Investigations of extracellular vesicles in triple-negative breast cancer

Abstract

Breast cancer is the most commonly diagnosed cancer worldwide accounting for 11.7% of the newly diagnosed 19.3 million cases in 2020. In Ireland breast cancer accounted for 10.8% of cancer cases and 23.8% of female cancers. Triple-negative breast cancer (TNBC), a subtype of breast cancer that lacks expression of the oestrogen receptor (ER), the progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2), accounts for 10-20% of all breast cancers. It is heterogeneous in nature, and unlike the other subtypes, has no identified molecular targets for improving treatment options. TNBC is often an aggressive cancer, commonly associated with younger women, and with poor overall survival. The main focus of this project was the role of extracellular vesicles (EVs) in TNBC. EVs are described as mini-maps of their cell of origin and are involved in cell-to-cell communication. In TNBC, initially our group and subsequently others have shown that EVs were able to pass on aggressive traits to recipient cells, prevent cancer cell death by exporting chemotherapeutic drugs, re-programme the tumour microenvironment, support pre-metastatic niche formation, and enhance the metastasis of TNBC cells. Thus, this project started by separating four EV sub-populations from the heterogeneous pool of EVs released from TNBC cells, in order to identify which sub-populations are most responsible for the transfer of the negative effects. We found that all four sub-populations played a part in the transfer of increased cell proliferation, migration, invasion and anoikis resistance. No one sub-population was responsible; rather the total heterogeneous population. Therefore, the next logical step was to investigate ways which we might block the release of EVs from the TNBC cells, in an effort to prevent the spread of these undesirable traits. Using proposed EV inhibitors including calpeptin, Y27632, manumycin A, and GW4869- at non-toxic concentrations- we quantified and characterised any EVs that continued to be released after inhibitor treatment. We found that EV quantities were substantially inhibited after treatment. Specifically, calpeptin (p<0.0001), Y27632 (p<0.0001), combo 1 (p<0.0001), manumycin A (p=0.001), GW4869 (p<0.0001) and combo 2 (p<0.0001) all significantly decreased EV release. GW4869 caused the greatest levels of decrease with 98% reduction in EV release compared to the control. Furthermore, the TNBC EVs that were still released after inhibitor treatment did not stimulate migration of recipient TNBC cells when compared to EVs released from TNBC cells not treated with an inhibitor. Specifically, BT549 migration was significantly reduced when cells were treated with the highest dose of EVs released following calpeptin (p=0.036), Y27632 (p=0.007), combo 1 (p=0.005), manumycin A (p=0.005), GW4869 (p=0.005) and combo 2 (p=0.017) treatment. Venous thromboembolism (VTE), a subset of thromboembolism, is the second leading cause of cancer-related fatality, with cancer patients accounting for 20% of all VTE cases. We identified that two TNBC cell line variants and their corresponding EVs can induce platelet aggregation. Our global proteomic profiling of the EVs cargo identified a number of proteins (including platelet derived growth factor receptor &#946; (PDGFR&#946;), Protein Cyr61, mucin 18 (MUC18), urokinase plasminogen activator receptor (uPAR), CD97 and glypican-1) that may be involved in this platelet aggregation. Additionally, EVs may be used as diagnostic tools. However, the methods typically used for EV separation and characterisation, by the EV Community, are not realistically translatable to clinical utility. Therefore, we compared multiple methods of EV separation from serum (breast cancer patients and age-matched healthy controls) with the intention of finding a method that would result in reasonably pure, intact, EVs that were not contaminated with serum-derived proteins; but with a particular interest in potential future clinical utility. To do this, we evaluated differential ultracentrifugation, magnetic immunobeads capture, size exclusion chromatography, nickel-based separation, and precipitation with poly(ethylene glycol) (PEG). To conclude, we found that a magnetic immunobead approach provided an EV separation method that resulted in a higher purity when compared to other methods tested. Although, every method has its disadvantages, this method provides an easy, reliable, and fast method of EV separation that could be realistically translated into a hospital setting. In conclusion, our results demonstrate that TNBC EVs may play a role in the many facets of TNBC disease, but that the increased migration associated with the TNBC EVs may be reduced through their inhibition. Although our results are preliminary, some EV protein cargo was identified that may be targetable to inhibit VTE in cancer patients or help identify patients who are most at risk of developing VTE. There are many different methods of EV separation for patient-derived blood-based samples. We have demonstrated that EVs from serum can be separated using a user-friendly, easy method that would be realistically translated to patient care.