Exosomes from triple-negative breast cancer cells can transfer phenotypic traits representing their cells of origin to secondary cells

https://doi.org/10.1016/j.ejca.2013.01.017Get rights and content

Abstract

Background

Triple-negative breast cancer (TNBC) accounts for 15–20% of breast cancers but is responsible for a disproportionate number of deaths. We investigated the relevance, in TNBC, of nano-sized exosomes expelled from cells. Specifically, we compared effects of exosomes derived from the claudin-low TNBC cell line Hs578T and its more invasive Hs578Ts(i)8 variant, as well as exosomes from TNBC patient sera compared to normal sera.

Methods

Exosomes were isolated from conditioned media (CM) of Hs578T and Hs578Ts(i)8 cells and from sera by filtration and ultracentrifugation. Successful isolation was confirmed by transmission electron microscopy and immunoblotting. Subsequent analysis, of secondary/recipient cells in response to exosomes, included proliferation; motility/migration; invasion; anoikis assays and endothelial tubule formation assays.

Results

Hs578Ts(i)8-exosomes versus Hs578T-exosomes significantly increased the proliferation, migration and invasion capacity of all three recipient cell lines evaluated i.e. SKBR3, MDA-MB-231 and HCC1954. Exosomes from Hs578Ts(i)8 cells also conferred increased invasiveness to parent Hs578T cells. Hs578Ts(i)8-exosomes increased sensitivity of SKBR3, MDA-MB-231 and HCC1954 to anoikis when compared to the effects of Hs578T-exosomes reflecting the fact that Hs578Ts(i)8 cells are themselves innately more sensitive to anoikis. In relation to vasculogenesis and subsequent angiogenesis, Hs578Ts(i)8-exosomes versus Hs578T-exosomes stimulated significantly more endothelial tubules formation. Finally, our pilot translational study showed that exosomes from TNBC patients’ sera significantly increased recipient cells’ invasion when compared to those derived from age- and gender-matched healthy control sera.

Conclusion

This study supports the hypothesis that TNBC exosomes may be involved in cancer cell-to-cell communication, conferring phenotypic traits to secondary cells that reflect those of their cells of origin.

Introduction

Exosomes and microvesicles expelled from eukaryotic cells have attracted much recent interest since the realisation that these are not merely debris, but rather miniature maps of their cells of origin with both physiological and pathological relevance.1 Accumulating evidence indicates that exosomes (∼30–120 nm) and microvesicles (up to 1000 nm) may play an important role in cell-to-cell communication.2, 3, 4 In cancer, these entities have been implicated in carcinogenesis and tumour growth; moulding the tumour microenvironment; promoting angiogenesis and modulating immune response.5, 6, 7, 8, 9

Triple-negative breast cancers (TNBCs), although constituting a minority of breast cancers (15–20%), cause a disproportionate number of metastatic cases and breast cancer deaths. TNBCs, which have ∼70–80% overlap with the basal subtypes,10, 11 are classified by their lack of expression of oestrogen receptor (ER) and progesterone receptor (PR) and neither over-expression nor amplification of human epidermal growth factor receptor 2 (HER2) gene. This substantially limits treatment options because of their insensitivity to widely used targeted approaches such as endocrine therapies and trastuzumab that have proven effective at reducing breast cancer mortality. Additionally, compared to other breast tumours, TNBCs occur most frequently in younger patients (<50 yrs), and those tumours tend to be of higher-grade at presentation with more aggressive behaviour. These cancers typically relapse with distant metastases rather than with local, more manageable, recurrence and have increased likelihood of developing visceral metastases including central nervous involvement. TNBC is associated with shortened disease-free interval post-surgery and post-adjuvant treatment and, ultimately, a shortened overall survival.12, 13, 14, 15 Furthermore, TNBCs are themselves a heterogeneous group within which the claudin-low subtype is associated with worst prognosis.16

Compared to the overall prevalence of breast cancer, a limited number of reports have indicated exosomes and/or microvesicles to be of relevance in breast cancer.17, 18, 19 The focus, however, has typically been on HER2-overexpressing cells and their exosomes. Here, in what we believe to be the first study specifically considering exosomes in the context of TNBC, we have included analysis of exosomes from the claudin-low TNBC cell line Hs578T20 and its isogenic subclone, Hs578Ts(i)8,21 as well as exosomes from TNBC patients compared to healthy controls. Hs578Ts(i)8, compared to Hs578T parent cells, has the same genetic background but significantly increased capacity to proliferate, migrate, invade through extracellular matrix and produce tumours in mice. This cell line pair is, therefore, very useful for investigating the capabilities of exosomes to transfer phenotypic traits representative of their cells of origin to secondary recipient cells.

Section snippets

Cell culture

All cell lines are from breast tissue and were obtained from ATCC and cultured at 37 °C/5% CO2. Hs578T and it’s isogenic subclone, Hs578Ts(i)8 were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma–Aldrich), 10% foetal bovine serum (FBS) (PAA), 2 mM l-glutamine (Sigma–Aldrich), and 10 μg/ml of insulin (Sigma–Aldrich). SKBR3, MDA-MB-231, and HCC1954 were grown in RPMI-1640 (Sigma–Aldrich), 10% FBS, 2 mM l-glutamine.

Conditioned medium (CM)

Hs578T and Hs578Ts(i)8 were seeded (1 × 105 cells/flask) in 9xT75 flasks in

Exosomes were successfully isolated and taken up by recipient/secondary cells

After performing the isolation procedure, the first step in our analysis was to establish if we had successfully isolated exosomes. To assess this we performed immunoblot analysis to determine the presence of three proteins accepted as markers of exosomes. These include PDC6I/Alix, TSG101 and CD63. We also elected to perform transmission electron microscopy (TEM) analysis to identify if the vesicles isolated were typically with the expected size-range (30–120 nm) for exosomes. Comparative total

Discussion

A limited number of studies have indicated breast cancer cell-derived exosomes and/or microvesicles to have functional relevance. Lau and Wong19 assessed the effects of MDA-MB-231 ‘exosome-like microvesicles’ on human submandibular gland (HSG) cells, using lysed exosome-like microvesicles as controls. Intact exosome-like particles, compared to controls, increased the total amount of RNA in HSG cells and resulted in increased levels of 88 proteins in subsequent HSG-derived exosome-like

Role of funding source

Student scholarships, test materials, equipment, etc. are supported by these funds. The sponsors had no involvement in the study design through to manuscript preparation.

Conflict of interest statement

None of the authors have any conflict of interest to declare.

Acknowledgements

The authors wish to thank Mr. Neal Leddy Chief Technical Officer Specialist for his microscopy expertise. We also gratefully acknowledge funding support in the form of the Marie Keating Foundation PhD Scholarship at Trinity College Dublin (LOD); Trinity Foundation, Trinity College Dublin (LOD); Science Foundation Ireland’s funding of Strategic Research Cluster, Molecular Therapeutics for Cancer Ireland 08/SRC/B1410 (JC, LOD); the Irish Research Council for Science, Engineering & Technology (RW,

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