Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods.

Assessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals.

The Challenges and Possibilities of Extracellular Vesicles as Therapeutic Vehicles.

Extracellular vesicles (EVs) are small lipid-enclosed particles that can carry various types of cargo, including proteins, nucleic acids and metabolites. They are known to be released by all cell types and can be taken up by other cells, leading to the transfer of the cargo they carry. As such, they represent an important type of intercellular signalling and a natural mechanism for transferring macromolecules between cells. This ability to transfer cargo could be harnessed to deliver therapeutic molecules. Indeed, a growing body of work has described the attempt by the field to utilise EVs to deliver a range of therapeutics including RNAi, CRISPR/Cas9 and chemotherapeutics, to a specific target tissue. However, there are numerous barriers associated with the use of EVs as therapeutic vehicles, including the challenge of efficiently loading therapeutics into EVs, avoiding clearance of the EVs from circulation, targeting the correct tissue type and the inefficiency of internalisation and functional delivery of the cargo. Despite these difficulties, EVs represent a tremendous therapeutic opportunity, both for the delivery of exogenous cargo, as well as the therapeutic benefit of targeting aberrant EV signalling or treating patients with natural EVs, such as those released by mesenchymal stem cells. This review describes current knowledge on the therapeutic potential of EVs and the challenges faced by the field. Many of these challenges are due to a lack of complete understanding of EV function, but further research in this area should continue to yield new solutions that will lead to the use of EVs in the clinic.

The development of cisplatin resistance in neuroblastoma is accompanied by epithelial to mesenchymal transition in vitro.

Neuroblastoma is a challenging childhood malignancy, with a very high percentage of patients relapsing following acquisition of drug resistance, thereby necessitating the identification of mechanisms of drug resistance as well as new biological targets contributing to the aggressive pathogenicity of the disease. In order to investigate the molecular pathways that are involved with drug resistance in neuroblastoma, we have developed and characterised cisplatin resistant sublines SK-N-ASCis24, KellyCis83 and CHP-212Cis100, integrating data of cell behaviour, cytotoxicity, genomic alterations and modulation of protein expression. All three cisplatin resistant cell lines demonstrated cross resistance to temozolomide, etoposide and irinotecan, all of which are drugs in re-initiation therapy. Array CGH analysis indicated that resistant lines have acquired additional genomic imbalances. Differentially expressed proteins were identified by mass spectrometry and classified by bioinformatics tools according to their molecular and cellular functions and their involvement into biological pathways. Significant changes in the expression of proteins involved with pathways such as actin cytoskeletal signalling (p = 9.28E-10), integrin linked kinase (ILK) signalling (p = 4.01E-8), epithelial adherens junctions signalling (p = 5.49E-8) and remodelling of epithelial adherens junctions (p = 5.87E-8) pointed towards a mesenchymal phenotype developed by cisplatin resistant SK-N-ASCis24. Western blotting and confocal microscopy of MYH9, ACTN4 and ROCK1 coupled with invasion assays provide evidence that elevated levels of MYH9 and ACTN4 and reduced levels of ROCK1 contribute to the increased ROCK1-independent migratory potential of SK-N-ASCis24. Therefore, our results suggest that epithelial-to-mesenchymal transition is a feature during the development of drug resistance in neuroblastoma.