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. 2024 Mar 20;16(6):1223.
doi: 10.3390/cancers16061223.

Proteomic Profiling of Cerebrospinal Fluid and Its Extracellular Vesicles from Extraventricular Drainage in Pediatric Pilocytic Astrocytoma, towards Precision Oncology

Sonia Spinelli  1 , Xhuliana Kajana  1 , Andrea Garbarino  1 , Martina Bartolucci  2 , Andrea Petretto  2 , Marco Pavanello  3 , Enrico Verrina  4 , Giovanni Candiano  1 , Isabella Panfoli  5 , Maurizio Bruschi  1   6
Affiliations

Proteomic Profiling of Cerebrospinal Fluid and Its Extracellular Vesicles from Extraventricular Drainage in Pediatric Pilocytic Astrocytoma, towards Precision Oncology

Sonia Spinelli et al. Cancers (Basel). .

Abstract

Pediatric pilocytic astrocytoma (PA) is the most common brain tumor in children. Complete resection provides a favorable prognosis, except for unresectable PA forms. There is an incomplete understanding of the molecular and cellular pathogenesis of PA. Potential biomarkers for PA patients, especially the non-BRAF-mutated ones are needed. Cerebrospinal fluid (CSF) is a valuable source of brain tumor biomarkers. Extracellular vesicles (EVs), circulating in CSF, express valuable disease targets. These can be isolated from CSF from waste extraventricular drainage (EVD). We analyzed the proteome of EVD CSF from PA, congenital hydrocephalus (CH, non-tumor control), or medulloblastoma (MB, unrelated tumoral control) patients. A total of 3072 proteins were identified, 47.1%, 65.6%, and 86.2% of which were expressed in the unprocessed total and in its large-EV (LEV), and small-EV (SEV) fractions. Bioinformatics identified 50 statistically significant proteins in the comparison between PA and HC, and PA and MB patients, in the same fractions. Kinase enrichment analysis predicted five enriched kinases involved in signaling. Among these, only Cyclin-dependent kinase 2 (CDK2) kinase was overexpressed in PA samples. PLS-DA highlighted the inactive carboxypeptidase-like protein X2 (CPXM2) and aquaporin-4 (AQP4) as statistically significant in all the comparisons, with CPXM2 being overexpressed (validated by ELISA and Western blot) and AQP4 downregulated in PA. These proteins were considered the most promising potential biomarkers for discriminating among pilocytic astrocytoma and unrelated tumoral (MB) or non-tumoral conditions in all the fractions examined, and are proposed to be prospectively validated in the plasma for translational medicine applications.

Keywords: cerebrospinal fluid; extracellular vesicles; extraventricular drainage; large extracellular vesicles; pilocytic astrocytoma; proteomics; small extracellular vesicles.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Multidimensional scaling with k-means of whole proteomic dataset of EVD CSF samples. Two-dimensional scatter plot of unsupervised cluster analysis of whole-protein dataset. The plot shows the absence of any outlier and the presence of three distinct clusters corresponding to unprocessed-total (square), small-EV (circle), and large-EV (triangle) fractions. On the other hand, there was no discrimination between the congenital hydrocephalus (CH) (gray), pilocytic astrocytoma (PA) (red), and medulloblastoma (MB) (blue) clinical groups.
Figure 2
Figure 2
Heatmap of highly discriminant proteins for the different samples. Heatmap of 50 proteins highlighted by statistical analysis. In the heatmap, each row represents a protein, and each column corresponds to a condition. Normalized Z-scores of protein abundance are depicted by a pseudo color scale, with red indicating positive expression, white indicting equal expression, and blue indicating negative expression compared to each protein value, whereas the dendrogram displays the outcome of unsupervised hierarchical clustering analysis, placing similar protein profile values near each other. In addition, the diagram on the right of the heatmap reports the statistical significance (red) of each protein in each comparison. Visual inspection of the dendrogram and heatmap demonstrates the ability of these proteins to distinguish between pilocytic astrocytoma (PA), congenital hydrocephalus (CH), and medulloblastoma (MB) in unprocessed-total (Tot), small-EV (SEV), and large-EV (LEV) fractions (see detail in Supplementary Table S1). Carboxypeptidase-like protein X2 (CPXM2) was statistically more abundant in all PA samples compared to all other samples, whereas aquaporin-4 (AQP4) showed the opposite protein profile.
Figure 3
Figure 3
Partial least squares discriminant analysis (PLS-DA) of proteomic dataset. Two-dimensional scatter plot of PLS-DA score of CSF EVD protein-profile label-free quantitation intensity from pilocytic astrocytoma (PA) (red), congenital hydrocephalus (CH) (gray), and medulloblastoma (MB) (blue) in unprocessed-total (square), small-EV (circle), and large-EV (triangle) samples. Ellipses represent the 95% confidence intervals. The plot shows a clear separation between PA and non-PA samples.
Figure 4
Figure 4
Inactive carboxypeptidase-like protein X2 (CPXM2) ELISA validation. (a) Box plots of direct homemade ELISA assay for CPXM2 protein in unprocessed total fraction (Tot) of EVD CSF obtained from congenital hydrocephalus (CH), pilocytic astrocytoma (PA), and medulloblastoma (MB) patients. CPXM2 is statistically more abundant in PA (p < 0.0001) samples compared to CH and MB samples. (b) ROC curve analysis for the CPXM2 ELISA assay.
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References

    1. Damodharan S., Puccetti D. Pediatric Central Nervous System Tumor Overview and Emerging Treatment Considerations. Brain Sci. 2023;13:1106. doi: 10.3390/brainsci13071106. - DOI - PMC - PubMed
    1. Ganz J.C. Low grade gliomas. Prog. Brain Res. 2022;268:271–277. doi: 10.1016/bs.pbr.2021.10.036. - DOI - PubMed
    1. Ryall S., Tabori U., Hawkins C. Pediatric low-grade glioma in the era of molecular diagnostics. Acta Neuropathol. Commun. 2020;8:30. doi: 10.1186/s40478-020-00902-z. - DOI - PMC - PubMed
    1. Salles D., Laviola G., Malinverni A.C.M., Stavale J.N. Pilocytic Astrocytoma: A Review of General, Clinical, and Molecular Characteristics. J. Child. Neurol. 2020;35:852–858. doi: 10.1177/0883073820937225. - DOI - PubMed
    1. Bauman M.M.J., Harrison D.J., Giesken M.B., Daniels D.J. The evolving landscape of pilocytic astrocytoma: A bibliometric analysis of the top-100 most cited publications. Childs Nerv. Syst. 2022;38:1271–1280. doi: 10.1007/s00381-022-05503-w. - DOI - PubMed