The role of exosomes in tumour-stroma interaction in colorectal cancer

Abstract

Colorectal cancer is a global problem with rising incidence. Death is usually due to metastatic dissemination, and therefore, biological factors which influence disease progression are an important focus of study.

The tumour microenvironment is a functional ecosystem of cancer and stromal cells. The stroma plays a critical role in tumour proliferation, invasion and chemoresistance. Cancer-associated fibroblasts, the most abundant stromal cells, are associated with multiple pleiotropic processes, including tumorigenesis, proliferation, angiogenesis, epithelial-mesenchymal transition and resistance to treatment. The bidirectional transfer of information between tumour and stromal compartments is therefore important to elucidate.

One mechanism of paracrine signalling between cancer cells and stromal fibroblasts is by exosomes. These sub-100 nm nanoparticles have a lipid bilayer structure, contain lipid, protein and nucleic acid cargos, and are secreted by all cells. Importantly, these cargos are functional, such that one cell can alter the phenotype of another by exosome transfer. The most stable cargo is microRNA, small non-coding RNA which post-transcriptionally regulates over one-third of all human genes.

This thesis investigates the reciprocal transfer of exosomal microRNAs between cancer cells and stromal fibroblasts, and the effect of this on colorectal cancer progression. The first part demonstrates techniques to isolate, characterise, label and transfer exosomes from cancer cells and fibroblasts. Exosome transfer resulted in miRNA alterations in recipient cells, activation of ERK/ Akt pathways, and functional consequences for proliferation and apoptosis. In vivo exosome transfer was demonstrated by generating CRC cells expressing the CD63-GFP fusion protein, which transmitted GFP-positive exosomes to fibroblasts in murine tumour xenografts.

The second part investigates stroma to tumour exosome transfer. Here, exosomal microRNAs were profiled from paired patient-derived normal and cancer-associated fibroblasts. A colorecta
cancer stromal exosome panel consisting of microRNAs 329, 181a, 199b, 382, 215 and 21 was identified. Of these, miR-21 had highest abundance and was enriched in exosomes compared to parent cells. Transfer of stromal exosomes to colorectal cancer cells increased miR-21 levels in recipient cells. Orthotopic xenografts, established with miR-21-overexpressing fibroblasts and colorectal cancer cells, led to increased liver metastases, compared to those established with control fibroblasts, highlighting the role of stromal miR-21 in colorectal cancer progression.

The third part investigates tumour to stroma exosome transfer, specifically the influence of epithelial-mesenchymal transition on fibroblast phenotype. Here, exosomes from a panel of epithelial and mesenchymal colorectal cancer cells were used to condition fibroblasts. Epithelial exosomes, rich in miR-200, increased this microRNA in recipient fibroblasts, repressing fibroblast Zinc finger E-box-binding homeobox 1, and reducing transforming growth factor-β-induced myofibroblast transdifferentiation. The converse was true of mesenchymal exosomes, which allowed unattenuated myofibroblast differentiation. Fitting with this, mesenchymal colorectal cancer xenografts contained fibroblasts with less miR-200, expressing more α-SMA and fibronectin, compared to fibroblasts from epithelial xenografts. This provides a mechanism for the accumulation of activated fibroblasts in mensenchymal (metastatic) tumours.

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