Imaging exosome transfer from breast cancer cells to stroma at metastatic sites in orthotopic nude-mouse models☆
Graphical abstract
Introduction
Exosomes are small (30–100 nm) membrane vesicles that originate from the endosomal membrane compartment [1]. Recently, it has been demonstrated that cells shed exosomes containing significant amounts of mRNAs, miRNAs and proteins for transferring genetic and proteomic information to target cells as a way of cell-to-cell communication [1], [2], [3]. Cancer cells actively release their exosomes into the tumor microenvironment and peripheral blood of cancer patients [1], [4].
Cancer-cell-derived exosomes stimulate secretion of growth factors, cytokines and angiopoietic factors by stroma cells, induce proliferation of endothelial cells, and promote angiogenesis in metastatic organs [1], [2], [5], [6]. Thus, cancer-cell-derived exosomes are able to alter the cellular environment to form a metastatic niche and promote metastasis [7], [8]. Hence, the tracking fate of cancer-cell-derived exosomes in a metastatic models may help us understand the mechanism of cancer metastasis.
Breast cancer is the most common cancer and the leading cause of cancer death among women worldwide [9]. Metastasis is the major cause of death in breast cancer [9]. Therefore novel biomarkers for detection of early metastasis and therapeutic strategies as well as elucidation of the molecular mechanisms underlying the metastatic process, are urgently required.
In the present report, we describe imaging of transfer of cancer-cell-derived GFP-labeled exosomes in vitro and in mouse models of metastatic breast cancer.
Section snippets
Markers of exosomes
Several proteins may serve as markers of exosomes [10], [11]. Cytoplasmic proteins such as molecular chaperon/heat shock proteins Hsp70 and Hsp90, and ESCRT (endosomal sorting complex required for transport)-associated proteins Alix and Tsg101 exist in exosomes [10], [11]. Some membrane-associated proteins such as tetraspanins CD9, CD63 and CD81, which are involved in adhesion and targeting, are found on the surface of exosomes [10], [11].
For imaging of exosomes, we used the well-known exosomal
Cell-to-cell transfer of cancer-cell-derived exosomes
Cancer-cell-shed exosomes/microvesicles which transfer genetic and proteomic information to target cells as a way of cell-to-cell communication. For example, primary human glioblastoma cells were observed to secrete microvesicles, including exosomes, into the culture medium by scanning electron microscopy [2]. Glioblastoma microvesicles purified from conditioned medium were internalized by human brain microvascular endothelial (HBMVEC) and delivered functional RNA to the recipient cells [2].
Cancer cells secrete exosomes into the tumor microenvironment
It has been suggested that cancer cells shed exosomes and condition their niche via exosomal material to promote tumor growth and metastasis. We examined whether cancer cells secrete exosomes into the surrounding tissue in breast cancer models using CLSM imaging. We established an orthotopic mouse model of breast cancer metastasis to the lung by orthotopic implantation of MMT mouse breast cancer cells into the mammary fat pad (MFP) of nude mice (Fig. 3A, C). MMT orthotopic model forms primary
Conclusions and perspectives
The combination of GFP-tagged exosomes and CLSM is useful for imaging exosomes in vitro and in vivo. Imaging of exosomes can contribute to understanding the role of intercellular communication in tumor progression.
The cancer stem cell (CSC) theory proposes that a small subset of cells within a heterogeneous tumor have stem cell-like properties and produce new heterogeneous tumor cells [21], [22], [23]. This theory suggests that CSCs lead to metastasis while closely interacting with non-CSCs and
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This review is part of the Advanced Drug Delivery Reviews theme issue on “Exosomes: a key to delivering genetic materials”.
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These authors contributed equally to this work.