A molecularly distinct subset of glioblastoma requires serum-containing media to establish sustainable bona fide glioblastoma stem cell cultures

Glioblastoma (GBM) is the most frequent and deadly primary malignant brain tumor. Hallmarks are extensive intra- and inter-tumor heterogeneity and highly invasive growth, which provide great challenges for treatment. Efficient therapy is lacking for GBM and the majority of patients survive less than one year from diagnosis. GBM progression and recurrence is caused by treatment-resistant glioblastoma stem cells (GSCs). GSC cultures are considered important models to use in target identification and drug screening. The current state of the art method to isolate and maintain GSC cultures that faithfully mimic the primary tumor, is to use serum-free (SF) media conditions developed for neural stem cells (NSCs). Here we have investigated the outcome of explanting 230 consecutively collected GBM patient samples under both SF and standard, serum-containing media conditions. The establishment of maintainable SF cultures (SFCs) was most frequent, but for a subgroup of GBM specimens a viable culture could only be established in serum-containing media, called exclusive serum culture (ESC). ESCs expressed nestin and SOX2, and displayed all functional characteristics of GSCs, i.e. extended proliferation, sustained self-renewal and orthotopic tumor initiation. Once adapted to the in vitro milieu they were also sustainable in SF media. Molecular analyses showed that ESCs formed a discrete group that was most related to the mesenchymal GBM subtype. This distinct subgroup of GBM that had evaded modeling under SF conditions provide unique cell models of GBM intertumor heterogeneity. Transcriptome analysis of 2 ESC (U3040MG, U3097MG) and 6 ESC_to_SFC (U3040MG, U3045MG, U3097MG, U3112MG, U3138MG, U3158MG) cell cultures on Affymetrix Human Transcriptome 2.0 arrays. ESC cultures are patient-derived glioblastoma cell cultures that were exclusively established in serum-containing media and not viable in serum-free defined NSC media. ESC_to_SFC cultures are ESC cultures that were cultured in serum-free defined NSC media for at least two passages. All cells were cultured adherently.