Multi-omics characterization of IDH-mutant astrocytoma-derived cell lines reveals NOTCH-regulated plastic quiescent astrocyte-like state and insights into progression.

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 limited by the scarcity of relevant in vitro models. Here, we established and extensively characterized four astrocytoma cell lines (LGG275, LGG336, LGG85, LGG349) derived from IDH-mutant astrocytomas of different grades and analyzed them using multi-omics approaches. These lines display growth rates in vitro and in vivo consistent with tumor grade and recapitulate some key molecular alterations observed in patient tumors, including IDH1, ATRX, and TP53 mutations, ALT (alternative lengthening of telomeres) activation and, in more aggressive lines, MET and PDGFRA alterations. Single-cell RNA sequencing revealed three major transcriptional states (astrocyte-like, oligodendrocyte-like, and NPC-like), consistent with those described in patient tumors. A hallmark of higher-grade-derived lines (LGG85, LGG349) is the persistence of NPC-like populations without growth factors possibly 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, respectively, quiescent and activated neural stem (NSC) cells from the adult subventricular zone (SVZ). Finally, we provide evidence that NOTCH signaling contributes to the regulation of cell state balance, promoting transitions toward an astrocyte-like transcriptional program while DLL3, an anti-Notch protein, 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.