Introduction Exosomes are nano-sized (40–100 nm) extracellular vesicles which are exchanged by cancer and stromal cells. They contain proteins and nucleotides (DNA, mRNA, microRNA) and are therefore capable of conveying genetic information. Exosome transfer affects cellular function but this is not well defined in the colorectal cancer (CRC) setting. We aimed to isolate, label and transfer exosomes from colorectal cancer cells to fibroblasts and vice versa. Moreover, we investigated the effect of exosome transfer at the cellular level, focussing on two key pathways: ERK and AKT.
Method Exosomes were isolated from conditioned media from DLD-1 CRC cells or MRC5 fibroblasts by selective centrifugation and validated by transmission electron microscopy, western blotting and flow cytometry. The resulting exosome pellet was labelled with a lipophilic (green) fluorescent dye and co-cultured with the reciprocal target cell (MRC5 or DLD-1). The cells were then washed thoroughly to remove all extracellular particles and visualised by fluorescence microscopy. Cell lysate was analysed by western blotting for activation of key signalling pathways such as Ras (ERK) and AKT.
Results Microscopy revealed presence of fluorescent labelled exosomes within both DLD-1 and MRC5 cells following exosome co-culture. There was marked increase in AKT pathway activation in MRC5 fibroblasts after co-culture with DLD-1 CRC exosomes. As a result, significant changes to anti-apoptotic stimuli have been observed. There was a marked increase in ERK pathway activation and moderate increase in AKT activation in DLD-1 CRC cells after co-culture with MRC5 fibroblast exosomes. Again, significant changes to downstream anti-apoptotic signals were observed.
Conclusion We have shown that CRC exosomes can be transferred to fibroblasts and vice versa. This transfer induces selective phosphorylation of AKT and resistance to apoptosis in recipient fibroblasts and CRC cells. Stromal and tumour-derived exosomes promote resistance to apoptosis and can create an apoptosis resistant niche in the tumour microenvironment.
Disclosure of interest None Declared.
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