Unveiling Neuroblastoma: characterizing the Proteome and Surface Markers of Extracellular Vesicle to elucidate Tumor Microenvironment and Premetastatic Niche Formation

Background

Neuroblastoma (NB), a tumor derived from neuroectodermal embryonic creast cells, is responsible for 11% of cancer-related deaths among children. We are investigating NB-secreted extracellular vesicles (EVs) regarding their crucial impact on the tumor microenvierment, their role in premetastatic niche formation and their potential as biomarkers and/or therapeutic targets.  

 

Methods

First, we isolated EVs from different NB cell lines and from plasma of high-risk NB patients. After standard EV characterization (nanoparticle tracking analysis, electron microscopy, visualization of EV-markers) the proteome of the EVs were analyzed. Flow cytometry methods allowed us to display the surface marker profiles of NB-EVs by using different MACSPlex Kits (Miltenyi Biotec). To evaluate the impact of NB-EVs on angiogenesis, endothelial cells were treated with NB-EVs in an in vivo model designed to mimic angiogenesis. 

 

Results

Analyzing the vesicle’s proteome revealed EV-protein profiles crucial for distinguishing between three prominent genetic subgroups of neuroblastoma donor cell types. Additionally, we were able to indicate the cell state (adrenergic vs. mesenchymal) of the donor cells by analyzing their EV-proteome. 

Preliminary flow cytometry data of EVs isolated from plasma indicates a different profile of EV surface markers for NB-high risk patients compared to healthy controls. Isolated EVs from different NB-cell lines indicate distinct EV-marker profiles.

NB-EV-treated endothelial cells during angiogenesis show a modified cell growth.

 

Conclusion

Our findings will provide clinicians with a novel, non-invasive tool to obtain important tumor information about genotype and cell states. Both information, cell state and genotype, are crucial for finding the best therapy for patients. Particularly for underage patients, non-invasive methods for diagnosing and monitoring cancer diseases are important, as these tools are less traumatizing for body and mind.

The extensive characterization of surface markers and protein cargos of EVs from different NB origins will help to understand the EV uptake of potential recipient cells for pre-metastatic niche formation and shed light on possible tumor-microenvironment processes. Both processes are potential targets for new drugs.

 

Keywords

Neuroblastoma, proteome, liquid biopsy, premetastatic niche formation

 

Funding/Acknowledgments

DFG, SFB, CRC1588 

 

Authors

Martin Auber1 (Corresponding Author: martin.auber[at]charite[dot]de), Maddalena Grimaldi1, Marieluise Kirchner2, Philipp Mertins2, Holger Gerhardt3, Jennifer Peach3 and Hedwig Deubzer1

 

1Department of Pediatric Oncology and Hematology, Charité – University Medicine, Berlin, Germany
2Department of Proteomics, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany
3Department of Integrative Vascular Biology, Max Delbrueck Center for Molecular Medizin

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