Due to limited availability of pharmacological therapies, triple-negative breast cancer (TNBC) is the subtype with worst outcome. We hypothesised that 2-Deoxy-D-Glucose (2-DG), a glucose analogue, may hold potential as a therapy for particularly aggressive TNBC. We investigated 2-DG’s effects on TNBC cell line variants, Hs578T parental cells and their isogenic more aggressive Hs578Ts(i) 8 variant, using migration, invasion and anoikis assays. We assessed their bioenergetics by Seahorse. We evaluated metabolic alterations using a Seahorse XF Analyzer, citrate synthase assay, immunoblotting and flow cytometry. We assessed the cancer stem cell (CSC) phenotype of the variants and 2-DG’s effects on CSCs. 2-DG significantly inhibited migration and invasion of Hs578Ts(i) 8 versus Hs578T and significantly decreased their ability to resist anoikis . Investigating 2-DG’s preferential inhibitory effect on the more aggressive cells, we found Hs578Ts(i) 8 also had significantly decreased oxidative phosphorylation and increased glycolysis compared to Hs578T. This is likely due to mitochondrial dysfunction in Hs578Ts(i) 8 , shown by their significantly decreased mitochondrial membrane potential. Furthermore, Hs578Ts(i) 8 had a significantly increased proportion of cells with CSC phenotype, which was significantly decreased by 2-DG. 2-DG may have benefit as a therapy for TNBC with a particularly aggressive phenotype, by targeting increased glycolysis. Studies of more cell lines and patients’ specimens are warranted.

Extracellular vesicles (EVs) have great potential as drug delivery vehicles. While mesenchymal/stromal stem cell (MSC) conditioned medium (CM) and milk are potentially safe and scalable sources of EVs for this purpose, the suitability of MSC EVs and milk EVs as drug delivery vehicles has never been compared and so was the objective of this study. Here EVs were separated from MSCs’ CM and from milk and were characterised by nanoparticle tracking analysis, transmission electron microscopy, total protein quantification, and immunoblotting. An anti-cancer chemotherapeutic drug, doxorubicin (Dox), was then loaded into the EVs by one of three methods: by passive loading or by active loading by either electroporation or sonication. Dox-loaded EVs were analysed by fluorescence spectrophotometer, high-performance liquid chromatography (HPLC), and imaging flow cytometer (IFCM). Our study showed that EVs were successfully separated from the milk and MSC CM, with significantly (p < 0.001) higher yields of milk EVs/mL starting material compared to MSC EVs/mL of starting material. Using a fixed amount of EVs for each comparison, electroporation achieved significantly more Dox loading when compared to passive loading (p < 0.01). Indeed, of 250 µg of Dox made available for loading, electroporation resulted in 90.1 ± 12 µg of Dox loading into MSC EVs and 68.0 ± 10 µg of Dox loading into milk EVs, as analysed by HPLC. Interestingly, compared to the passive loading and electroporation approach, after sonication significantly fewer CD9+ EVs/mL (p < 0.001) and CD63+ EVs/mL (p < 0.001) existed, as determined by IFCM. This observation indicates that sonication, in particular, may have detrimental effects on EVs. In conclusion, EVs can be successfully separated from both MSC CM and milk, with milk being a particularly rich source. Of the three methods tested, electroporation appears to be superior for achieving maximum drug loading while not causing damage to EV surface proteins.

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Background Cancer cells release heterogeneous populations of extracellular vesicles (EVs) that transmit aggressive phenotypic traits to recipient cells. We aimed to establish if the heterogenous EVs population or a sub-population is responsible, if we could block undesirable cell-to-cell communication by EVs, and, if some EVs continued to be released, would their undesirable influences on recipient cells continue. Methods Three triple-negative breast cancer (TNBC) cell lines were used. Non-toxic concentrations of calpeptin, Y27632, manumycin A, GW4869 and combinations thereof were tested to block EVs. Ultracentrifugation-based methods collected EVs, which were then characterised by nanoparticle tracking analysis, immunoblotting, and transmission electron microscopy. A quick screening flow cytometry method evaluated EVs in solution. The influences of EVs on recipient cells’ migration was investigated. Results All EV sub-populations were apparently involved in transmitting undesirable phenotypic characteristics. All compounds/combinations significantly (64–98%) reduced EVs’ release. Our quick screening broadly reflected our more comprehensive EVs analysis. The 2–36% of EVs that continued to be released caused less transmission to recipient cells, but not on a comparable scale to the reduction of EVs release achieved. Conclusion Up to 98% inhibition of EVs’ release was achieved. To prevent the transmission of undesirable phenotypic traits by EVs, their total inhibition may be necessary.

Background Cancer patients have an increased risk of developing venous thromboembolism, with up to 30% dying within a month of their development. Some cancer cells are known to induce platelet aggregation, and this interaction is understood to contribute to thrombosis and haematogenous metastasis. Many researchers have reported on extracellular vesicles (EVs) released from platelets. However, less is known about how cancer cells’ EVs may affect platelet function. Here EVs released by triple-negative breast cancer (TNBC) cell line variants were extensively investigated in this regard. Methods EVs were separated from conditioned media of TNBC Hs578T and Hs578Ts(i)8 cells using filtration and ultracentrifugation and were characterised by nanoparticle tracking analysis, immunoblots, and transmission electron microscopy. Blood samples from consenting donors were procured, and their platelets collected by differential centrifugation. Light transmission aggregometry and optical microscopy evaluated the potential interaction of TNBC cells and their EVs with platelets. Global proteomic analysis was performed on the EVs, by in-solution digestion and mass spectrometry. Data analysis included the use of Perseus, FunRich, and Vesiclepedia. Immunoblotting was used as a secondary method to investigate some key EV cargo proteins identified by the global proteomics approach. Results Both TNBC cell variants induced platelet aggregation. Increasing cell numbers significantly reduced the time taken for platelet aggregation to occur. EVs released by the cells also resulted in platelet aggregation. The time to induce platelet aggregation was EV dose-dependent. Proteomics profiling and immunoblotting of the EVs’ cargo identified candidate proteins (including uPAR and PDGFRβ) that may be involved during this process. Conclusions TNBC cells induce platelet aggregation. Furthermore, the cell-free EVs induced this undesirable effect. A number of EV cargo proteins were identified that may be relevant as therapeutic targets.