.5. PKH26 labelling and analysis

3. Results

Exosomes were labelled using the PKH26 Red Fluorescent Cell Linker Kit according to the manufacturer’s instructions (Sigma). The labelled exosomes were incubated for 3 h with cells in eight well chamber slides and viewed on a Leica DMIRE2 confocal microscope.

3.1. Time course of exosome production by human mammary epithelial cells and breast cancer cells

2.6. Statistical analysis An unpaired t test was used for statistical analysis where the variances were equal and an unpaired test with Welch’s correction when the variances were not equal. Error bars represent s.e.m.

Table 1 The percentage increase in exosomes released in two consecutive 12 h periods compared to a 24 h collection period for four independent experiments. Experiment number

1 2 3 4

% increase in exosomes released in two consecutive 12 h collections compared to a single 24 h collection B42 human mammary epithelial cells (HMECs) (%)

B42 clone 16 cells (%)

77 71 40 38

67 70 55 30

The number of exosomes released with time after incubating the cells in exosome depleted medium was evaluated in three 75 cm2 flasks. The number of exosomes per flask increased with time for 9 h for both the HMEC B42 cells and B42 clone 16 cells. After this time, the number remained constant up to 16 h (Fig. 1). The release of exosomes was further assessed by incubating the cells with exosome depleted medium for 24 h or for two consecutive 12 h periods. The total number of exosomes per flask produced after 24 h was consistently less than the sum of those produced in two consecutive 12 h periods per flask for both the HMEC B42 cells (Fig. 2A) and for the B42 clone 16 cells (Fig. 2B). There was no difference in the number of cells per flask at the end of the 24 h period and two consecutive periods of 12 h (HMEC B42 cell line, P = 0.56, n = 3; B42 clone 16 cell line P = 0.78, n = 3). The breast cancer cell line produced ((53.2 ± 1.6)  108 exosomes per 106 cells) in the 24 h period whereas the normal mammary epithelial cell line produced ((4.5 ± 2.3)  108 exosomes per 106 cells) in the same period. The results from four independent experiments consistently demonstrated increased production in the two 12 h periods. There was an increase

Fig. 3. Exosomes in the extracellular environment regulate exosome release. Regulation of exosome release by human mammary epithelial cell (HMEC) B42 mammary epithelial cells (normal cells). The number of exosomes produced in vitro was quantified using NTA. Three separate flasks were used per treatment and the exosome numbers quantified from three video recordings. Replicate experiments are presented in Supplementary Figs. S1 and S2. Error bars are plotted as s.e.m. (a). Number of exosomes produced in a 12 h collection period by HMEC B42 mammary epithelial cells in the presence and absence of added HMEC B42 exosomes ((12.95 ± 1.25)  108 per ml) to the culture. Control represents the number of exosomes produced in 12 h. The expected number of exosomes is plotted (i.e. number produced in 12 h plus number added to the culture) and the number of exosomes released following subtraction of the number added. There was no difference in the cell number per flask between the two groups (P = 0.8 using the two tailed unpaired t test). (B) Number of exosomes produced in a 12 h collection period by HMEC B42 mammary epithelial cells in the presence and absence of added B42 clone 16 exosomes ((10.80 ± 0.24)  108 per ml) to the culture. Control represents the number of exosomes produced in 12 h. The expected number of exosomes is plotted (i.e. number produced in 12 h plus number added to the culture) and the number of exosomes released following subtraction of the number added. There was no difference in the cell number per flask between the two groups (P = 0.28, n = 3, using the two tailed unpaired t test).

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of (56.5 ± 10.2)% in the number of exosomes released in two consecutive 12 h periods compared to a single 24 h collection period for the HMEC B42 normal mammary epithelial cells and (55.5 ± 9.1)% for the B42 clone 16 breast cancer cells (Table 1). This temporal difference in the number of exosomes released suggested that the production of exosomes might be regulated by the presence of pre-existing exosomes in the extracellular environment. 3.2. Exosomes in the extracellular environment regulate exosome release To test this hypothesis, exosomes were added to the medium to investigate whether this would modify exosome production. If it is assumed that there is no interaction between the added exosomes and the cells, then the number of exosomes in the supernatant should be the number produced in 12 h plus the number of exosomes added. The observed number of exosomes released in a 12 h collection period was measured in both HMEC B42 and B42 clone 16 cultures to which concentrated suspensions of exosomes were added. The number of exosomes produced in the presence of added exosomes was markedly inhibited in both cases

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(Figs. 3A and 4A, Supplementary Fig. S1). This is plotted as the expected value and is significantly different from the measured value when exosomes are added (Fig. 3A comparing the number of exosomes produced by HMEC B42 cells with HMEC B42 exosomes added to the expected value, P = 0.0001, n = 3, using the two tailed unpaired t test; Fig. 4A comparing the number of exosomes produced by B42 clone 16 cells with B42 clone 16 exosomes added to the expected value, P = 0.0023, n = 3, using the two tailed unpaired t test). Experiments were then repeated adding exosomes from normal mammary epithelial cells to breast cancer cells and vice versa. The tumour derived exosomes also inhibited exosome release from normal mammary epithelial cells (Fig. 3B, Supplementary Fig. S2; comparing the number of exosomes produced by HMEC B42 cells with B42 clone 16 exosomes added to the expected value, P = 0.0001, n = 3, using the two tailed unpaired t test). However when exosomes from normal mammary epithelial cells were added to breast cancer cells there was a marked suppression in exosome production (Fig. 4B, Supplementary Fig. S2; comparing the number of exosomes produced by B42 clone 16 cells with HMEC B42 exosomes added to the expected value, P = 0.0004, n = 3,using the two tailed unpaired t test).

Fig. 4. Exosomes in the extracellular environment regulate exosome release. Regulation of exosome release by B42 clone 16 breast cancer cells (tumour cells). The number of exosomes produced in vitro was quantified using NTA. Three separate flasks were used per treatment and the exosome numbers quantified from three video recordings. Replicate experiments are presented in Supplementary Figs. S1 and S2. Error bars are plotted as s.e.m. (A) Number of exosomes produced in a 12 h collection period by B42 clone 16 breast cancer cells in the presence and absence of added B42 clone 16 exosomes ((34.00 ± 0.84)  108 per ml) to the culture. Control represents the number of exosomes produced in 12 h. The expected number of exosomes is plotted (i.e. number produced in 12 h plus number added to the culture) and the number of exosomes released following subtraction of the number added. There was no difference in the cell number per flask between the two groups (P = 0.73 using the two tailed unpaired t test). (B) Number of exosomes produced in a 12 h period by B42 clone 16 breast cancer cells in the presence and absence of added HMEC B42 exosomes ((13.90 ± 0.70)  108 per ml) to the culture. Control represents the number of exosomes produced in 12 h. The expected number of exosomes is plotted (i.e. number produced in 12 h plus number added to the culture) and the number of exosomes released following subtraction of the number added. There was no difference in the cell number per flask between the two groups (P = 0.07, n = 3, using the two tailed unpaired t test).

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In fact fewer exosomes were produced than the untreated controls (Fig. 4B, Supplementary Fig. S2; comparing the number of exosomes produced by B42 clone 16 cells only with B42 clone 16 cells with HMEC B42 exosomes added, P = 0.0006, n = 3, using the two tailed unpaired t test). In all cases, the number of cells per flask after the 12 h period was the same for each pair of treatments with and without exosomes added. Different concentrations of either B42 clone 16 exosomes or HMEC B42 exosomes were added to B42 clone 16 cells and the number of exosomes determined after 12 h incubation in exosome free medium. The number of exosomes added was subtracted from the number of exosomes per ml and plotted against the percentage of exosomes added relative to the largest concentration (Fig. 5). A dose response relationship was observed with increasing exosome concentrations added. Examples of the size distribution profiles derived from NTA analysis indicate that they were similar for all treatment combinations as might be expected (Fig. 6A). The exosomes released expressed well established exosome markers in the form of Tsg101 and Alix (Fig. 6B). The levels of expression of Tsg101 and Alix at different times after changing to exosome free medium correlates with the concentration of exosomes in the medium at the same time points (Fig. 6C).

3.3. Specificity of the inhibition of exosome release The specificity of the regulatory response was investigated by adding exosomes from a human bladder cancer cell line (MGH-U1). Similar numbers of exosomes added had no effect on exosome release by the breast cancer cells suggesting that the response is tissue specific. (Fig. 7, Supplementary Fig. S3). There was no difference between the measured exosome numbers when exosomes were added and the expected numbers by adding the number produced without exosome addition to the number of exosomes added (Fig. 7, comparing the number of exosomes produced by B42 clone 16 cells with MGH exosomes added to the expected value, P = 0.15, n = 3, using the two tailed t test with Welch’s correction). If the number of exosomes added is subtracted from the number produced with B42 clone 16 cells with exosomes added, this should equal the number of exosomes produced by B42 clone 16 cells. The number of exosomes produced was similar to that with no MGH exosomes added to the culture (Fig. 7. comparing the number of exosomes produced by B42 clone 16 cells alone with the number produced after subtraction of the number of MGH exosomes added to B42 clone 16 cells, P = 0.015, n = 3, using the two tailed t test with Welch’s correction). In a further three independent experiments,

Fig. 5. Dose response relationship with added exosomes. Regulation of exosome release by B42 clone 16 breast cancer cells (tumour cells). The number of exosomes produced in vitro was quantified using NTA in the presence of different numbers of added exosomes. Three separate flasks were used per treatment and the exosome numbers quantified from three video recordings. (A) Number of exosomes produced in a 12 h collection period by B42 clone 16 breast cancer cells in the presence of different numbers of added B42 clone 16 exosomes. The results are plotted as the number of exosomes per ml at 12 h minus the number of exosomes added against the percentage number added. (B) Number of exosomes produced in a 12 h collection period by B42 clone 16 breast cancer cells in the presence of different numbers of added human mammary epithelial cell (HMEC) B42 exosomes. The results are plotted as the number of exosomes per ml at 12 h minus the number of exosomes added against the percentage number added.

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Fig. 6. Properties of exosomes derived from human mammary epithelial cell (HMEC) B42 cells and B42 clone 16 breast cancer cells. (A) Estimated size distribution profiles of exosomes from HMEC B42 cells and B42 clone 16 cells in the absence and presence of added HMEC B42 exosomes and B42 clone 16 exosomes quantified using NTA. (B) Western blot of HMEC B42 and B42 clone 16 exosomes demonstrating the presence of well established exosome markers (Alix and Tsg 101). (C) Western blot of B42 clone 16 exosomes demonstrating the levels of expression of the exosome markers (Alix and Tsg 101) at different times after incubating cells in exosome free medium. The table shows the number of exosomes per ml at the same time points.

the trends were similar demonstrating that MGH exosomes had a markedly less inhibitory effect on exosome release by breast cancer cells (Supplementary Fig. S3). 3.4. Incorporation of exosomes into cells Exosomes, derived from normal mammary epithelial cells labelled with PKH26 (red fluorescence) in vitro, were incubated with tumour cells and viewed by a confocal microscope. Aggregates of PKH26 labelled exosomes were incorporated into the tumour cells (Fig. 8). Labelled exosomes from the different normal and tumour cell combinations also were incorporated into the cells (Supplementary Fig. S4). Adding dye alone produced no intracellular incorporation. 4. Discussion How the number of exosomes in body fluids is controlled and whether exosome release is regulated is unknown. We have used a cell culture model using a human mammary epithelial cell line (HMEC B42) and a breast cancer cell line (B42 clone 16) derived from it to investigate this regulatory process. We have shown that exosomes in the extracellular environment of cells exert a feedback mechanism regulating the release of

exosomes from normal mammary epithelial cells and from breast cancer cells. The breast cancer cells produce increased numbers of exosomes relative to the same number of normal mammary epithelial cells. Evidence from patients with ovarian cancer and lung cancer indicate that there are increased numbers of exosomes in the blood of these patients [33]. Both the normal mammary epithelial cells and the breast cancer cells showed a plateau of exosome production over a 24 h period. In both cases the number of exosomes released in a 24 h collection period was less than that from two consecutive 12 h collections. Thus by removing the exosomes from the extracellular environment allowed the cells to produce a further cohort of exosomes. This suggested that a feedback mechanism existed. To test this hypothesis, concentrated suspensions of exosomes were added to exosome depleted medium to investigate whether this influenced exosome production. The exosomes were added in volumes of less than 200 ll to 3 ml of exosome depleted medium to eliminate any changes that might be attributed to spent medium. Exosomes derived from autologous cells had an inhibitory effect on exosome release from cells of the same type. The presence of exosomes in the extracellular environment of these cells inhibited exosome release by their own cells.

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Fig. 7. Specificity of the inhibition of exosome release. The number of exosomes produced in vitro was quantified using NTA. Three separate flasks were used per treatment and the exosome numbers quantified from three video recordings. Replicate experiments are presented in Supplementary Fig. S3. Error bars are plotted as s.e.m. Number of exosomes produced in a 12 h collection period by B42 clone 16 breast cancer cells in the presence and absence of added MGH (bladder cancer) exosomes ((7.19 ± 0.05)  108 per ml) to the culture. Control represents the number of exosomes produced in 12 h. The expected number of exosomes is plotted (i.e. number produced in 12 h plus number added to the culture) and the number of exosomes released following subtraction of the number added. There was no difference in the cell number per flask between the two groups (P = 0.18, n = 3, using the two tailed unpaired t test).

However, exosomes derived from tumour cells also inhibited exosome release from normal cells. Tumour cells produced more exosomes per cell than normal mammary epithelial cells and as exosomes from breast cancer cells could inhibit exosome release from mammary epithelial cells at concentrations of 10  108 exosomes per ml, this would suggest that tumour cells exert a dominant effect on the regulation of exosomes from normal cells. Exosomes derived from normal mammary epithelial cells had a dramatic inhibitory effect on breast cancer cell production of exosomes. Indeed less exosomes were detected when normal exosomes were added to the culture compared to control tumour cultures. The specificity of the interaction of exosomes with breast cancer cells was investigated by using exosomes from a bladder cancer cell line. Bladder cancer derived exosomes had little effect on exosome release by breast cancer cells suggesting that the response is tumour type specific. Exosomes from normal mammary epithelial cells at concentrations of 10–13  108 per ml had a dramatic inhibitory effect on exosome production by breast

cancer cells whereas exosomes from bladder cancer cells at a similar range of concentrations had little effect on exosome release from breast cancer cells. Several groups have demonstrated that tumour exosomes can be incorporated into tumour cells. Escrevante et al. [24] investigated exosomes from an ovarian cancer cell line (SKOV3) and showed that several endocytic pathways were involved in the uptake of exosomes. In a further study, breast cancer cells were used to stably express a GFP-tagged CD63 construct so that secreted exosomes were GFP labelled [27]. Transplantation of these cells into nude mice established that exosomes were transferred to other tumour cells and were circulating in the blood. Phenotypic traits of triple negative breast cancer cells were transferred by exosomes. Labelled exosomes were shown to be taken up by tumour cells [26]. Using optical sectioning with confocal microscopy, we have demonstrated that labelled exosomes from either normal or breast cancer cells are incorporated into both normal and cancer cells. Thus there is a clear interaction of exosomes derived from the extracellular environment with tumour cells and normal cells. Presumably a dynamic equilibrium exists between exosome release and uptake and is influenced by exosome concentration in the extracellular environment. Regulation of tumour exosome release could provide a new therapeutic strategy. Tumour exosomes induce a variety of deleterious responses in the patient. Inhibition of these responses would be advantageous. It has been proposed that removal of HER2 expressing exosomes in breast cancer by affinity plasmapheresis might prove a useful therapeutic adjuvant [16]. Thus having demonstrated a tissue specific response by exosomes derived from normal human mammary epithelial cells inhibiting specifically the release of exosomes from breast cancer cells opens up the potential for identifying the pathway of interaction and thus the possibility for developing novel small molecule inhibitors. Furthermore, it is possible that exosomes from normal cells may be attempting to suppress exosomeinduced activities of tumour cells. That this process may ultimately break down in the local microenvironment surrounding emerging tumour foci may identify a further change in progression of cancer in a patient. As tumour cells produce more exosomes than normal cells, it is also likely that tumour exosomes will dominate and inhibit exosome production by normal cells as the tumour increases in size. We have demonstrated that exosome release by human mammary epithelial cells and breast cancer cells is regulated by the presence of exosomes in the extracellular environment. Exosomes from normal mammary epithelial cells markedly inhibit exosome release from breast cancer cells. This represents a new regulatory pathway for the feedback control of exosome concentrations. It also seems likely that this

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Fig. 8. Incorporation of exosomes into tumour cells. B42 clone 16 breast tumour cells incubated with PKH26 labelled human mammary epithelial cell (HMEC) B42 exosomes for 3 h. Exosomes have been incorporated into the tumour cells.

is a generic mechanism and may be important for other tumour types. While the mechanism is unknown, Christianson et al. [34] have demonstrated that heparin exerts a dose dependent inhibition on cancer exosome uptake and cancer exosomes depend on cell-surface heparin sulphate proteoglycans for internalisation and functional activity. Similarly Yuyama et al. [35] have shown that annexin V blocks phosphatidylserine on neuron derived exosomes and prevents uptake into microglia. Conflict of interest statement None declared. Acknowledgements EB was funded by Alzheimers Research UK. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.ejca.2013.12.019.

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Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway.

Exosomes are small 50-100nm sized extracellular vesicles released from normal and tumour cells and are a source of a new intercellular communication p...
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