GBM Mouse Model Research Articles

Abstract IA07: The effect of cell of origin on GBM phenotype

Abstract Current thinking about solid tumor biology is largely influenced by analysis of the large data sets afforded by modern genomic capabilities. In GBM, classification of tumors into four categories (proneural, neural, mesenchymal, and classic) relies on differential transcriptional profiles coupled with copy numbers, and mutational profiles. These approaches for example, imply EGFR receptor as a major driver of Classic sub-type whereas NF1 mutations are preferentially found in mesenchymal subtypes. Functional evidence that these molecular categories indeed reflect biologically different tumors remains to be proved. We have generated mouse models of GBM by driving loss of function of in the NF1, p53 and Pten tumor suppressors. We find that two phenotypically and biologically different GBMs emerge that reflect tumor initiation different adult CNS progenitor populations. Thus, in a mouse model, distinct progenitor cell populations dictate different tumors created by mutation in identical drivers. Citation Format: Luis F. Parada. The effect of cell of origin on GBM phenotype. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA07.

Abstract IA27: Glioma stem cells: What are they?

Abstract We have generated mouse models of GBM by driving loss of function of in the NF1, p53 and Pten tumor suppressors in the adult mouse brain. Using genetic cell labeling and lineage tracing approaches, our models support the hypothesis that stem/progenitor cells are the most likely cells to accumulate mutations over time and become the source of GBM. We have succeeded in reporter labeling the putative “Cancer Stem Cell” population and demonstrate their requirement for effectively mediating endogenous tumor recurrence after chemotherapy and in successfully supporting serial transplantation of tumors in intracranial allografts. The availability of enriched Cancer Stem cells should provide a powerful tool for identifying critical molecular properties and novel therapeutic targets. Citation Format: Luis F. Parada. Glioma stem cells: What are they? [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA27.

Abstract A63: Identification of small molecules for GBM study

Abstract Glioblastoma Multiforme (GBM) is the most frequent of malignant tumors of the brain. It is fatal usually within fifteen months after diagnosis. The Cancer Genome Atlas (TCGA) has surveyed the genomes of hundreds of human GBMs for presence of mutations in genes previously associated with cancer. Among those identified, three of the five most frequently mutated genes encode the well-known tumor suppressors: P53, NF1, and Pten (TCGA, 2008). We have generated genetic mouse models of GBM based on mutation in these same three tumor suppressors and they develop GBM with 100% incidence. These mouse GBMs resemble the human counterparts by all commonly used criteria for diagnosis. Extensive study of these mice and tumors has provided novel insights into the natural history of GBM development and moreover pointed at the adult neural stem cell/progenitor compartment as the most likely and frequent source of these tumors (Kwon CH, et al., 2008). We have thus isolated a cancer stem cell and demonstrated a hierarchical growth for these tumors in vivo. (Chen J, et al., 2012) In an effort to identify novel GBM specific molecular pathways that might serve as therapeutic targets, we undertook a 200,000 small compound high throughput screen using primary low passage GBM cells generated by pooling cells from multiple mouse tumors (Mut6 cells). A critical counter screen was designed to minimize compounds that targeted the cell division and DNA replication machinery of cells since many normal cells divide and this property is not unique to cancer cells. We therefore ruled out all compounds that displayed toxicity to primary mouse embryo fibroblasts (MEFs) or to primary astrocytes. At the end of the screen, we selected three lead compounds with amenable physical-chemical properties for further evaluation and development as research discovery reagents and possible translational tools for drug development. We verified the activity of one of these compounds, termed Compound X, on mouse and human GBM primary cells as well as on a subset of established cancer cell lines, but not on MEFs or primary astrocytes. We identified a rapid cellular stress response accompanied by the robust activation of a transcription factor and eventual apoptotic response that can be mimicked by glucose deprivation. Through intensive structure-activity relationship study, analogs were successfully developed for in vivo activity and also for biochemical pull-down assay. Another compound (Compound Y) also induced apoptosis, however displaying response through completely different pathway, which indicates our screen identified multiple vulnerabilities in GBM tumor cells. Citation Format: Sang Kyun Lim, Yufeng Shi, Qiren Liang, Inga Nazarenko, Shuguang Wei, Maya Palnitkar, Yanjiao Li, Woosung Cho, Noelle Williams, Bruce Posner, Jef De Brabander, Luis F. Parada. Identification of small molecules for GBM study. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A63.

Abstract LB-140: Investigating cancer stem cells in glioblastoma initiation and recurrence

Abstract In this study, I examine the unique features of quiescent cancer stem cells (CSCs) in glioblastoma initiation and recurrence in genetically engineered mouse models. A nestin-TK-GFP transgene was used to label CSCs with GFP and to render them susceptible to ganciclovir (GCV) treatment in a fully penetrant mouse model of GBM (M7: hGFAP-Cre; Nf1fl/+; p53fl/fl; Ptenfl). Using food-mediated GCV delivery (GCV chow) to optimize the drug delivery, Doublecortin-positive and NG2-positive cells derived from the nestin-TK-GFP transgene labeled neural stem cells were significantly reduced after six weeks. In addition, on GCV chow the tumor-bearing mice containing the nestin-TK-GFP transgene live significantly longer than the ones without the transgene. Isolation and transplantation of the GFP+/GFP- tumor cells demonstrated that the GFP+ cells have enhanced tumorigenic potential over the GFP- cells. These results support the hypothesis that the nestin-TK-GFP transgene targets the CSCs that are responsible for the sustained GBM growth. In the M7 model, the hGFAP transgene has a broader expression, and thus broader Cre targeting, than the nestin transgene. To eradicate the problem of differing scopes of Cre expression versus targeting of CSCs, I generated a new transgene (CGD: nestin-CreERT2-H2BeGFP-hDTR) that enables Cre-mediated recombination, specific cell labeling and targeted cell ablation. The CGD is designed to express a fusion protein that consists of CreERT2, H2B-eGFP, and hDTR (human diphtheria toxin receptor). Containing P2A linkers between the three gene cassettes, the fusion protein can be efficiently cleaved into three active. I have screened for a transgenic line that can induce GBM formation upon tamoxifen induction. Six to eight week-old mice with the following genetic configuration: CGD; Nf1fl/+; p53fl/+; Ptenfl/+, were given one dose of tamoxifen and all mice died of high grade gliomas three to six months later. The analysis of the CGD-induced tumors demonstrated that the CGD transgene labels tumor cells that are relatively quiescent, compared to the highly proliferative Ki67+ cells. Thus our CGD transgene labels putative CSCs within the tumor. In this model, the same cells initiating tumor formation (Cre-expressing) are also expressing H2B-eGFP and hDTR. Thus, all the tumor cells are derived from the CGD transgene and all the CSCs should be labeled with the H2B-eGFP protein. This dramatically facilitates the sorting of the tumor-initiating cells. Transplantation of the GFP+/GFP- cells in these tumors revealed that the quiescent GFP+ cells are more tumorigenic than the proliferating GFP- cells. Furthermore, diphtheria toxin-mediated ablation of the hDTR-expressing cells significantly elongates the lives of tumor-bearing mice. To mimic the endogenous environment for GBM growth, wild-type mice were also used to prove the differential tumorigenic ability of the GFP+/- cells. These results validate the essential roles of CSCs in GBM initiation and recurrence. Citation Format: xuanhua xie, Xiuping Zhou, German B. Sánchez, Luis F. Parada. Investigating cancer stem cells in glioblastoma initiation and recurrence. [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 LB-140. doi:10.1158/1538-7445.AM2015-LB-140

Abstract 3243: Notch inhibitors and chemoradiation in an orthotopic glioblastoma model

Abstract Glioblastoma multiforme (GBM) is the most common and malignant brain tumor in adults. Aggressive multimodal treatment using surgery followed by radiotherapy and chemotherapy extends the median survival of GBM patients to approximately one year after diagnosis. Treatment is not curative because of the intrinsic and acquired radiation resistance of a subpopulation of tumor cells. Notch inhibition has been shown to impair the tumorigenic capacity of these cells as well as enhance their sensitivity towards radiation. Therefore, we investigated if a highly potent and clinically approved Notch pathway inhibitor (g-secretase inhibitor) improves tumor control when combined with radiotherapy and chemotherapy in an orthotopic GBM mouse model. To investigate the treatment efficacy of combinations treatments between standard of care treatment (radiotherapy and temozolomide) and Notch inhibitors, we used a three dimensional spheroid growth assay using both established and primary human glioma cell lines in which spheroid volume growth delay was quantitatively monitored. In addition, we assessed the expression of the putative glioma stem cell marker CD133 using flow cytometry. Furthermore, therapeutic efficacy of these combination treatments was als assessed in vivo in an orthotopic glioma tumor model (U87-luc) wherein tumor progression was evaluated using bioluminescence (BLI) and contrast-enhanced micro-computed tomography (CT) imaging. A small animal precision irradiation platform (PXI, XRAD 225Cx, CT, USA) was used for micro-CT imaging and irradiation delivery of conformal doses between 2- 10 Gy to intracranial tumors in mice receiving single or combination treatments with Notch inhibitors and chemotherapy. Results: Notch blockade alone did not affect the sphere volume compared to control, whereas combination treatment with radiation and / or temozolomide resulted in a substantial spheroid growth delay (p = 0.004). Irradiation enhanced the expression of the stem cell marker CD133, while Notch blockade reduced CD133 expression. In intracranial tumours we found a strong correlation between CT and BLI imaging of tumor growth (Pearson coefficient (r) = 0.85, p = 0.001). The potential of Notch inhibition combined with precision radiotherapy and chemotherapy in our orthotopic GBM model is currently being determined and the outcome of this study will be presented. Citation Format: Sanaz Yahyanejad, Patrick Granton, Stefan van Hoof, Lydie Barbeau, Jan Theys, Frank Verhaegen, Marc Vooijs. Notch inhibitors and chemoradiation in an orthotopic glioblastoma model. [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 3243. doi:10.1158/1538-7445.AM2015-3243

Myeloid derived suppressor cell infiltration of murine and human gliomas is associated with reduction of tumor infiltrating lymphocytes.

Myeloid derived suppressor cells (MDSCs) are bone marrow derived cells with immunosuppressive properties. We have shown previously that MDSCs numbers are elevated in the circulation of GBM patients and that they produce reversible T cell dysfunction. Here, we evaluated whether MDSCs infiltrate human GBM tissues, and whether a commonly used mouse model of GBM reproduces the biology of MDSCs that is observed in patients. We evaluated tumor specimens from patients with newly diagnosed GBM. We harvested and evaluated normal brain, tumors and hematopoietic tissues from control, vehicle and sunitinib-treated mice. In human GBM tumors, MDSCs represented 5.4 ± 1.8 % of total cells. The majority of MDSCs (CD33+HLADR-) were lineage negative (CD14-CD15-), followed by granulocytic (CD15+CD14-) and monocytic (CD15-CD14+) subtypes. In murine GBM tumors, MDSCs were 8.06 ± 0.78 % of total cells, of which more were monocytic (M-MDSC, CD11b+ Gr1-low) than granulocytic (G-MDSC, CD11b+ Gr1-high). Treatment with the tyrosine kinase inhibitor sunitinib decreased the infiltration of both granulocytic and monocytic MDSCs in murine GBM tumors. In the hematopoietic tissues, circulating G-MDSC blood levels were reduced after sunitinib treatment. In tumors, both CD3(+) and CD4(+) T cell counts increased following sunitinib treatment (p ≤ 0.001). Total T cell proliferation (p < 0.001) and interferon gamma production (p = 0.004) were increased in the spleens of sunitinib treated mice. Sunitinib-treated mice survived longer than vehicle-treated mice (p = 0.002). MDSCs are present in both human and mouse GBM tumors. Sunitinib may have an immunostimulatory effect, as its use is associated with a reduction in G-MDSCs and improvement in anti-tumor immune function.

Abstract B03: Synergistic control of GBM growth by MEK and PI3kinase signaling in a RAS-driven preclinical orthotopic model for human glioblastoma multiforme

Abstract PI3K, RTK/RAS, and Rb signaling are commonly altered in human GBM. We utilized a genetically engineered mouse (GEM) model for GBM, designated “TRP,” that expresses GFAP-T121 to suppress Rb (T), the KrasG12D mutation (R), and is heterozygous for a PTEN null allele (P), to develop an efficient and tractable orthotopic mouse model for GBM treatment preclinical evaluation. The orthotopic brain tumors presented linear foci of necrosis with peudopalisading by neoplastic cells described in human GBM, and were highly proliferative, invasive, and vascular. Immunohistochemistry analysis of TRP orthotopic tumors identified markers characteristic of human GBM, and tumor progression was readily examined by serial MRI. Both PI3K and MAPK pathway inhibitors inhibited growth of TRP GEM-derived primary tumor cells, but did not result in significant apoptosis. However, when GBM derived cells were treated with a combination of pathway inhibitors such as BKM120 (a pan-PI3K inhibitor currently in clinical trials for solid tumors) and PD0325901 (a MEK inhibitor), potency was enhanced with a substantial increase in cell death. Analysis of downstream targets revealed a synergistic effect on target downregulation in the PI3K pathway. In the orthotopic GBM model, the PI3K/MAPK targeted combination delayed tumor growth by increasing GBM cell apoptosis and resulted in significantly increased survival. Taken together, our results validate this new orthotopic model for the assessment of targeted therapeutic regimens for human GBM, and show that the feedback loops and incomplete pathway suppression, observed in vivo with targeted single agents, can be overcome with a combination treatment strategy. Citation Format: Rajaa El Meskini, Anthony Iacovelli, Alan Kulaga, Michelle Gumprecht, Philip Martin, Maureen Baran, Deborah Householder, Terry van Dyke, Zoe Weaver Ohler. Synergistic control of GBM growth by MEK and PI3kinase signaling in a RAS-driven preclinical orthotopic model for human glioblastoma multiforme. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B03. doi: 10.1158/1557-3125.RASONC14-B03

An in vitro mouse model of GBM with abrupt and predictable onset

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Abstract 524: Analysis of long non-coding RNA expression and function in a mouse model of glioblastoma

Abstract Glioblastoma (GBM) is the most common adult brain tumor and represents one of the most treatment refractory cancers. Although significant progress has been made in understanding the coding genomic alterations associated with GBM, patient survival rarely exceeds 12-14 months following diagnosis. Long non-coding RNAs (lncRNAs) are RNA species generally classified as &amp;gt; 200 base-pairs in length that lack protein coding potential and therefore exert their function as RNA. Several lncRNAs have been shown to play important roles in cancer biology. Despite this, the role of lncRNAs in cancer, and GBM in particular, remains relatively uncharacterized. We hypothesize that lncRNAs are differentially expressed in GBM and contribute to the pathogenesis of this disease. Using next generation sequencing, we have identified lncRNAs expressed in our mouse model of GBM, initiated by lentivirus-mediated expression of oncogenic Ras and a shRNA targeting p53. This resulted in the identification of 818 putative lncRNAs, the majority of which represent novel, uncharacterized transcripts. The majority of the lncRNAs identified were intergenic and not associated with any known protein coding gene. Of the total lncRNAs, 44% were differentially expressed in tumor tissue compared to normal mouse brain. Expression of a subset of these differentially expressed lncRNAs was validated by real-time PCR. Expression of several of these validated lncRNAs could be altered by direct activation of oncogenic signaling in normal mouse neuroprogenitor cells and astrocytes. Using a combination of computational and molecular biology approaches, we will identify candidate lncRNAs to test in functional assays for their role in glioblastoma biology. Citation Format: Chad Myskiw, Sabah Kadri, Eugene Ke, Alex Shishkin, Dinorah Friedmann-Morvinski, Yasushi Soda, Mitchell Guttman, Inder Verma. Analysis of long non-coding RNA expression and function in a mouse model of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 524. doi:10.1158/1538-7445.AM2014-524

Abstract 3137: Combined inhibition of MEK and PI3 kinase signaling results in improved survival in a preclinical orthotopic model for human glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM; astrocytoma grade IV) is the most frequent and aggressive brain tumor for which no effective therapy is currently available. It has been shown that PI3K, RTK/RAS, and Rb signaling are commonly altered in human GBM. We utilized a genetically engineered mouse (GEM) model for GBM, designated “TRP,” that expresses GFAP-T121 (T; for Rb suppression), the KrasG12D mutation (R), and is heterozygous for a PTEN null allele (P), to develop an orthotopic mouse model for the preclinical evaluation of potential therapeutics for GBM treatment. Although the de novo TRP brain tumor GEM model recapitulates features of human GBM including tissue invasion, pseudopalisading necrosis and dense vascularization, the latency to tumorigenesis (4-6 months) makes its use as a preclinical model for drug screening challenging. Therefore we isolated primary GBM cells from TRP GEMs and injected cells intracranially (IC) into syngeneic mouse brains. Recipient mice developed grade IV astrocytomas and recapitulated TRP GEM tumor histopathology. The orthotopic tumors presented linear foci of necrosis with peudopalisading by neoplastic cells that are hallmarks of human GBM, and were highly proliferative, invasive, and vascular. In addition, immunohistochemistry analysis of TRP orthotopic tumors identified markers characteristic of human GBM, and tumor progression was readily examined by serial MRI. We used primary tumor cells derived from the TRP model in cell proliferation assays and found that PI3K and MAPK pathway inhibitors used as single agents inhibited cell growth alone, but did not result in significant apoptosis. However, when cells were treated with a combination of agents such as BKM120 (a pan-PI3K inhibitor currently in clinical trials for solid tumors) and PD0325901 (a MEK inhibitor), potency was enhanced and there was a substantial increase in cell death. Analysis of downstream targets revealed a synergistic effect on target downregulation in the PI3K pathway. The drug combination was also evaluated in vivo in the orthotopic model. We found that it delayed tumor growth by increasing GBM cell apoptosis, resulting in significantly increased survival. Our results show that combined targeting of the PI3K and MAPK pathways can have a synergistic effect in glioblastoma, and that our primary cells were a valuable tool to predict the in vivo outcome. We also validated this new orthotopic model of GBM and showed that it is tractable for the assessment of potential therapeutic regimens for human GBM. Citation Format: Rajaa El Meskini, Anthony Iacovelli, Alan Kulaga, Michelle Gumprecht, Philip Martin, Maureen Baran, Deborah Householder, Terry Van Dyke, Zoe Weaver Ohler. Combined inhibition of MEK and PI3 kinase signaling results in improved survival in a preclinical orthotopic model for human glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3137. doi:10.1158/1538-7445.AM2014-3137

Abstract 3726: Inhibition of MET overcomes invasive resistance to Bevacizumab and prolongs survival in orthotopic mouse models of glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is the most malignant brain tumor. Despite recent therapeutic improvements, the median overall survival remains poor. Due to its high grade of vascularity, GBM is an ideal candidate for anti-angiogenic therapy. In 2009, US Food and Drug Administration granted accelerated approval for Bevacizumab, a humanized monoclonal antibody directed against VEGF-A, for patients with recurrent disease. Its use in newly diagnosed GBM is currently being evaluated. In the recurrent setting, Bevacizumab provides a transient improvement in PFS that unfortunately does not translate into increased OS. In fact, most tumors become resistant to Bevacizumab and patients invariably experience tumor regrowth. Moreover, a large number of recurrent tumors display an infiltrative and invasive phenotype, thus leading to extremely aggressive tumor growth. Understanding the molecular mechanisms underlying ‘invasive resistance’ to Bevacizumab represents a key step towards the development of improved therapeutic strategies for GBM. We hypothesized that Bevacizumab-induced tumor hypoxia could be responsible for the activation of pro-invasive pathways. We tested this hypothesis using different human GBM cell lines, both in vitro and in orthotopic mouse models of GBM. In vitro, hypoxia promoted increased expression of MET, the high affinity receptor for Hepatocyte Growth Factor(HGF), in all GBM cells analyzed. Increased MET expression resulted in higher sensitivity to HGF stimulation and in enhanced invasiveness. Higher MET levels were not due to transcriptional activation of c-met, but rather to post-transcriptional redistribution of the MET protein from the cytoplasm to the plasma membrane. In vivo, Bevacizumab did marginally improve survival of mice implanted orthotopically with human GBM tumors, but exacerbated tumor hypoxia and resulted in MET activation. Histological analysis of Bevacizumab-treated tumors revealed a highly invasive and infiltrative phenotype. Combined treatment with Bevacizumab and JNJ-38877605, a highly selective, orally available small molecule MET tyrosine kinase inhibitor, significantly prolonged mouse survival compared to both placebo and Bevacizumab alone. Histological examination showed no infiltrative phenotype in the combined treatment arm. Treatment with JNJ-38877605 alone also improved mouse survival in both tumors displaying HGF-MET autocrine stimulation (implanted in nude mice) and in tumors requiring paracrine HGF stimulation (implanted in human HGF knock-in SCID mice). These results unveil a new mechanism by which GBM may become resistant to Bevacizumab, generating proof-of-concept for conducting clinical trials using angiogenesis inhibitors in combination with MET inhibitors. Citation Format: Manuela Cazzanti, Paolo Michieli. Inhibition of MET overcomes invasive resistance to Bevacizumab and prolongs survival in orthotopic mouse models of glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3726. doi:10.1158/1538-7445.AM2014-3726

Abstract LB-41: Prolonged survival upon ultrasound-enhanced doxorubicin delivery in two syngenic glioblastoma mouse models

Abstract Glioblastoma multiforme (GBM) is the most common and most aggressive malignant primary brain tumor, with a very poor prognosis. Chemotherapeutical treatment of GBM is limited by the blood-brain barrier (BBB). This physical and metabolic barrier separates the blood from the brain parenchyma and prevents the entry of toxins but also of potentially useful chemotherapeutics from the blood into the brain. Microbubble-enhanced focused ultrasound (MB-FUS) has been proposed to disrupt locally and reversibly the BBB to facilitate diffusion of drugs from the micro vasculature into brain tissue. The present study investigates the feasibility and the safety of such an approach in two syngenic mouse models of GBM (GL261 and SMA-560). Local doxorubicin (DOX) concentration in MB-FUS sonicated normal brain tissue as well as in brain tumor tissue was increased as compared to the unsonicated control tissue in the contralateral hemisphere. Moreover, ultrasound mediated BBB disruption, in combination with DOX therapy, resulted in a significant increase of survival and in a slower disease progression in the two syngenic GBM mouse models. In conclusion, our results confirm that MB-ultrasound might ultimately be an effective technology to improve the therapy of GBM, and they provide for the first time evidence that combining MB-FUS with DOX treatment is effective in syngenic mouse models for GBM which can serve as preclinical models to study the impact of immune system on the therapeutic application of MB-FUS chemotherapy. Citation Format: Zsofia Kovaks, Beat Werner, Ernst Martin-Fiori, Michele Bernasconi. Prolonged survival upon ultrasound-enhanced doxorubicin delivery in two syngenic glioblastoma mouse models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-41. doi:10.1158/1538-7445.AM2014-LB-41

Abstract 1484: CD57 defines a novel maker of glioblastoma stem cells that have greater invasive potential than CD133+ tumor cells

Abstract Glioblastoma multiforme (GBM) is the most aggressive and lethal brain tumor that occurs both in children and adults. Diffuse invasion into normal brain tissue is one of the important biologic features that make GBM refractory to conventional therapies. While existing studies on GBM invasion are primarily conducted using tumor core tissues from surgical resections, it is unclear whether unresectable, infiltrative GBM cells would be more informative for studying their invasive nature compared to those in the resected tumor cores. More importantly, little is known if and which cancer stem cell populations are driving glioma invasion. To address these fundamental issues, we utilized our panel of 7 (6 pediatric and 1 adult) patient tumor-derived orthotopic xenograft mouse models of GBM to isolate invasive GBM cells (infiltrating normal mouse brain parenchyma) and tumor core GBM cells and directly compared their biological differences. Our result showed that the invasive cells have stronger neurosphere forming efficiency in vitro in a serial dilution assay and increased tumorigenic capacity after in vivo transplantation (particularly at 100 cells/mouse) compared to the tumor core cells. A screening of putative cancer stem cell markers (CD133, CD15, CD24/CD44, CD57 and CD117) showed that invasive GBM cells are enriched (&amp;gt;2 folds) with CD57+ cells compared to the tumor core cells, and these infiltrating cells were predominantly CD57+/CD133-. Even the CD133+ cells were frequently dual-positive with CD57 (CD33+CD57+), not only in the xenograft tumors but also in a separate set of patient GBM samples. Mechanistically, we found that CD57+ cells expressed high levels of self-renewal genes and tend to stay in G0/G1 phases. In conclusion, we showed that invasive GBM cells are biologically deferent from the matched tumor core cells and identified CD57 as a novel stem cell marker that is associated with GBM infiltration. Our findings suggest that new anti-invasion therapies should target CD57+ cells in addition to CD133+ cells in GBM. Citation Format: Lin Qi, Yu-lun Huang, Mari Kogiso, Hua Mao, Patricia Baxter, Jack MF Su, Laszlo Perlaky, Ching C. Lau, Murali Chintagumpala, Xiao-Nan Li. CD57 defines a novel maker of glioblastoma stem cells that have greater invasive potential than CD133+ tumor cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1484. doi:10.1158/1538-7445.AM2014-1484

Abstract 1914: Functional characterization of oncogenic-induced dedifferentiation in neurons and astrocytes using DP-seq

Abstract Glioblastoma (GBM) is the most common and lethal form of intracranial tumor. Using a Cre-inducible lentiviral GBM mouse model we recently showed that gliomas can originate from dedifferentiation of mature neurons and astrocytes. Expression analysis of the known markers (of the differentiated cell-types) showed diminished expression while the expression of the undifferentiated state markers were significantly up-regulated in the dedifferentiated transformed neurons and astrocytes. In this study, we performed whole transcriptome analysis of these cells along with mouse pluripotent embryonic stem cells (mESC), neural stem cells (NSC), neurons and astrocytes to characterize the undifferentiated state of these cells. Our analysis revealed that dedifferentiated cell-types shared traits with neurons and NSCs at the global transcriptome level suggesting that they have retained memory of their cell-of-origin and share markers of undifferentiated cells with NSCs. Functional analysis of the transcriptomics data revealed involvement of the Wnt signaling, cell cycle and the focal adhesion pathways in defining the state of the dedifferentiated cell-types. Our analysis further revealed conservation of a gene interaction network in both dedifferentiated cell-types. This network exhibited a modular architecture, connecting components of the cell cycle pathway to Wnt signaling and the focal adhesion pathways. One of these components, Osteopontin (OPN), also known as Spp1, is highly expressed and secreted by the tumors and dedifferentiated transformed neurons and astrocytes. Attempts to inhibit the action of OPN, either by using a neutralizing antibody or silencing the OPN gene (shRNA), blocked the formation of tumorspheres and diminished their proliferating capacity. We are currently assessing the effects of the inhibition of OPN function in vivo. Further genetic perturbation of additional interacting partners and/or the abolishment of the interactions can help elucidate the regulatory mechanism of this network in maintaining the dedifferentiated state of the transformed neurons and astrocytes. Citation Format: Dinorah Friedmann-Morvinski, Vipul Bhargava, Shakti Gupta, Inder M. Verma, Shankar Subramaniam. Functional characterization of oncogenic-induced dedifferentiation in neurons and astrocytes using DP-seq. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1914. doi:10.1158/1538-7445.AM2014-1914

Assessing the role of STAT3 in DC differentiation and autologous DC immunotherapy in mouse models of GBM.

Cellular microenvironments, particularly those found in tumors, elicit a tolerogenic DC phenotype which can attenuate immune responses. Central to this process is the STAT3-mediated signaling cascade. As a transcription factor and oncogene, STAT3 promotes the expression of genes which allow tumor cells to proliferate, migrate and evade apoptosis. More importantly, activation of STAT3 in tumor infiltrating immune cells has been shown to be responsible, in part, for their immune-suppressed phenotype. The ability of STAT3 to orchestrate a diverse set of immunosuppressive instructions has made it an attractive target for cancer vaccines. Using a conditional hematopoietic knockout mouse model of STAT3, we evaluated the impact of STAT3 gene ablation on the differentiation of dendritic cells from bone marrow precursors. We also assessed the impact of STAT3 deletion on phagocytosis, maturation, cytokine secretion and antigen presentation by GM-CSF derived DCs in vitro. In addition to in vitro studies, we compared the therapeutic efficacy of DC vaccination using STAT3 deficient DCs to wild type counterparts in an intracranial mouse model of GBM. Our results indicated the following pleiotropic functions of STAT3: hematopoietic cells which lacked STAT3 were unresponsive to Flt3L and failed to differentiate as DCs. In contrast, STAT3 was not required for GM-CSF induced DC differentiation as both wild type and STAT3 null bone marrow cells gave rise to similar number of DCs. STAT3 also appeared to regulate the response of GM-CSF derived DCs to CpG. STAT3 null DCs expressed high levels of MHC-II, secreted more IL-12p70, IL-10, and TNFα were better antigen presenters in vitro. Although STAT3 deficient DCs displayed an enhanced activated phenotype in culture, they elicited comparable therapeutic efficacy in vivo compared to their wild type counterparts when utilized in vaccination paradigms in mice bearing intracranial glioma tumors.

AMPK regulated metabolic programing: oncogenic or growth suppressive? Evolving lessons from genetic and pharmacologic studies

Background Cancer cells reprogram their metabolism for optimal growth and survival. Identifying the genes and their functions crucial for cancer metabolic reprograming might have therapeutic implications. The multifunctional kinase AMPK is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because while on one hand active AMPK inhibits mTOR and lipogenesis - two crucial arms of cancer growth, AMPK also ensures viability during metabolic stress. Many studies have shown that AMPK activation by two indirect AMPK agonists AICAR and metformin (now in many cancer clinical trials) reduces cancer cell proliferation - an effect that is rescued by an AMPK inhibitor Compound C in some studies. We used genetic models to scrutinize the specificity of these reagents and examine whether AMPK is a growth suppressor in glioblastoma. Materials and methods We used silencing RNA against AMPK in established GBM cell lines and examined viability in the presence or absence of the reagents to define their mechanisms of action. Results Surprisingly, we found that compared to normal tissue, AMPK is constitutively active in both human and mouse gliomas in vivo. We found that both AMPK agonists inhibited proliferation, but through discrete AMPK-independent mechanisms and both reagents reduced GBM growth in vivo independent of AMPK. Importantly, A769662, a new and direct AMPK activator had no effect on GBM cell proliferation. Metformin directly inhibited mTOR by enhancing PRAS40 association with RAPTOR, while AICAR blocked cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. In another surprise, Compound C itself proved to be a potent anti-GBM agent; however, its cell killing effects were pleiotropic and also independent of AMPK. We next examined if AMPK is required for viability of freshly established patient-derived GBM cells and found that many of these cells lose viability in vitro when AMPK was silenced. Conclusions Our results suggest that AICAR, metformin and Compound C are all AMPK-independent anti-proliferative agents. Importantly, physiologically active AMPK in GBM could be a growth promoter rather than a growth suppressor in vivo. We are taking multiple genetic approaches to determine this in mouse models of human GBM.

Abstract C11: Combined inhibition of MEK and PI3 kinase signaling results in improved survival in a preclinical orthotopic model for human glioblastoma multiforme.

Abstract Glioblastoma multiforme (GBM; astrocytoma grade IV) is the most frequent and aggressive brain tumor for which no effective therapy is currently available. It has been shown that PI3K, RTK/RAS, and Rb signaling are commonly altered in human GBM. We utilized a genetically engineered mouse (GEM) model for GBM, designated “TRP,” that expresses GFAP-T121 (T; for Rb suppression), the KrasG12D mutation (R), and is heterozygous for a PTEN null allele (P), to develop an orthotopic mouse model for the preclinical evaluation of potential therapeutics for GBM treatment. Although the de novo TRP brain tumor GEM model recapitulates features of human GBM including tissue invasion, pseudopalisading necrosis, and dense vascularization, the latency to tumorigenesis (4-6 months) makes its use as a preclinical model for drug screening challenging. Therefore we isolated primary GBM cells from TRP GEMs and injected cells intracranially (IC) into syngeneic mouse brains. Recipient mice developed grade IV astrocytomas and recapitulated TRP GEM tumor histopathology. The orthotopic tumors presented linear foci of necrosis with peudopalisading by neoplastic cells that are hallmarks of human GBM, and were highly proliferative, invasive, and vascular. In addition, immunohistochemistry analysis of TRP orthotopic tumors identified markers characteristic of human GBM, and tumor progression was readily examined by serial MRI. We used primary tumor cells derived from the TRP model in cell proliferation assays and found that PI3K and MAPK pathway inhibitors used as single agents inhibited cell growth alone, but did not result in significant apoptosis. However, when cells were treated with a combination of agents such as BKM120 (a pan-PI3K inhibitor currently in clinical trials for solid tumors) and PD0325901 (a MEK inhibitor), potency was enhanced and there was a substantial increase in cell death. Analysis of downstream targets revealed a synergistic effect on target downregulation in the PI3K pathway. The drug combination was also evaluated in vivo in the orthotopic model. We found that it delayed tumor growth by increasing GBM cell apoptosis, resulting in significantly increased survival. Our results show that combined targeting of the PI3K and MAPK pathways can have a synergistic effect in glioblastoma, and that our primary cells were a valuable tool to predict the in vivo outcome. We also validated this new orthotopic model of GBM and showed that it is tractable for the assessment of potential therapeutic regimens for human GBM. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C11. Citation Format: Rajaa El Meskini, Anthony Iacovelli, Alan Kulaga, Michelle Gumprecht, Philip L. Martin, Maureen L. Baran, Deborah B. Householder, Terry Van Dyke, Zoe Weaver Ohler. Combined inhibition of MEK and PI3 kinase signaling results in improved survival in a preclinical orthotopic model for human glioblastoma multiforme. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C11.

F11R Is a Novel Monocyte Prognostic Biomarker for Malignant Glioma

ObjectiveBrain tumors (gliomas) contain large populations of infiltrating macrophages and recruited microglia, which in experimental murine glioma models promote tumor formation and progression. Among the barriers to understanding the contributions of these stromal elements to high-grade glioma (glioblastoma; GBM) biology is the relative paucity of tools to characterize infiltrating macrophages and resident microglia. In this study, we leveraged multiple RNA analysis platforms to identify new monocyte markers relevant to GBM patient outcome.MethodsHigh-confidence lists of mouse resident microglia- and bone marrow-derived macrophage-specific transcripts were generated using converging RNA-seq and microarray technologies and validated using qRT-PCR and flow cytometry. Expression of select cell surface markers was analyzed in brain-infiltrating macrophages and resident microglia in an induced GBM mouse model, while allogeneic bone marrow transplantation was performed to trace the origins of infiltrating and resident macrophages. Glioma tissue microarrays were examined by immunohistochemistry, and the Gene Expression Omnibus (GEO) database was queried to determine the prognostic value of identified microglia biomarkers in human GBM.ResultsWe generated a unique catalog of differentially-expressed bone marrow-derived monocyte and resident microglia transcripts, and demonstrated that brain-infiltrating macrophages acquire F11R expression in GBM and following bone-marrow transplantation. Moreover, mononuclear cell F11R expression positively correlates with human high-grade glioma and additionally serves as a biomarker for GBM patient survival, regardless of GBM molecular subtype.SignificanceThese studies establish F11R as a novel monocyte prognostic marker for GBM critical for defining a subpopulation of stromal cells for future potential therapeutic intervention.

Abstract IA13: Glioma stem cells and cancer

Abstract Malignant GBMs are incurable tumors that progress within a year's time. Genome wide studies have pinpointed prevalent cancer associated mutions in GBM and among these, the tumor suppressors p53, Pten and NF1 are among the most frequently found. We have developed mouse models of GBM via mutation of p53, Pten and NF1 in the brain. Among the many key insights gleaned from studies with our mouse models have been: 1) The first demonstration that stem cells are the major source of these tumors. 2) The demonstration that all tumor cells are not equivalent and that the Cancer Stem Cells, a subpopulation of cells, are the root of tumor progression. 3) The discovery of a distinct form of GBM that arises in a different cell population and that has unique properties. This novel form of GBM has until now been grouped together with the predominant form of GBM. Our work will lead to a separation of prognosis and treatment for the different forms. In addition, using high throughput screens, we have small compounds in development that specifically target GBM cells but not normally dividing cells. I will discuss these advances and the implications for glioma, solid tumors and cancer in general. Citation Format: Luis F. Parada. Glioma stem cells and cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA13.

Abstract 4322: A preclinical orthotopic mouse model for human GBM: Recapitulation of features of GEM model of origin and potency of PI3K inhibitors

Abstract Glioblastoma (GBM; grade IV) is the most frequent and aggressive brain tumor for which no effective therapy is currently available. It has been shown that PI3 kinase, involving both PI3 kinase – Ras activation and PTEN inhibition, and RB signaling are commonly altered in human GBM. We utilized a GEM model expressing GFAP-T121, KrasG12D and heterozygous mutant in PTEN +/- (TRP+/-) to develop an orthotopic mouse model for the preclinical evaluation of potential therapeutics for GBM treatment. The TRP+/- GEM model recapitulates features of human GBM including tissue invasion, pseudopalisading necrosis and dense vascularisation, however the latency to tumorigenesis (4-6 months) makes its use as a preclinical model for drug screening challenging. Therefore we isolated primary tumor cells from induced TRP+/- GEMs with Grade III or Grade IV astrocytoma and injected cells intracranially (IC) into syngeneic mouse brains. Primary cells induced grade IV tumors and recapitulated TRP+/- GEM tumor histopathology. Highly proliferative, invasive, and vascular, the orthotopic grade IV tumors presented linear foci of necrosis with peudopalisading by neoplastic cells that is characteristic of human GBM. Immunohistochemistry characterization of TRP+/- orthotopic tumors revealed similar T121, GFAP and PTEN expression profile to TRP+/- GEM grade IV tumors. Other cellular markers known to be expressed in neuronal progenitors, glial cells or found to be associated with human GBM aggressiveness were assessed in the orthotopic mouse model GBM tumors in comparison with the TRP+/- GEM model. By adjusting the number of primary cells IC injected, the time period of tumor growth and assessment by MRI was increased from 3 weeks to 6 weeks allowing reasonable time for tumor growth assessment, before potential drug efficacy testing. We used primary cells of grade IV tumor origin from TRP+/- GEM to evaluate the effect of PI3 kinase pathway inhibition on cell proliferation. BEZ235, PI103 and PIK75, compounds that target the PI3 kinase pathway, are effective inhibitors of primary tumor cell proliferation with an EC50 in the 10-100 nM range. MK2206, an Akt inhibitor, was found to be a potential inhibitor of tumor cell proliferation. Although MAPK pathway activation through RB inactivation has been found to be key in triggering cell proliferation in TRP+/- GEM model, MEK inhibitors, namely AZD6244, PD0325901 and UO126 did not have a strong effect on cell proliferation. In vitro inhibition of cell proliferation assays and target validation in TRP+/- GEM primary cells are useful tools to choose compounds for orthotopic mouse efficacy studies. Preliminary in vivo mouse pharmacokinetic studies are underway to insure potential drug distribution in the brain tissue. Using the orthotopic model, we found that PI3 kinase inhibitors may be effective against brain tumors in the mouse orthotopic model for human GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4322. doi:10.1158/1538-7445.AM2011-4322