Small extracellular vesicle communication and cisplatin resistance in the neuroblastoma microenvironment
Creators: name: Frawley, Thomas
Publisher: name: Royal College of Surgeons in Ireland
Year: 2023
Subjects: classificationCode: 321199
subject: Oncology and carcinogenesis not elsewhere classified
classificationCode: 310199
subject: Biochemistry and cell biology not elsewhere classified

description: Neuroblastoma is a paediatric cancer that accounts for approximately 15% of all childhood cancers deaths. The heterogeneous nature of neuroblastoma means that some patient’s disease will spontaneously regress while others will develop distant metastases and resistance to chemotherapy. Therefore, discovery of new mechanisms involved in the acquisition of these aggressive traits is essential for overcoming these therapeutic obstacles. Exosomes or small extracellular vesicles (sEV) are a population of membrane bound vesicles that mediate cell-cell communication by transferring bioactive cargo between cells. The molecular cargo of sEV is partly cell type dependent and determines their function. In cancer, sEV derived from tumour cells can aid in the acquisition of several hallmarks of cancer. It has also been reported that the development of resistance to chemotherapy drugs can alter the cargo and function of tumour derived sEV, in numerous cancers. Accordingly, we hypothesise that the development of cisplatin resistance in neuroblastoma alters the proteome and function of tumour derived sEV resulting in enhanced tumour progression in the neuroblastoma microenvironment. To test our hypothesis, we utilized a cisplatin resistant model of neuroblastoma developed in our lab, comprising the MYCN amplified neuronal cell line Kelly and its cisplatin resistant subline KellyCis83. First, we characterised a new method to produce larger amounts of sEV from Kelly and KellyCis83 for downstream analysis, improving the efficiency of our experimental setup. Next, we implemented a standardised approach to characterise sEV for size, protein content and morphology, using the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines as a framework. Additionally, we characterised Kelly and KellyCis83 sEV by the number of secreted sEV, tetraspanin surface expression and co-localisation and by mass spectrometry to characterise the full sEV proteome. Mass spectrometry analysis of sEV derived from Kelly (cisplatin sensitive) and KellyCis83 (cisplatin resistant) cells revealed 243 differentially expressed proteins, out of 2,625 detected proteins. Demonstrating that the development of cisplatin resistance alters the proteome of sEV. Functional enrichment analysis of differentially expressed proteins in KellyCis83 sEV identified several biological processes related to metabolism, regulation of cell cycle and regulation of Wnt signalling. To facilitate the investigation of the role of sEV, derived from our cisplatin resistant model of neuroblastoma, we bioengineered Kelly sEV to express increased levels of EGFR. We also characterised and validated the endogenous labelling of sEV with the near infrared fluorophore, NIR-AZA 1, to enable visualisation of sEV uptake in the neuroblastoma microenvironment. To build upon the knowledge gained from proteomic analysis of Kelly and KellyCis83 sEV, we investigated their functional properties in the neuroblastoma microenvironment. First, we demonstrated sEV derived from cisplatin resistant KellyCis83 cells increase tubule formation in HUVECs compared to the untreated negative control and Kelly sEV. We determined EGFR does not contribute to KellyCis83 sEV mediated tubule formation in HUVECs. Next, we demonstrated sEV derived from both Kelly and KellyCis83 cells partially inhibit HUVEC proliferation and metabolically reprogram HUVECs by increasing lactate secretion, a by-product of glycolysis. Finally, we determined that both Kelly and KellyCis83 sEV have no effect on HUVEC migration. We identified an enrichment of proteins in KellyCis83 sEV related to Wnt signalling, which regulates several biological processes in neuroblastoma, including proliferation, migration and cell survival. Therefore, we tested if KellyCis83 could transfer these traits, or hallmarks of cancer, to other neuroblastoma cells through sEV signalling. Firstly, we demonstrated that Kelly and KellyCis83 sEV have no effect on the migration of Kelly, KellyCis83 or SH-SY5Y cells. Secondly, we identified that the ability of sEV to transfer a proliferative and cisplatin resistant phenotype is not exclusive to KellyCis83 sEV. In conclusion, this study demonstrates the development of cisplatin resistance changes the protein composition and function of neuroblastoma derived sEV. We identified a new mechanism that promotes tubule formation in endothelial cells in neuroblastoma that is enhanced after resistance to cisplatin has developed. We demonstrated that neuroblastoma derived sEV promote the acquisition of several hallmarks of cancer, including proliferation, dysregulated metabolism and cell survival.