Extracellular Vesicles carrying Tenascin-C are clinical biomarkers and improve tumor-derived DNA analysis in Glioblastoma patients |
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| Background Extracellular vesicles (EVs) can transfer biological information from tumors to the bloodstream, enabling the detection of circulating tumor material and tracking of disease progression. This is particularly crucial in glioblastoma, a highly aggressive and heterogeneous tumor that is challenging to monitor. |
| Methods & Results EVs were isolated by differential ultracentrifugation from plasma samples of 37 newly diagnosed glioblastoma patients (pre- and post-surgery), 11 matched glioblastoma relapses, and 22 healthy donors (HD). EVs were characterized by NTA, TEM and SEM. Using imaging flow cytometry (IFCM), we also immunophenotyped the tetraspanins (CD9, CD81 and CD63) and eight glioma-associated antigens (TNC, ITGB1, CD44, CD131, PFN1, SPARC, GPNMB and HLA class-II) on EVs. Corrected values per milliliter of plasma of all combinations of EV populations were compared among the groups by Mann-Whitney and ROC analysis. Tenascin-C (TNC) positive EVs displayed the strongest differences in newly diagnosed and recurrent glioblastoma, when compared to non-tumor subjects. Among dual-positive subpopulations, TNC+/CD9+ EVs were the most elevated in newly diagnosed (FC=7.6, p<.0001, AUC=81%) and recurrent patients (FC=16.5, p<.0001; AUC=90%) than HD. Additionally, TNC+/CD9+ EV levels were 3.3-fold elevated in cerebrospinal fluid from glioblastoma patients (n=6) than controls (p<.05). Immunohistochemical analysis revealed high levels of TNC in tumor tissues. Spatial transcriptomic analysis indicated a TNC overexpression in malignant cell populations of glioblastoma resections, particularly with mesenchymal-like signatures and chromosomal aberrations. Lastly, we purified TNC+ EVs from plasma of 21 glioblastoma patients by magnetic sorting and analyzed the oncogenic mutation TERT*C228T by droplet digital PCR. The mutant allele frequency was higher in TNC+ EVs versus TNC-negative EVs (FC=32, p<.001), total EVs (FC=5.3, p<.001) or cell-free DNA (FC=5.3, p<.01). |
| Conclusion Circulating TNC+ EVs are clinical biomarkers in glioblastoma, and their purification could potentially improve the identification of tumor-specific mutations in liquid biopsies. |
| Keywords Tenascin-C, extracellular vesicles, glioblastoma, biomarkers, liquid biopsies. |
| Funding/Acknowledgments We acknowledge the patients enrolled in this study and the staff of the UKE Neurosurgery department. This study was supported by grants from the Deutsche Forschungsgemeinschaft and from the Rudolf Bartling Stiftung. |
Authors Amanda Salviano-Silva1* (Corresponding Author: a.salvianodasilva[at]uke[dot]de), Kathrin Wollmann1, Santra Brenna2, Rudolph Reimer3, Julia E. Neumann4,5, Matthias Dottermusch4,5, Laura Woythe6, Cecile L. Maire1, Berta Puig2, Ulrich Schüller4,7,8, Manfred Westphal1, Richard Drexler1, Lasse Dührsen1, Jens Gempt1, Dieter H. Heiland9, Katrin Lamszus1, Franz L. Ricklefs1*† (Corresponding Author: f.ricklefs[at]uke[dot]de)
1Department of Neurosurgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 2 Neurology Department, Experimental Research in Stroke and Inflammation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 3 Leibniz Institute for Experimental Virology, Hamburg, Germany 4 Institute of Neuropathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 5 Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg-Eppendorf, Hamburg, Germany 6 Oxford Nanoimaging Limited (ONI), Oxford OX2 8TA, United Kingdom 7 Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 8 Children's Cancer Research Center Hamburg, Hamburg, Germany 9 Department of Neurosurgery, Medical Center University of Freiburg, Freiburg, Germany † Senior author |