Daniela Marubbi

SOX2 silencing in glioblastoma tumor-initiating cells causes stop of proliferation and loss of tumorigenicity.

Glioblastoma, the most aggressive cerebral tumor, is invariably lethal. Glioblastoma cells express several genes typical of normal neural stem cells. One of them, SOX2, is a master gene involved in sustaining self‐renewal of several stem cells, in particular neural stem cells. To investigate its role in the aberrant growth of glioblastoma, we silenced SOX2 in freshly derived glioblastoma tumor‐initiating cells (TICs). Our results indicate that SOX2 silenced glioblastoma TICs, despite the many mutations they have accumulated, stop proliferating and lose tumorigenicity in immunodeficient mice. SOX2 is then also fundamental for maintenance of the self‐renewal capacity of neural stem cells when they have acquired cancer properties. SOX2, or its immediate downstream effectors, would then be an ideal target for glioblastoma therapy. STEM CELLS 2009;27:40–48

NK Cells Recognize and Kill Human Glioblastoma Cells with Stem Cell-Like Properties

In this study, cancer cells were isolated from tumor specimens of nine glioblastoma patients. Glioblastoma cells, cultured under suitable culture conditions, displayed markers typical of neural stem cells, were capable of partial multilineage differentiation in vitro, and gave origin to infiltrating tumors when orthotopically injected in NOD/SCID mice. These cells, although resistant to freshly isolated NK cells, were highly susceptible to lysis mediated by both allogeneic and autologous IL-2 (or IL-15)-activated NK cells. Indeed, all stem cell-cultured glioblastoma cells analyzed did not express protective amounts of HLA class I molecules, while expressing various ligands of activating NK receptors that triggered optimal NK cell cytotoxicity. Importantly, glioblastoma stem cells expressed high levels of PVR and Nectin-2, the ligands of DNAM-1-activating NK receptor.

Different Response of Human Glioma Tumor-initiating Cells to Epidermal Growth Factor Receptor Kinase Inhibitors

Because a subpopulation of cancer stem cells (tumor-initiating cells, TICs) is believed to be responsible for the development, progression, and recurrence of many tumors, we evaluated the in vitro sensitivity of human glioma TICs to epidermal growth factor receptor (EGFR) kinase inhibitors (erlotinib and gefitinib) and possible molecular determinants for their effects. Cells isolated from seven glioblastomas (GBM 1-7) and grown using neural stem cell permissive conditions were characterized for in vivo tumorigenicity, expression of tumor stem cell markers (CD133, nestin), and multilineage differentiation properties, confirming that these cultures are enriched in TICs. TIC cultures were challenged with increasing concentrations of erlotinib and gefitinib, and their survival was evaluated after 1-4 days. In most cases, a time- and concentration-dependent cell death was observed, although GBM 2 was completely insensitive to both drugs, and GBM 7 was responsive only to the highest concentrations tested. Using a radioligand binding assay, we show that all GBM TICs express EGFR. Erlotinib and gefitinib inhibited EGFR and ERK1/2 phosphorylation/activation in all GBMs, irrespective of the antiproliferative response observed. However, under basal conditions GBM 2 showed a high Akt phosphorylation that was completely insensitive to both drugs, whereas GBM 7 was completely insensitive to gefitinib, and Akt inactivation occurred only for the highest erlotinib concentration tested, showing a precise relationship with the antiproliferative effects of the drug. Interestingly, in GBM 2, phosphatase and tensin homolog expression was significantly down-regulated, possibly accounting for the insensitivity to the drugs. In conclusion, glioma TICs are responsive to anti-EGFR drugs, but phosphatase and tensin homolog expression and Akt inhibition seem to be necessary for such effect.

Emx2 in adult neural precursor cells

Emx2 is a vertebrate homeobox gene involved in the control of the central nervous system development. In the formation of cerebral cortex, Emx2 expression is restricted mainly to the germinal ventricular zone fading away in the first postmitotic neurons. This expression pattern, the severe impairment of cortex organization and the size in mutant mice suggest a role of Emx2 in the control of proliferation and migration of neural precursor cells. The observed persistence of Emx2 expression in adult neurogenic areas in vivo is here confirmed at later stages. We also find that Emx2 is expressed at high levels in adult neural stem cells (ANSCs) in vitro and is down modulated upon differentiation. Overexpression of Emx2 gene in ANSCs has an anti-proliferative effect but it does not influence a particular differentiation pathway. Our results suggest that Emx2 may act promoting an asymmetric mode of cell division thereby increasing the size of a transit amplifying population.

Sorafenib selectively depletes human glioblastoma tumor-initiating cells from primary cultures

Glioblastomas are grade IV brain tumors characterized by high aggressiveness and invasiveness, giving patients a poor prognosis. We investigated the effects of the multi-kinase inhibitor sorafenib on six cultures isolated from human glioblastomas and maintained in tumor initiating cells-enriching conditions. These cell subpopulations are thought to be responsible for tumor recurrence and radio- and chemo-resistance, representing the perfect target for glioblastoma therapy. Sorafenib reduces proliferation of glioblastoma cultures, and this effect depends, at least in part, on the inhibition of PI3K/Akt and MAPK pathways, both involved in gliomagenesis. Sorafenib significantly induces apoptosis/cell death via downregulation of the survival factor Mcl-1. We provide evidence that sorafenib has a selective action on glioblastoma stem cells, causing enrichment of cultures in differentiated cells, downregulation of the expression of stemness markers required to maintain malignancy (nestin, Olig2 and Sox2) and reducing cell clonogenic ability in vitro and tumorigenic potential in vivo. The selectivity of sorafenib effects on glioblastoma stem cells is confirmed by the lower sensitivity of glioblastoma cultures after differentiation as compared with the undifferentiated counterpart. Since current GBM therapy enriches the tumor in cancer stem cells, the evidence of a selective action of sorafenib on these cells is therapeutically relevant, even if, so far, results from first phase II clinical trials did not demonstrate its efficacy.

Glioma immunotherapy by IL-21 gene-modified cells or by recombinant IL-21 involves antibody responses

Most tumors of the central nervous system, especially glioblastoma, are refractory to treatment and invariably lethal. The aim of this study was to assess the ability of different interleukins (IL), IL‐2, IL‐12 and IL‐21, produced by transduced glioma cells to activate an immune response and trigger intracranial tumor rejection. Such experiments were performed by the use of a slow‐growing clone of GL261 (GL D2‐60) that was used as orthotopic glioma model. Using GL D2‐60‐transduced cells, all cytokines elicited an immune response against the tumor. Most notably 100% of the animals receiving a primary implant of IL‐21‐transduced cells rejected the implant, and 76% of these animals survived to a subsequent rechallenge with GL261 parental cells, while the other transduced cytokine genes were not as effective. Rejection responses were also obtained by admixing wild‐type tumor cells with IL‐21‐producing GL D2‐60 cells, indicating a local bystander effect of IL‐21. More importantly, IL‐21‐secreting GL D2‐60 cells or 1 μg of rIL‐21 protein stereotactically injected into established GL D2‐60 tumors were able to trigger glioblastoma rejection in 90 and 77% of mice, respectively. Again most of these mice survived to GL261 rechallenge. Immune mice showed antibody responses to glioma antigens, predominantly involving IgG2a and IgG2b isotypes, which mediated complement‐ or cell‐dependent glioma cell lysis. Antibody responses were crucial for glioma immunotherapy by IL‐21‐secreting GL D2‐60 cells, as immunotherapy was uneffective in syngeneic μMT B‐cell‐deficient mice. These results suggest that IL‐21 should be considered as a suitable candidate for glioma immunotherapy by local delivery.

Effects of Emx2 inactivation on the gene expression profile of neural precursors

Emx2 plays a crucial role in the development of the diencephalon and dorsal telencephalon. Thus, Emx2‐null mutants have abnormal cortical lamination and a reduction in size of the caudal and medial areas of the prosencephalon. Emx2 is expressed in neural precursors of the subventricular zone in vivo and in cultured neurospheres in vitro where it controls the size of the transit‐amplifying population, affecting proliferation and clonal efficiency of neural stem cells. To identify the cellular processes mastered by Emx2, and possibly the molecular mechanisms by which the gene exerts its action, we compared the expression profile of cultured neurospheres derived from wild‐type and Emx2‐null mouse embryos. The differential expression of several genes was also confirmed by semiquantitative RT‐PCR, real‐time PCR and cytofluorimetric analysis in different preparations of neurospheres, and by in situ hybridization. The gene expression profile suggested a role for Emx2 in regulating the differentiation and migration properties of neural precursor cells. This involvement was confirmed in vitro, where the altered clonogenicity and impaired migration of Emx2‐null cells were partially corrected by transduction of the Emx2 gene. Taken together, our results indicate that Emx2 is indeed involved in the transition between resident early progenitors (perhaps stem cells) and more mature precursors capable of migrating out of the ventricular zone, becoming postmitotic and differentiating into the appropriate cell type, and help explain the alterations observed in the brains of knock‐out mice.

Predictability, efficacy and safety of radiosensitization of glioblastoma-initiating cells by the ATM inhibitor KU-60019.

We have previously shown that pharmacological inhibition of ataxia telangiectasia mutated (ATM) protein sensitizes glioblastoma‐initiating cells (GICs) to ionizing radiation (IR). Herein, we report the experimental conditions to overcome GIC radioresistance in vitro using the specific ATM inhibitor KU‐60019, two major determinants of the tumor response to this drug and the absence of toxicity of this treatment in vitro and in vivo. Repeated treatments with KU‐60019 followed by IR substantially delayed GIC proliferation in vitro and even eradicated radioresistant cells, whereas GIC treated with vehicle plus radiation recovered early and expanded. The tumor response to the drug occurred under a cutoff level of expression of TP53 and over a cutoff level of expression of phosphatidylinositol 3‐kinase (PI3K). No increased clastogenicity or point mutagenicity was induced by KU‐60019 plus radiation when compared to vehicle plus radiation. No significant histological changes to the brain or other organs were observed after prolonged infusion into the brain of KU‐60019 at millimolar concentrations. Taken together, these findings suggest that GIC‐driven tumors with low expression of TP53 and high expression of PI3K might be effectively and safely radiosensitized by KU‐60019.

Pharmacokinetics, pharmacodynamics and efficacy on pediatric tumors of the glioma radiosensitizer KU60019

We have recently reported that glioblastoma (GB)‐initiating cells (GIC) with low expression and/or mutation of TP53 and high expression of PI3K (“responder” genetic profile) can be effectively and safely radiosensitized by the ATM inhibitor KU60019. We report here on drugs diffusion and elimination from the animal body and brain, its effects on orthotopic GB and efficacy toward pediatric GIC. Healthy mice were infused by convection enhanced delivery (CED) with KU60019 and the drug kinetics followed by high performance liquid chromatography–mass spectrometry. Already at the end of CED, KU60019 had diffused from the injection site to the ipsilateral and, to a lower extent, controlateral hemisphere. After 24 hr, no drug could be detected all over the brain or in other organs, indicating rapid draining and excretion. After intraperitoneal injection, traces only of KU60019 could be detected in the brain, indicating inability to cross the brain–blood barrier. Consistent with the induction of cell cycle progression previously observed in vitro, KU60019 stimulated proliferation of orthotopic GB cells with the highest effect observed 96 hr after drug delivery. Adult GIC with high expression of TP53 and low expression of PI3K could be radiosensitized by KU60019, although less promptly than GIC bearing the “responder” profile. Consistent with the kinetics of proliferation induction, the highest radiosensitizing effect was observed 96 hr after delivery of KU60019 to GIC. Pediatric GIC could be similarly radiosensitized after exposure to KU60019. The results indicate that ATM inhibition may allow to radiosensitize a wide range of adult and pediatric high‐grade gliomas.

In vitro and in vivo characterization of highly purified Human Mesothelioma derived cells

BackgroundMalignant pleural mesothelioma is a rare disease known to be resistant to conventional therapies. A better understanding of mesothelioma biology may provide the rationale for new therapeutic strategies. In this regard, tumor cell lines development has been an important tool to study the biological properties of many tumors. However all the cell lines established so far were grown in medium containing at least 10% serum, and it has been shown that primary cell lines cultured under these conditions lose their ability to differentiate, acquire gene expression profiles that differ from that of tissue specific stem cells or the primary tumor they derive from, and in some cases are neither clonogenic nor tumorigenic. Our work was aimed to establish from fresh human pleural mesothelioma samples cell cultures maintaining tumorigenic properties.MethodsThe primary cell cultures, obtained from four human pleural mesotheliomas, were expanded in vitro in a low serum proliferation-permissive medium and the expression of different markers as well as the tumorigenicity in immunodeficient mice was evaluated.ResultsThe established mesothelioma cell cultures are able to engraft, after pseudo orthotopic intraperitoneal transplantation, in immunodeficient mouse and maintain this ability to after serial transplantation. Our cell cultures were strongly positive for CD46, CD47, CD56 and CD63 and were also strongly positive for some markers never described before in mesothelioma cell lines, including CD55, CD90 and CD99. By real time PCR we found that our cell lines expressed high mRNA levels of typical mesothelioma markers as mesothelin (MSLN) and calretinin (CALB2), and of BMI-1, a stemness marker, and DKK1, a potent Wingless [WNT] inhibitor.ConclusionsThese cell cultures may provide a valuable in vitro and in vivo model to investigate mesothelioma biology. The identification of new mesothelioma markers may be useful for diagnosis and/or prognosis of this neoplasia as well as for isolation of mesothelioma tumor initiating cells.