The use of methylation fingerprints to investigate the cell-of-origin of gliomas

Aviel Iluz ^1,2 Hanna Charbit ^1,2 Masha Idelson ³ Debora Steiner ³ Yakov Fellig ⁴ Samuel Moscovici ⁵ Yigal Shoshan ⁵ Anat Mordechai ⁶ Alexander Lossos ^1,2,6 Benjamin Reubinoff ^3,7 Iris Lavon ^1,2

¹Leslie and Michael Gaffin Center for Neuro-Oncology, Department of Neurology, Hadassah Medical Center, Faculty of Medicine, Hebrew University of Jerusalem, Israel
²Agnes Ginges Center for Human Neurogenetics, Department of Neurology, Hadassah Medical Center, Faculty of Medicine, Hebrew University of Jerusalem, Israel
³The Hadassah Human Embryonic Stem Cell Research Center, The Goldyne Savad Institute of Gene Therapy, Hadassah Medical Center, Faculty of Medicine, Hebrew University of Jerusalem, Israel
⁴Department of Pathology, Hadassah Medical Center, Faculty of Medicine, Hebrew University of Jerusalem
⁵Department of Neurosurgery, Hadassah Medical Center, Faculty of Medicine, Hebrew University of Jerusalem, Israel
⁶Department of Oncology, Hadassah Medical Center, Faculty of Medicine, Hebrew University of Jerusalem, Israel
⁷Department of Obstetrics & Gynecology, Hadassah Medical Center, Faculty of Medicine, Hebrew University of Jerusalem, Israel

Gliomas are the most common primary malignant brain tumors with poor prognoses. The cellular origin of gliomas remains a challenging topic of controversy in cancer research, especially in high-grade gliomas which characterized by remarkable intra-tumor heterogeneity. Previous works have shown similarities of histological markers, transcriptome, and biological behavior features between gliomas and neural progenitor cells (NPC). Our study has shown a similarity of miRNA expression profiles between glioma and NPC. This evidence implies that the latter are glioma`s cell-of-origin. Studies have shown that brain tumors can be subclassified reliably by their methylation profiles. Moreover, it is known that every cell type has its unique methylation fingerprint.

Based on this knowledge, we used the Oxford Nanopore device to obtain methylation sequences that we considered a potential method to investigate glioma cell-of-origin. We then sequenced 19 glioma and 5 in-vitro human embryonic stem cell-derived NPC samples at various differentiation stages of oligodendrocyte lineage. Using different unsupervised clustering methods (hierarchical clustering, heatmap, PCA), and brain tumors classifier, based on Random Forest (RF) algorithm, we applied comparative analysis of these cells methylation profiles.

Our results show that NPCs of late oligodendrocyte lineage - oligodendrocyte progenitor cells (OPC) - are clustered together with histopathologically classified glioblastomas, based on methylation profiles similarity, and are also classified by RF as glioblastomas. Our results imply that OPCs at later stages might be the glioblastoma`s cells of origin. These results should be further investigated as they might be biased due to small sample size and the shallow sequencing depth.