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Glioma-propagating cells as an in vitro screening platform: PLK1 as a case study

ABSTRACT: Gliomas are the most devastating of primary adult malignant brain tumors. These tumors are highly infiltrative and can arise from cells with extensive self-renewal capability and chemoresistance, frequently termed glioma-propagating cells (GPCs). GPCs are thus the plausible culprits of tumor recurrence. Treatment strategies that eradicate GPCs will greatly improve disease outcome. Such findings support the use of GPCs as in vitro cellular systems for small molecule screening. However, the nuances in utilizing GPCs as a cellular screening platform are not trivial. These slow-growing cells are typically cultured as suspension, spheroid structures in serum-free condition supplemented with growth factors. Consequently, replenishment of growth factors throughout the screening period must occur to maintain cells in their undifferentiated state, as the more lineage-committed, differentiated cells are less tumorigenic. We will present a case study of a small molecule screen conducted with GPCs and explain how unique sphere activity assays were implemented to distinguish drug efficacies against the long-term, self-renewing fraction, as opposed to transient-amplifying progenitors, latter of which are detected in conventional viability assays. We identified Pololike kinase 1 as a regulator of GPC survival. Finally, we leveraged on public glioma databases to illustrate GPC contribution to disease progression and patient survival outcome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE36782 | GEO | 2012/08/02

SECONDARY ACCESSION(S): PRJNA156711

REPOSITORIES: GEO

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Project description:Aims: We explore the role of elevated O2-:H2O2 ratio as a prosurvival signal in glioma-propagating cells (GPCs). We hypothesize that depleting this ratio sensitizes GPCs to apoptotic triggers. Results: We observed that elevated O2-:H2O2 ratio conferred enhanced resistance in GPCs, and depletion of this ratio by pharmacological and genetic methods sensitized cells to apopotic triggers. We established the ROS Index as a quantitative measure of normalized O2-:H2O2 ratio and determined its utility in predicting chemosensitivity. Importantly, mice implanted with GPCs of reduced ROS Index demonstrated extended survival. Analysis of tumor sections revealed effective targeting of CD133- and nestin-expressing neural precursors. Furthermore, we established the Connectivity Map to interrogate a gene signature derived from varied ROS Index for patterns of association with individual patient gene expression in 2 clinical databases. We showed that patients with reduced ROS Index demonstrate better survival. These data provide clinical evidence for the viability of our O2-:H2O2-mediated chemosensitivity profiles. Innovation and Conclusion: Gliomas are notoriously recurrent and highly infiltrative, and have been shown to arise from stem-like cells. We implicate elevated O2-:H2O2 ratio as a prosurvival signal in GPC self-renewal and proliferation. The ROS Index provides quantification of O2-:H2O2-mediated chemosensitivity, an advancement in a previously qualitative field. Intriguingly, glioma patients with reduced ROS Index correlate with longer survival and the Proneural molecular classification, a feature frequently associated with tumors of better prognosis. These data emphasize the feasibility of manipulating the O2-:H2O2 ratio as a therapeutic strategy. Total RNA from primary neurosphere culture of brain tumor specimens treated with DPI and DDC as well as non-treated CD133+ and CD133- fractions were compared. Specimens were obtained from 3 patients and replicate arrays were performed for all 3 neurosphere cultures.

Project description:Brain tumors are among the most malignant cancers and can arise from neural stem cells or oligodendrocyte progenitor cells (OPCs). Glioma-propagating cells (GPCs) that have stem-like properties have been derived from tumor variants such as glioblastoma multiforme (GBM) and oligodendroglial tumors, the latter being more chemosensitive with better prognosis. It has been suggested that such differences in chemosensitivity arise from the different profiles of OPCs versus neural stem cells. We thus explored if GPCs derived from these glioma variants can serve as reliable in vitro culture systems for studies. We utilized gene expression analyses, since GBM and oligodendrogliomas can be molecularly classified. Accordingly, we derived a gene signature distinguishing oligodendroglial GPCs from GBM GPCs collated from different studies, which was enriched for the Wnt, Notch and TGF-beta pathways. Using a novel method in glioma biology, the Connectivity Map, we mapped the strength of gene signature association with patient gene expression profiles in 2 independent glioma databases [GSE16011, http://caintegrator-info.nci.nih.gov/rembrandt]. Our gene signature consistently stratified survival in glioma patients. This data would suggest that in vitro low passage GPCs are similarly driven by transcriptomic changes that characterize the favorable outcome of oligodendrogliomas over GBM. Additionally, the gene signature was associated with the 1p/19q co-deletion status, the current clinical indicator of chemosensitivity. Our gene signature detects molecular heterogeneity in oligodendroglioma patients that cannot be accounted for by histology or the 1p/19q status alone, and highlights the limitation of morphology-based histological analyses in tumor classification, consequently impacting on treatment decisions. Total RNA obtained from primary neurosphere culture from brain tumor specimens of 6 patients were compared. Replicate arrays were performed for all 6 neurosphere cultures.

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Glioma-propagating cells as an in vitro screening platform: PLK1 as a case study

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