Jean-Philippe Hugnot

Diffuse gliomas are primary brain tumors including glioblastomas (GB), astrocytomas, and oligodendrogliomas, the latter two harboring IDH1 mutations and exhibiting slower progression. Gliomas display cellular plasticity, with transitions between astrocyte‐like, oligodendrocyte‐like, progenitor‐like, and mesenchymal‐like states driven by genetic alterations and microenvironmental signals. The proneural‐to‐mesenchymal transition (PMT), associated with increased malignancy, is tightly regulated by the tumor microenvironment, notably through cytokine signaling and non‐tumor cell interactions. Endothelins (ET‐1, ET‐2, ET‐3), vasoactive peptides mainly produced by vascular cells, signal through the G‐protein‐coupled receptors EDNRA and EDNRB and were previously suggested to promote glioma proliferation based on serum‐based models. Here, we revisited endothelin signaling using eleven serum‐free glioma lines and tumor samples. Multi‐omics and electrophysiological analyses identified EDNRB as the predominant receptor, enriched in astrocyte‐like cells, increased by BMPs or growth factor withdrawal, and repressed by interferons, IL‐6 family cytokines, endothelins, and Hippo/YAP signaling. EDNRA was confined to a perivascular tumor subpopulation and induced by Notch signaling selectively in GB. Functionally, endothelins reduced proliferation while promoting migration and PMT via EDNRB‐dependent Ca ²⁺ signaling, ERK/STAT3 activation, and apamin‐sensitive SK2/SK3 potassium channel activity. Collectively these findings establish endothelin signaling as an important regulator of glioma cell plasticity and behavior.

Diffuse gliomas, the most prevalent primary brain tumors, are categorized into low and high grades based on histopathological criteria and genetic mutations. Diffuse low-grade gliomas constitute a subset, predominantly afflicting young adults and predisposing to high-grade gliomas. There are two main types of diffuse low-grade gliomas: astrocytomas and oligodendrogliomas, each defined by distinct genetic and histological features. These tumors characteristically exhibit a mutated variant of the metabolic enzyme IDH1, disrupting cellular differentiation. They are heterogeneous, comprising both proliferative stem cells and more differentiated, quiescent glial cells. Patient-specific, in vitro tumor models hold significant potential for unraveling oncogenic mechanisms and tailoring drug selection for personalized therapy. However, the absence of reliable in vitro models specifically for IDH1-mutant low-grade gliomas has hindered fundamental and translational research. Here, we present a simple method for culturing IDH1-mutant tumors as explants in defined media without needing enzymatic dissociation. These explants can be sustained for extended periods, maintaining IDH1-mutant protein expression as well as markers for oligodendrocyte progenitor cells such as ASCL1 (MASH1), OLIG1, and OLIG2 and for astrocytes (GFAP, SOX9). They can also be established from frozen tumors in DMSO. These cultures can serve as a valuable platform for drug screening, videomicroscopy, and gene studies, thus facilitating advancements in understanding and treating these challenging tumors. They also offer the potential to derive IDH1-mutant cell lines.

Single-cell transcriptomics has unveiled a vast landscape of cellular heterogeneity in which the cell cycle is a significant component. We trained a high-resolution cell cycle classifier (ccAFv2) using single cell RNA-seq (scRNA-seq) characterized human neural stem cells. The ccAFv2 classifies six cell cycle states (G1, Late G1, S, S/G2, G2/M, and M/Early G1) and a quiescent-like G0 state (Neural G0), and it incorporates a tunable parameter to filter out less certain classifications. The ccAFv2 classifier performed better than or equivalent to other state-of-the-art methods even while classifying more cell cycle states, including G0. We demonstrate that the ccAFv2 classifier effectively generalizes the S, S/G2, G2/M, and M/Early G1 states across cell types derived from all three germ layers. While the G0, G1, and Late G1 states perform well in neuroepithelial cell types, their accuracy is lower in other cell types. However, misclassifications are confined to the G0, G1, and Late G1 states. We showcased the versatility of ccAFv2 by successfully applying it to classify cells, nuclei, and spatial transcriptomics data in humans and mice, using various normalization methods and gene identifiers. We provide methods to regress the cell cycle expression patterns out of single cell or nuclei data to uncover underlying biological signals. The classifier can be used either as an R package integrated with Seurat or a PyPI package integrated with SCANPY. We proved that ccAFv2 has enhanced accuracy, flexibility, and adaptability across various experimental conditions, establishing ccAFv2 as a powerful tool for dissecting complex biological systems, unraveling cellular heterogeneity, and deciphering the molecular mechanisms by which proliferation and quiescence affect cellular processes.

Ependymal cells lining the central canal of the spinal cord play a crucial role in providing a physical barrier and in the circulation of cerebrospinal fluid. These cells express the FOXJ1 and SOX2 transcription factors in mice and are derived from various neural tube populations, including embryonic roof and floor plate cells. They exhibit a dorsal–ventral expression pattern of spinal cord developmental transcription factors (such as MSX1, PAX6, ARX, and FOXA2), resembling an embryonic-like organization. Although this ependymal region is present in young humans, it appears to be lost with age. To re-examine this issue, we collected 17 fresh spinal cords from organ donors aged 37–83 years and performed immunohistochemistry on lightly fixed tissues. We observed cells expressing FOXJ1 in the central region in all cases, which co-expressed SOX2 and PAX6 as well as RFX2 and ARL13B, two proteins involved in ciliogenesis and cilia-mediated sonic hedgehog signaling, respectively. Half of the cases exhibited a lumen and some presented portions of the spinal cord with closed and open central canals. Co-staining of FOXJ1 with other neurodevelopmental transcription factors (ARX, FOXA2, MSX1) and NESTIN revealed heterogeneity of the ependymal cells. Interestingly, three donors aged > 75 years exhibited a fetal-like regionalization of neurodevelopmental transcription factors, with dorsal and ventral ependymal cells expressing MSX1, ARX, and FOXA2. These results provide new evidence for the persistence of ependymal cells expressing neurodevelopmental genes throughout human life and highlight the importance of further investigation of these cells.

Metabolites produced by GSCs following MSC mitochondria acquisition and TMZ treatment

Metabolite detection in resected GBM from 8 patients, at 1rst resection and at 2nd resection post-TMZ treatment. For each metabolite, the C12/C13 ratios normalized to tissue protein concentrations are indicated.

MSC mitochondria induce a higher orotate turnover in GSCs which supports resistance to TMZ. A and B, [U-13C]-glucose and [U-13C]-glutamine orotate isotope profiling in GSCs with and/or without MSC mitochondria and treated or not with TMZ (48 hours; n = 3). Mean + SEM. Statistical analysis by Student t test. ***, P < 0.001. C, Schematic of BRQ inhibition of DHODH-dependent orotate production (BioRender). D, Effect of BRQ (100 nmol/L) on the survival of GSCs (GB4 and GB5, from 2 donors) to TMZ treatment (50 μmol/L) following acquisition of MSC mitochondria (3 MSC donors for each GSC). Two-way ANOVA, *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 4, each dot represents a culture well.

Cocultures of red MitoTracker-labeled MSCs and green CellTracker-labeled GSCs