Investigations into the dynamic uptake of extracellular vesicles and their role in breast cancer metastasis after chemotherapy
Extracellular vesicles (EVs) are small lipid bound spherical structures released by almost all types of cells. They are mediators of intercellular communication, however, the exact mechanisms of EV-cell interactions and uptake have not been fully characterised. EVs play key roles in cancer including all the stages of metastasis. Breast cancer is amongst the most common types of cancer for women and the primary cause of cancer related death. The high mortality rates are due to the metastatic spread of cancer cells and tumour recurrence after therapy. Studies have proposed chemotherapy to be a double-edged sword, which in some cases can promote metastasis. Here, it was hypothesised that EVs from chemotherapy treated cells were able to induce metastasis-related capacities to naïve cells. The aims of this project were: a) to explore the dynamics and specificity of EV uptake by recipient cells, and b) to investigate the effect of chemotherapy induced intercellular communication via EVs on breast cancer metastasis. To study EV uptake, different methods of EV labelling were tested. It was observed that PKH26 labelling may result in false-positive signals due to micelle formation. EV labelling with CFSE also produced non-specific effects, while dual labelling with both PKH26 and CFSE resulted in EV populations with heterogeneous labelling. EVs bearing a fluorescently tagged EV marker (mEmerald-CD81) were preferential for experiments investigating EV dynamics, as labelling appeared to be more specific than the dyes previously tested. Using this type of labelled EVs, it was shown that EV uptake differs across cell lines and the EV origin affects docking on the plasma membrane. Using a range of uptake inhibitors, it was observed that cholesterol may be involved in early EV-plasma membrane interactions, thus affecting EV uptake. Single molecule tracking revealed two patterns of such interactions: short and fast, and long and slow. To study the effect of EVs after chemotherapy, docetaxel and mitomycin C, two anti-cancer agents used for the treatment of breast cancer, were employed. Chemotherapy-treated cells released significantly higher numbers of EVs compared to non-treated cells. EVs from chemotherapy-treated cells did not induce any pro-metastatic effects (increased motility and invasiveness, EMT, and ability to adhere to endothelial cells), although changes in the glycosylation patterns of recipient cells were observed. Overall, these results: highlight the variability amongst different EV labelling methods; suggest a role for cholesterol in early EV-membrane interactions and specificity of uptake; and indicate that chemotherapy-derived EVs may confer glycosylation changes but not pro-metastatic effects in recipient cells.
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