Abstract
PurposeExtracellular vesicles, small vesicles carrying inter alia proteins, miRNA and RNA, are important mediators of intercellular communication. The purpose of this study was to assess the distribution of extracellular vesicles from highly malignant breast cancer and their subsequent effect on the immune cell infiltrate in target organs of metastasis.ProceduresExtracellular vesicles were isolated from the tissue culture supernatant of highly malignant 4T1 breast cancer cells or the serum of healthy BALB/c mice. The purity of the isolate was verified by electron microscopy and western blotting. Extracellular vesicles were additionally subjected to proteome analysis. After labeling with the fluorescent dye DiR, extracellular vesicles were injected into healthy BALB/c mice and their in vivo distribution was assessed using fluorescence reflectance imaging (FRI). Following ex vivo imaging of the organs, lung tissue samples were analyzed for extracellular vesicle-mediated changes of myeloid cells and T cell numbers, using flow cytometry. Proteome analysis revealed major differences in the cargo of tumor cell–derived versus extracellular vesicles from healthy serum.ResultsIn contrast to control extracellular vesicles, DiR-labeled extracellular vesicles from tumor cells preferentially accumulated in lung, liver, and spine. Subsequent flow cytometry of the immune cell composition of lung tissue samples revealed an increase of cytotoxic CD8+ T cells and a decrease of CD4+ T-helper cells as well as an increase in mature macrophages in response to tumor cell EV.ConclusionsIn conclusion, distribution of tumor cell–derived extracellular vesicles follows a specific pattern and can be monitored, using dedicated imaging. Extracellular vesicles alter the immune cell composition in target organs of metastasis, using a specific proteome cargo.
Highlights
Cancer cells rely on the recruitment and activation of various benign, physiological cells, most prominently immune cells, to facilitate and support growth, invasion, and metastasis
We further examined the biological effects of tumor cell– derived extracellular vesicles in the lungs of extracellular vesicle-treated animals
Tracking of DiR-labeled extracellular vesicles in vivo using fluorescence reflectance imaging enables the in vivo visualization of their distribution and specific accumulation in target organs of subsequent metastasis
Summary
Cancer cells rely on the recruitment and activation of various benign, physiological cells, most prominently immune cells, to facilitate and support growth, invasion, and metastasis. Tumor cells exhibit an enhanced secretion of extracellular vesicles, which are of 50–150-nm diameter in size [3]. They contain inter alia (mi)RNA, proteins, and peptides and their individual content largely reflects the parental cell [4]. As the transport medium of a variety of molecules, extracellular vesicles are thought to contribute to metastatic spread via aid in induction of a premetastatic niche. This premetastatic niche forms as a tumor-supportive inflammatory environment, detectable in the target tissue before the establishment of metastases [5]. After fusion of multivesicular bodies with the cell membrane and release into the blood stream, extracellular vesicles distribute systemically, where they exert their effects in one of three different ways: (1) binding of extracellular vesicles to a membrane protein of the target cell activates a dedicated signal pathway inside the cell; (2) a protease in the extracellular matrix cleaves the extracellular vesicle membrane proteins, which afterwards bind to receptors on the cell membrane, activating a signaling pathway; (3) the extracellular vesicle membrane fuses with the target cell membrane, causing nonselective release of its content [6]
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