GAUTHIER, L. R. | bioRxiv Science

bioRxiv2025

Multi-omics characterization of IDH-mutant astrocytoma-derived cell lines reveals NOTCH-regulated plastic quiescent astrocyte-like state

Diffuse IDH-mutant astrocytomas are brain tumors typically diagnosed as low-grade but capable of progressing to higher grades. They exhibit three cellular states resembling astrocytes, oligodendrocytes, and neural progenitor (NPC) cells. Understanding their biology has been challenging due to the lack of relevant in vitro models. Here we established and extensively characterized four astrocytoma cell lines (LGG275, LGG336, LGG85, LGG349) derived from IDH-mutant astrocytoma at different grades, cultured in defined media and analyzed by multi-omics. These lines display growth rates in vitro and in vivo consistent with tumor grade and recapitulate key molecular alterations observed in patient tumors, including IDH1, ATRX, and TP53 mutations, activation of the alternative lengthening of telomeres (ALT) pathway and, in the most aggressive line, amplification of MET and PDGFRA. Single-cell RNA sequencing showed that the 4 astrocytoma lines maintain the three major cellular states observed in patient tumors. A hallmark of higher-grade-derived lines (LGG85, LGG349) is the persistence of NPC-like populations without growth factors, reflecting tumor progression. The LGG275 line most accurately mirrors slow-growing astrocytomas. Using CD44 and GLAST, we isolated astrocyte-like (CD44+/GLAST+) cells from LGG275 that preferentially adopt a quiescent state yet retain remarkable plasticity, generating oligodendrocyte-like cells (CD44-/GLAST-). Transcriptomic and proteomic analyses revealed that astrocyte-like and oligodendrocyte-like cells populations resemble quiescent and activated neural stem (NSC) cells from the adult subventricular zone (SVZ). Finally, we found that NOTCH signaling regulates the balance between astrocytic and oligodendrocytic states, while DLL3, expressed by oligodendrocyte-like cells, modulates both proliferation and phenotype. These cell lines represent valuable resources for dissecting lineage dynamics, heterogeneity, and progression mechanisms in IDH-mutant astrocytomas. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/696808v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@953108org.highwire.dtl.DTLVardef@c6234aorg.highwire.dtl.DTLVardef@3703fcorg.highwire.dtl.DTLVardef@1e6c3a9_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightO_LIWe constituted a richly annotated biobank derived from 4 astrocytomas, showing similar characteristics to those found in patients, providing valuable tools to investigate cellular heterogeneity and plasticity, and link with tumor progression. C_LIO_LIscRNA-sequencing revealed three cell states (oligodendrocyte-like, astrocyte-like, and stem cell-like cells) akin to those found in tumors. C_LIO_LIAstrocyte-like cells are quiescent cells, plastic and similar to quiescent neural stem cell (qNSC) from the sub-ventricular zone (SVZ), while oligodendrocyte-like cells are similar to active NSC (aNSC). C_LIO_LIThe Notch pathway plays a role in cell plasticity, enabling a shift towards an astrocyte-like state. C_LI

cancer biology↗

bioRxiv2025

Endothelin Signaling via EDNRB receptor Reduces Proliferation and Promotes Proneural-to-Mesenchymal Transition in Gliomas

Diffuse gliomas are incurable primary brain tumors encompassing three histo-molecular subtypes: glioblastomas (GB), astrocytomas, and oligodendrogliomas. The latter two harbor IDH1 mutations and exhibit slower progression than glioblastomas. Diffuse gliomas are composed of highly plastic tumor cells capable of transitioning between astrocyte-like, oligodendrocyte-like, progenitor-like, and mesenchymal-like states, driven by genetic alterations and microenvironmental cues. The proneural-to-mesenchymal transition (PMT), associated with increased malignancy, is notably influenced by cytokines in the tumor microenvironment. Endothelin cytokines (ET-1, ET-2, ET-3), primarily secreted by vascular cells, regulate not only vascular tone but also astrocyte and neural stem cell proliferation via the G-protein-coupled receptors EDNRA and EDNRB. Prior studies using serum-cultured glioma lines suggested pro-proliferative effects of endothelins; however, such models poorly recapitulate the in vivo glioma context. In this study, we comprehensively revisited endothelin signaling - covering receptor expression, regulation, downstream pathways, and cellular responses-using eleven serum-free, patient-derived glioma lines (glioblastomas, IDH-wt and IDH-mutant oligodendrogliomas and astrocytomas), along with primary tumor samples. Multi-omics and electrophysiological analyses revealed EDNRB as the predominant receptor, enriched in astrocyte-like cells, upregulated by BMPs or growth factor withdrawal, and downregulated by interferons, IL-6 cytokines, endothelins, and Hippo/YAP activation. In contrast, EDNRA was expressed by a perivascular tumor subpopulation and induced by Notch signaling in glioblastomas but not in IDH1-mutant cells. Functionally, endothelins reduced proliferation across all models while promoting migration and PMT. Mechanistically, EDNRB activation increased intracellular Ca{superscript 2} and activated ERK, STAT3, and apamin-sensitive SK2/SK3 potassium channels. These findings identify endothelin signaling as an important regulator of glioma cell plasticity and behavior. HighlightsO_LIEDNRB is the predominant endothelin receptor expressed in glioma cells, with a small subset of tumor cells expressing EDNRA in close proximity to blood vessels C_LIO_LIEndothelin signaling reduces proliferation while promoting cell migration and Proneural-to-Mesenchymal transition C_LIO_LIEndothelin activates downstream Ca2+, K+, ERK, and STAT3 signaling pathways C_LIO_LIEDNRB expression is both positively and negatively regulated by inflammatory cytokines and the Hippo/YAP1 pathway, whereas EDNRA is upregulated by Notch signaling and hypoxia C_LI

cancer biology↗