Extracellular Vesicles Carrying Tenascin‑C are Clinical Biomarkers and Improve Tumor-Derived DNA Analysis in Glioblastoma Patients

Extracellular vesicles
(EVs) act as carriers of 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. Using imaging flow cytometry (IFCM),
we conducted an immunophenotyping analysis of eight glioma-associated
antigens and tetraspanins in plasma EVs from 37 newly diagnosed glioblastoma
patients (pre- and post-surgery), 11 matched individuals with recurrent
glioblastoma, and 22 healthy donors (HD). Tenascin-C (TNC) positive
EVs displayed the strongest differences in newly diagnosed and recurrent
glioblastoma patients, when compared to non-tumor subjects. Among
dual-positive subpopulations, TNC+/CD9+ EVs
were the most elevated in newly diagnosed (FC = 7.6, p <0.0001, AUC = 81%) and recurrent patients (FC = 16.5, p <0.0001; AUC = 90%) than HD. In comparison with other
CNS tumors (n = 25), this subpopulation was also
34.5-fold higher in glioblastoma than in meningioma cases (p <0.01). Additionally, TNC+/CD9+ EV levels were 3.3-fold elevated in cerebrospinal fluid from glioblastoma
patients (n = 6) than controls (p <0.05). Aberrant TNC levels were further observed in glioblastoma
EVs from different sources and purified via different methods. 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 in cells with mesenchymal-like
signatures and chromosomal aberrations. Lastly, we purified TNC+ EVs from plasma of 21 glioblastoma patients by magnetic sorting
and detected the oncogenic mutation TERT*C228T by
droplet digital PCR. The mutant allele frequency was higher in TNC+ EVs vs TNC-negative EVs (FC = 32, p <0.001), total EVs (FC = 5.3, p <0.001)
or cell-free DNA (FC = 5.3, p <0.01). In conclusion,
circulating TNC+ EVs may have potential as clinical biomarkers
in glioblastoma, and their purification could improve the identification
of tumor-specific mutations in liquid biopsies.