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A focus on Glioblastomamultiforme heterogeneity through a paired primary culture in-vitro model.

Elisabetta Stanzani, Department of Physiological science II, University of Barcelona; Teresa Martin Mateos, Department of Physiological science II, University of Barcelona; Fina Martinez Soler, Department of Physiological science II, University of Barcelona; Noemi Vidal, Hospital de Bellvitge, Barcelona; Alberto Villanueva, ICO-IDIBELL, Barcelona; Pepita Gimenez-Bonafé, Department of Physiological science II, University of Barcelona; Avelina Tortosa, Department of Physiological science II, University of Barcelona.


Intra-tumoral heterogeneity together with Glioblastoma Initiating Cells (GICs) presence within the tumor mass are considered, among others, responsible for the daunting prospect of new therapeutic strategies in Glioblastoma multiforme (GBM).
The aim of this study is to mimic the intrinsic heterogeneity between the bulk of the tumor and GICs through an in-vitro model. Four GBM post-surgical specimens were cultured in two different growth conditions: neurosphere primary culture and their monolayer counterpart.
The stemness of established GICs was confirmed by their self-renewal capability, extensive proliferation potential and capability to differentiate along the main lineages of the central nervous system. Tumor initiation capability was assessed by orthotopical injection of GICs in nude mice and the new-formed tumors closely resembled the original histological features. A pattern of markers expressed in GICs, but not in monolayer primary culture, was identified (CD133, CD44, L1CAM, ITGA6). Moreover, GICs were found to display a strong mesenchymal gene signature profile. After fractionated radiotherapy treatment (2Gy/fraction x 4 days), GICs were found to be more resistant than their monolayer counterparts assessed by adapted clonogenic assay (p<0.001). Interestingly, the response to radiotherapy of GICs, but not monolayer cultures, correlates with both disease free survival and overall survival of the patient they belong to. In addition, a preferential basal activation of CHK2 protein was found in GICs but not in monolayer cultures. In conclusion, the proposed in-vitro model could be effectively used to evaluate and predict intra-tumoral heterogeneous response to radiation therapy in GBM.

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