Serum-free culture success of glial tumors is related to specific molecular profiles and expression of extracellular matrix-associated gene modules

Abstract

Background: Recent molecular characterization studies have identified clinically relevant molecular subtypes to coexist within the same histological entities of glioma. Comparative studies between serum-supplemented and serum-free (SF) culture conditions have demonstrated that SF conditions select for glioma stem-like cells, which superiorly conserve genomic alterations. However, neither the representation of molecular subtypes within SF culture assays nor the molecular distinctions between successful and nonsuccessful attempts have been elucidated.

Methods: A cohort of 261 glioma samples from varying histological grades was documented for SF culture success and clinical outcome. Gene expression and single nucleotide polymorphism arrays were interrogated on a panel of tumors for comparative analysis of SF+ (successful cultures) and SF- (unsuccessful cultures).

Results: SF culture outcome was correlated with tumor grade, while no relation was found between SF+ and patient overall survival. Copy number-based hierarchical clustering revealed an absolute separation between SF+ and SF- parental tumors. All SF+ cultures are derived from tumors that are isocitrate dehydrogenase 1 (IDH1) wild type, chromosome 7 amplified, and chromosome 10q deleted. SF- cultures derived from IDH1 mutant tumors demonstrated a fade-out of mutated cells during the first passages. SF+ tumors were enriched for The Cancer Genome Atlas Classical subtype and intrinsic glioma subtype-18. Comparative gene ontology analysis between SF+ and SF- tumors demonstrated enrichment for modules associated with extracellular matrix composition, Hox-gene signaling, and inflammation.

Conclusion: SF cultures are derived from a subset of parental tumors with a shared molecular background including enrichment for extracellular matrix-associated gene modules. These results provide leads to develop enhanced culture protocols for glioma samples not propagatable under current SF conditions.

Keywords: IDH1; extracellular matrix; glioma; molecular subtype; serum-free cultures.

Fig. 1.

SF culture outcome is not associated with worse clinical outcome in GBM. Kaplan–Meier-based overall survival (OS) curves for SF culture results for indicated histological entities of malignant glioma. SF+ (black), SF− (gray), and SFnp (dotted). Log-rank (Mantel–Cox) P-values are based on SF+ vs SF− or SF+ vs SFnp. Note a trend in decreased OS for SF+ grades II–III samples, while GBM samples show no difference.

Fig. 2.

Low passage cultures from SFnp tumors retain CNAs as found in parental tumors, while SS cultures from SF− tumors are overgrown by nonneoplastic cells. Heat map plots of copy number analysis results demonstrated loss of aberrations in SS cultures derived from SF− tumors. Each band is composed of the genomic data of 1 sample, as indicated in the legend on the right. Color-coding indicates blue for losses, red for gains, and white for nonaffected. Numbers below indicate chromosome number.

Fig. 3.

Genomic analysis of parental tumors reveals complete separation of SF+ and SF−/SFnp samples. Copy number intensity–based unsupervised hierarchical clustering of SF+ and SF−/SFnp tumors. Heat map intensities are coded between copy number intensity varying 0–4n. Note the similarity between SF−/SFnp samples as indicated by the integration of the samples within the same cluster dendrogram tree.

Fig. 4.

Gene expression profiling and IDH1 sequencing data demonstrates SF culture selects for TCGA CLA/IGS-18 and IDH1 wild-type tumors. (A) Distribution of SF+ and SF− samples across the previously published TCGA and IGS classifiers. Samples were assigned according to the centroids as published in the original reports, on the right. Proneural samples are spread between IGS-9, -17, and -22. Noteworthy is the lack of IGS-9 and -22 in the SF+ group, as well as the overrepresentation of IGS-18/CLA samples in the SF+ population compared with the incidences reported in the original publications. (B) Sequencing illustrations of IDH1 codons 130 (ATA in shadow) through 134. Thundermarks indicate G to A or C mutation in codon R132, indicative of R132H. Note the loss of this mutation in GS276 and GS297 SF passages. In GS310 SFp2 the sequence reveals a minor A peak, demonstrating an ambiguous mutation pattern, indicative of mixed populations of mutated tumor cells and (nonneoplastic) IDH-WT cells. (C) Microscopic images of GS301 SS/SF cultures demonstrate loss of neoplastic cells. SF culture retains original morphology as found in p0 for a longer duration than SS; however, eventually (p12) proliferation of nonneoplastic stromal cells is abundant with typical accumulation of bands and halos suggestive for fibroblasts.