The role of Extracellular Vesicles produced by Neuroblastoma Cells in the Regulation of Proinflammatory Pathways
Background
Neuroblastoma is the most common solid extracranial tumor of the developing sympathetic nervous system accounting for 12-15% of cancer related deaths in children. Amplification of the protooncogene MYCN is one of the most reliable predictors of poor prognosis and is presumed to influence the interaction between NB and immune cells within the tumor microenvironment. This regulation occurs via cell-to-cell contact, secretion of molecules and release/uptake of extracellular vesicles
(EVs), loaded with their molecular cargo. We assume a pivotal role of NB-secreted EVs in altering the tumor microenvironment, thereby modulating the inflammatory context. Hence, the aim of this study is to screen EVs from several NB cell lines for inflammatory-related proteins and to evaluate a potential MYCN amplification dependency.
Methods
For this purpose, EVs were isolated from several stage 4 neuroblastoma cell lines
(n=3 MYCN amplified, n=3 MYCN non-amplified). Prior to protein profiling, EVs’ characterization and sample purity control were assessed by Western blotting, nanoparticle tracking analysis (NTA) and electron microscopy. EV-markers such as CD81, CD9 and TSG101 were used. Finally, mass spectrometry (MS) of isolated EVs as well as of whole cell lysates was performed for proteomics determination.
Results
As revealed by NTA, our group suggests differences in the EV production according to the MYCN amplification status. Moreover, we are able to show distinct expression levels of some EV markers among the considered cell lines. Accordingly, MS also depicts differential protein profiles.
Conclusion
The aforementioned results propose a differential EV production and constitution in neuroblastoma cell lines according to their molecular biology. Thus, further research on this emerging topic is needed to shed light onto the tumorigenic mechanisms behind EV-mediated cell signaling in neuroblastoma.
Keywords
Extracellular Vesicles, Neuroblastoma, Protein Profiling, Inflammatory Processes
Funding/Acknowledgments
TiPrime Graduiertenkolleg (Else Kröner-Fresenius-Stiftung)
Authors
Sina Marie Ceplis1 (Corresponding Author: sina.ceplis[at]stud.uke.uni-hamburg[dot]de), Sara Peggion2, Konrad Reinshagen1, Laia Pagerols Raluy1