The cancer stem cell model suggests that glioblastomas contain a subpopulation of stem-like tumor cells that reproduce themselves to sustain tumor growth. Targeting these cells thus represents a novel treatment strategy and therefore more specific markers that characterize glioblastoma stem cells need to be identified. In the present study, we performed transcriptomic analysis of glioblastoma tissues compared to normal brain tissues revealing sensible up-regulation of CD9 gene. CD9 encodes the transmembrane protein tetraspanin which is involved in tumor cell invasion, apoptosis and resistance to chemotherapy. Using the public REMBRANDT database for brain tumors, we confirmed the prognostic value of CD9, whereby a more than two fold up-regulation correlates with shorter patient survival. We validated CD9 gene and protein expression showing selective up-regulation in glioblastoma stem cells isolated from primary biopsies and in primary organotypic glioblastoma spheroids as well as in U87-MG and U373 glioblastoma cell lines. In contrast, no or low CD9 gene expression was observed in normal human astrocytes, normal brain tissue and neural stem cells. CD9 silencing in three CD133+ glioblastoma cell lines (NCH644, NCH421k and NCH660h) led to decreased cell proliferation, survival, invasion, and self-renewal ability, and altered expression of the stem-cell markers CD133, nestin and SOX2. Moreover, CD9-silenced glioblastoma stem cells showed altered activation patterns of the Akt, MapK and Stat3 signaling transducers. Orthotopic xenotransplantation of CD9-silenced glioblastoma stem cells into nude rats promoted prolonged survival. Therefore, CD9 should be further evaluated as a target for glioblastoma treatment.

Malignant gliomas are among the rarest brain tumours, and they have the worst prognosis. Grade IV astrocytoma, known as glioblastoma multiforme (GBM), is a highly lethal disease where the standard therapies of surgery, followed by radiation and chemotherapy, cannot significantly prolong the life expectancy of the patients. Tumour recurrence shows more aggressive form compared to the primary tumour, and results in patient survival from 12 to 15 months only. Although still controversial, the cancer stem cell hypothesis postulates that cancer stem cells are responsible for early relapse of the disease after surgical intervention due to their high resistance to therapy. Alternative strategies for GBM therapy are thus urgently needed. Nanobodies are single-domain antigen-binding fragments of heavy-chain antibodies, and together with classical antibodies, they are part of the camelid immune system. Nanobodies are small and stable, and they share a high degree of sequence identity to the human heavy chain variable domain, and these characteristics offer them advantages over classical antibodies or antibody fragments. We first immunised an alpaca with a human GBM stem-like cell line prepared from primary GBM cultures. Next, a nanobody library was constructed in a phage-display vector. Using nanobody phage-display technology, we selected specific GBM stem-like cell binders through a number of affinity selections, using whole cell protein extracts and membrane protein-enriched extracts from eight different GBM patients, and membrane protein-enriched extracts from two established GBM stem-like cell lines (NCH644 and NCH421K cells). After the enrichment, periplasmic extract ELISA was used to screen for specific clones. These nanobody clones were recloned into the pHEN6 vector, expressed in Escherichia coli WK6, and purified using immobilised metal affinity chromatography and size-exclusion chromatography. Specific nanobody:antigen pairs were obtained and mass spectrometry analysis revealed two proteins, TRIM28 and β-actin, that were up-regulated in the GBM stem-like cells compared to the controls.

BackgroundPatient-derived glioblastoma (GBM) stem-like cells (GSCs) represent a valuable model for basic and therapeutic research. GSCs are usually propagated in serum-free Neural Basal medium supplemented with bFGF and EGF. Yet, the exact influence of these growth factors on GSCs is still unclear. Recently it was suggested that GBM stem-like cells with amplified EGFR should be cultured in stem cell medium without EGF, as the presence of EGF induced rapid loss of EGFR amplification. However, patient biopsies are usually taken into culture before their genomic profiles are defined. Thus, an important question remains whether GBM cells without EGFR amplification also can be cultured in stem cell medium without EGF.Meterials and methodsTo address this question, we used two heterogeneous glioblastoma GSC lines (NCH421k and NCH644) that lack EGFR amplification.ResultsAlthough both cell lines showed very low EGFR expression under standard growth conditions, bFGF stimulation induced higher expression of EGFR in NCH644. In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF. Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect. We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644).ConclusionsWe demonstrate that GSC cultures without EGFR amplification can be maintained and expanded with bFGF, while the addition of EGF has no significant effect and therefore can be omitted.

BackgroundGlioblastoma stem cells (GSC) have been extensively recognized as a plausible cause of glioblastoma resistance to therapy and recurrence resulting in high glioblastoma mortality. Abnormalities in the DNA repair pathways might be responsible for the inability of the currently used chemotherapeutics to eliminate the (GSC) subpopulation.MethodsIn this work, we compared the expression of sixty DNA repair related genes between primary glioblastoma cell cultures and the glioblastoma enriched stem cell primary cultures. MTT test was used to analyze the effect of selected drugs and immunofluorescence to evaluate the load of DNA damage.ResultsWe found several differentially expressed genes and we identified topoisomerase IIβ (Top2β) as the gene with highest up-regulation in GSC. Also among the tested cell lines the expression of Top2β was the highest in NCH421k cells, a well-characterized glioblastoma cell line with all the stemness characteristics. On the other hand, Top2β expression markedly decreased upon the induction of differentiation by all trans-retinoic acid. Depletion of Top2β increased the sensitivity of NCH421k cells to replication stress inducing drugs, such as cisplatin, methyl-methanesulfonate, hydrogen peroxide, and temozolomide. Consistently, we found an increased load of DNA damage and increased Chk1 activation upon Top2β depletion in NCH421k cells.ConclusionWe suggest that Top2β may represent a new target for gene therapy in glioblastoma. In addition, the other genes that we found to be up-regulated in GSC versus glioblastoma primary cells should be further investigated as glioblastoma theranostics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-016-0339-9) contains supplementary material, which is available to authorized users.

Genetic and signaling pathways involved in the development of glioblastoma have been relatively well characterized. Gene/protein expression profiles of glioblastoma have been described that reflect stem-like phenotype of the tumor. However, no reliable individual markers exist up-to-date that differentiate glioblastoma stem-like cells from the neural stem cells. Glioblastoma appears to be of heterogenous cellular population, but detailed cellular origin of these tumors is unknown. Using a label-free proteomic approach, we analyzed two different glioma models with proven de novo tumorigenic properties, and compared them with primary neurospheres. Of almost 2000 identified proteins, we found 482 to be significantly differentially expressed among the three groups. Among the identified substrates, the clusters of proteins with highest density of protein-protein interactions were functionally enriched in RNA processing related to development and metabolic processes. We then focused on two putative quantitative markers of glioma stem-like cells, HP1-gamma (Heterochromatin Protein 1- gamma) - CBX3 (chrome box 3), an epigenetic regulator involved in RNA processing, and LDHA (Lactate Dehydrogenase A), a key metabolic enzyme of anaerobic glycolysis. We quantitated their expression in glioma tissue of different grades in a large set of patient samples and were able to show that the expression of HP1-gamma, highly upregulated in glioblastoma stem-like cells, correlates to glioma grade. Independent analysis of publicly available glioma patient data showed that LDHA expression is linked to IDH1 mutation status both in high grade and low grade gliomas. The same analysis reinforced our findings towards an increased involvement of CBX3/HP1-gamma in high grade gliomas. Altogether, CBX3/HP1-gamma and LDHA are novel biomarkers for cancer stem cell phenotype with different clinical importance in gliomas. Citation Format: Uros Rajcevic, Sabrina Fritah, Petr V. Nazarov, Mara Popovic, Rajko Kavalar, Tadej Strojnik, Neza Podergajs, Jaco C. Knol, Thang V. Pham, Sander R. Piersma, Connie R. Jimenez, Simone P. Niclou. HP1-gamma's expression correlates with glioma grade and survival and is a putative marker of glioma stem like cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1503. doi:10.1158/1538-7445.AM2015-1503