Murine Model Of Glioblastoma Multiforme Research Articles

The immune landscape of high-grade brain tumor after treatment with immune checkpoint blockade.

Despite the therapeutic success of immune checkpoint blockade (ICB) therapy against multiple tumors, many patients still do not benefit from ICB. In particular, high-grade brain tumors, such as glioblastoma multiforme (GBM), have a very low response rate to ICB, resulting in several failed clinical trials. This low response rate might be caused by a lack of understanding of the unique characteristics of brain immunity. To overcome this knowledge gap, macroscopic studies of brain immunity are needed. We use single cell RNA sequencing to analyze the immune landscape of the tumor microenvironment (TME) under anti-PD-1 antibody treatment in a murine GBM model. We observe that CD8 T cells show a mixed phenotype overall that includes reinvigoration and re-exhaustion states. Furthermore, we find that CCL5 induced by anti-PD-1 treatment might be related to an increase in the number of anti-inflammatory macrophages in the TME. Therefore, we hypothesize that CCL5-mediated recruitment of anti-inflammatory macrophages may be associated with re-exhaustion of CD8 T cells in the TME. We compare our observations in the murine GBM models with publicly available data from human patients with recurrent GBM. Our study provides critical information for the development of novel immunotherapies to overcome the limitations of anti-PD-1 therapy.

Immune profiling of human tumors identifies CD73 as a combinatorial target in glioblastoma.

Immune checkpoint therapy with anti-CTLA-4 and anti-PD-1/PD-L1 has revolutionized the treatment of many solid tumors. However, the clinical efficacy of immune checkpoint therapy is limited to a subset of patients with specific tumor types1,2. Multiple clinical trials with combinatorial immune checkpoint strategies are ongoing; however, the mechanistic rationale for tumor-specific targeting of immune checkpoints is elusive. To garner an insight into tumor-specific immunomodulatory targets, we analyzed 94 patients representing five different cancer types, including those that respond relatively well to immune checkpoint therapy and those that do not, such as glioblastoma multiforme, prostate cancer and colorectal cancer. Through mass cytometry and single-cell RNA sequencing, we identified a unique population of CD73hi macrophages in glioblastoma multiforme that persists after anti-PD-1 treatment. To test if targeting CD73 would be important for a successful combination strategy in glioblastoma multiforme, we performed reverse translational studies using CD73-/- mice. We found that the absence of CD73 improved survival in a murine model of glioblastoma multiforme treated with anti-CTLA-4 and anti-PD-1. Our data identified CD73 as a specific immunotherapeutic target to improve antitumor immune responses to immune checkpoint therapy in glioblastoma multiforme and demonstrate that comprehensive human and reverse translational studies can be used for rational design of combinatorial immune checkpoint strategies.

Spatiotemporal Tracking of Brain-Tumor-Associated Myeloid Cells in Vivo through Optical Coherence Tomography with Plasmonic Labeling and Speckle Modulation.

By their nature, tumors pose a set of profound challenges to the immune system with respect to cellular recognition and response coordination. Recent research indicates that leukocyte subpopulations, especially tumor-associated macrophages (TAMs), can exert substantial influence on the efficacy of various cancer immunotherapy treatment strategies. To better study and understand the roles of TAMs in determining immunotherapeutic outcomes, significant technical challenges associated with dynamically monitoring single cells of interest in relevant live animal models of solid tumors must be overcome. However, imaging techniques with the requisite combination of spatiotemporal resolution, cell-specific contrast, and sufficient signal-to-noise at increasing depths in tissue are exceedingly limited. Here we describe a method to enable high-resolution, wide-field, longitudinal imaging of TAMs based on speckle-modulating optical coherence tomography (SM-OCT) and spectral scattering from an optimized contrast agent. The approach's improvements to OCT detection sensitivity and noise reduction enabled high-resolution OCT-based observation of individual cells of a specific host lineage in live animals. We found that large gold nanorods (LGNRs) that exhibit a narrow-band, enhanced scattering cross-section can selectively label TAMs and activate microglia in an in vivo orthotopic murine model of glioblastoma multiforme. We demonstrated near real-time tracking of the migration of cells within these myeloid subpopulations. The intrinsic spatiotemporal resolution, imaging depth, and contrast sensitivity reported herein may facilitate detailed studies of the fundamental behaviors of TAMs and other leukocytes at the single-cell level in vivo, including intratumoral distribution heterogeneity and roles in modulating cancer proliferation.

EnLIGHTenment of tumor vessel normalization and immunotherapy in glioblastoma.

Glioblastoma multiforme (GBM) is a highly vascularized and aggressive brain tumor. Despite aggressive standard care, GBM remains predominantly fatal; hence, new innovative therapies are required. Recent research published in the Journal of Pathology has identified the CGKRK peptide as a promising tool with which to specifically target the tumor vasculature from high-grade glioma. This tumor vessel-homing peptide was fused to the tumor necrosis factor superfamily member LIGHT/TNFSF14, and injected intravenously into murine orthotopic GBM models. After treatment, the tumor vasculature appeared to be less abnormal, with normalized features such as increased endothelial barrier integrity, pericyte contractility, and tumor perfusion. Moreover, CGKRK-LIGHT induced the appearance of high endothelial venules (HEVs), which are specialized structures that play a role in lymphocyte trafficking and have been shown to increase T-cell infiltration in solid tumors. Combining CGKRK-LIGHT with anti-angiogenic and immune checkpoint blockade treatments boosted HEV induction and cytotoxic T-cell infiltration, leading to a reduction in tumor burden. In this Commentary, I highlight the therapeutic opportunities provided by and the current limitations of LIGHT-vascular targeting peptide as a new approach to target GBM and enhance tumor vessel delivery and immunotherapy efficacy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

C-Met-mediated endothelial plasticity drives aberrant vascularization and chemoresistance in glioblastoma.

Aberrant vascularization is a hallmark of cancer progression and treatment resistance. Here, we have shown that endothelial cell (EC) plasticity drives aberrant vascularization and chemoresistance in glioblastoma multiforme (GBM). By utilizing human patient specimens, as well as allograft and genetic murine GBM models, we revealed that a robust endothelial plasticity in GBM allows acquisition of fibroblast transformation (also known as endothelial mesenchymal transition [Endo-MT]), which is characterized by EC expression of fibroblast markers, and determined that a prominent population of GBM-associated fibroblast-like cells have EC origin. Tumor ECs acquired the mesenchymal gene signature without the loss of EC functions, leading to enhanced cell proliferation and migration, as well as vessel permeability. Furthermore, we identified a c-Met/ETS-1/matrix metalloproteinase-14 (MMP-14) axis that controls VE-cadherin degradation, Endo-MT, and vascular abnormality. Pharmacological c-Met inhibition induced vessel normalization in patient tumor-derived ECs. Finally, EC-specific KO of Met inhibited vascular transformation, normalized blood vessels, and reduced intratumoral hypoxia, culminating in suppressed tumor growth and prolonged survival in GBM-bearing mice after temozolomide treatment. Together, these findings illustrate a mechanism that controls aberrant tumor vascularization and suggest that targeting Endo-MT may offer selective and efficient strategies for antivascular and vessel normalization therapies in GBM, and possibly other malignant tumors.

HDAC7 inhibition resets STAT3 tumorigenic activity in human glioblastoma independently of EGFR and PTEN: new opportunities for selected targeted therapies.

To date, the mutational status of EGFR and PTEN has been shown as relevant for favoring pro- or anti-tumor functions of STAT3 in human glioblastoma multiforme (GBM). We have screened genomic data from 154 patients and have identified a strong positive correlation between STAT3 and HDAC7 expression. In the current work we show the existence of a subpopulation of patients overexpressing HDAC7 and STAT3 that has particularly poor clinical outcome. Surprisingly, the somatic mutation rate of both STAT3 and HDAC7 was insignificant in GBM comparing with EGFR, PTEN or TP53. Depletion of HDAC7 in a range of GBM cells induced the expression of tyrosine kinase JAK1 and the tumor suppressor AKAP12. Both proteins synergistically sustained the activity of STAT3 by inducing its phosphorylation (JAK1) and protein expression (AKAP12). In absence of HDAC7, activated STAT3 was responsible for significant imbalance of secreted pro-/anti-angiogenic factors. This inhibited the migration and sprouting of endothelial cells in paracrine fashion in vitro as well as angiogenesis in vivo. In a murine model of GBM, induced HDAC7-silencing decreased the tumor burden by threefold. The current data show for the first time that silencing HDAC7 can reset the tumor suppressor activity of STAT3, independently of the EGFR/PTEN/TP53 background of the GBM. This effect could be exploited to overcome tumor heterogeneity and provide a new rationale behind the development of specific HDAC7 inhibitors for clinical use.

The use of anti-GITR antibody treatment in a murine model of glioblastoma multiforme

Meeting abstracts Glioblastoma multiforme (GBM), is the most common adult brain tumor and despite many new therapies, remains largely incurable. Regulatory T cells (Treg) are highly immunosuppressive cells that accumulate within glioma microenvironment, and reduction of their function or

Glioma microenvironment-derived CCL2 recruits regulatory T cells and myeloid-derived suppressor cells

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults with a median survival of 14.6 months despite surgery, radiotherapy, and chemotherapy. One hallmark of GBM is the accumulation of potently immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). However, the factors underlying Treg and MDSC trafficking to GBM have not been well established. We identified C-C motif chemokine 2 (CCL2) as a glioma microenvironment-derived chemokine that recruits Tregs and MDSCs that express the cognate receptors CCR4 and CCR2, respectively. First, we found that CCL2 expression is a prognostic factor for GBM patients using data from The Cancer Genome Atlas. High CCL2 expression was correlated with a poor prognosis in both univariate and multivariate Cox regression analyses. We also confirmed the presence of CCL2 in GBM patient samples by immunohistochemical staining. In the syngeneic murine GL261 model of GBM, the major source of CCL2 was macrophages and microglia in the glioma microenvironment rather than tumor cells themselves. Treatment of ex vivo isolated microglia and bone marrow (BM)-derived macrophages with GL261 conditioned media resulted in CCL2 production, suggesting that a soluble GL261-derived factor was responsible for the production of CCL2 in microglia and macrophages. To identify potential candidates for this GL261-derived factor, a cytokine array was performed. We found that CCL20 was highly produced by the tumors and was sufficient for the stimulation of CCL2 production by BM-derived macrophages. Implantation of GL261 cells in Ccl2-/- mice resulted in reduced levels of Tregs and Ly-6C+ monocytic MDSCs but not Ly-6G+ granulocytic MDSCs. Finally, treatment of mice bearing intracranial GL261 tumors with the small molecule chemokine receptor antagonist C 021 increased median survival ~30%. Thus, the CCL2 chemokine-receptor axis is a potential therapeutic target in GBM as a major mechanism for the recruitment of Tregs and MDSCs.

Abstract 5222: Endothelial plasticity generates aberrant angiogenesis and therapy resistance in glioblastoma

Abstract Overgrown, abnormal vasculature characterizes the microenvironment that fuels cancer progression and induces therapeutic resistance. Here we reveal that endothelial plasticity drives excessive and abnormal tumor angiogenesis. Glioblastoma multiforme (GBM) is among the most lethal of human malignancies, distinguished by prominent vascularity and extreme vascular abnormality. We identify endothelial fibro-transformation (Endo-FT) in GBM, contributing significantly to aberrant vascularization, tumor progression, and therapeutic resistance. Utilizing human GBM specimens and allograft and genetic murine GBM models driven by RCAS/Ntv-a-mediated gene transfer of PDGF in Ink4a-Arf-/-PTEN-/- mice, we reveal robust Endo-FT in the tumor vasculature, characterized by endothelial cell (EC) expression of the fibroblast and mesenchymal cell markers including FSP-1, α-smooth muscle actin, N-cadherin. Moreover, genetic lineage tracing shows a prominent population of GBM-associated fibroblast-like cells with EC origin. Furthermore, glioma-conditioned medium induces EC in vitro acquisition of the fibroblast signature with decreased expression of EC-specific proteins including CD31, VE-cadherin, Tie2, VEGFR2, leading to enhanced cell proliferation, motility, and invasiveness, and vessel permeability. Interestingly, the fibroblast-like ECs retain the key EC-specific functions including Ac-LDL absorption and tube formation, suggesting Endo-FT without complete EC transition to fibroblasts. Proteomic analysis identifies c-Met is a regulator of Endo-FT, requisite for the vascular abnormality. Pharmacological inhibition and siRNA-mediated knockdown of c-Met or neutralization with a HGF antibody remarkably abrogate tumor-conditioned medium-induced Endo-FT and EC malfunctions, suggesting a critical role of HGF/c-Met in Endo-FT. Furthermore, c-Met activation induces ETS-1-dependent matrix metalloproteinase (MMP)-14 expression, leading to VE-cadherin cleavage and Endo-FT. Finally, we test the in vivo role of c-Met-mediated Endo-FT in GBM progression by using mice with Tie2-Cre-driven, EC-specific Met knockout. Met deletion in ECs significantly improves mouse survival and inhibits GBM growth after temozolomide (TMZ) treatment. Moreover, Met deletion in ECs abolishes Endo-FT and vascular abnormality, as shown by remarkable decreases in CD31+FSP-1+ cell population and intratumor hypoxia, and increases in vasculature-mediated perfusion and vascular pericyte coverage, suggesting that c-Met-mediated Endo-FT is required for vascular abnormality and GBM progression and therapeutic resistance. In sum, these findings illustrate a novel mechanism controlling aberrant vascularization and GBM progression, and suggest that targeting Endo-FT may offer selective and efficient therapeutic strategies for the treatment-resistant GBM, and possibly other malignant tumors. Citation Format: Menggui Huang, Yi Fan. Endothelial plasticity generates aberrant angiogenesis and therapy resistance in glioblastoma. [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 5222. doi:10.1158/1538-7445.AM2015-5222

Combined temozolomide and ionizing radiation induces galectin-1 and galectin-3 expression in a model of human glioma

AbstractBackground Despite aggressive treatment for glioblastoma multiforme (GBM), including surgical resection, radiotherapy and temozolomide (TMZ) chemotherapy, over 90% of patients experience tumor recurrence. Galectins are carbohydrate-binding proteins that are overexpressed in the stroma of GBM tumors, and are potent modulators of GBM cell migration and angiogenesis. The objective of this study was to analyze glioma and endothelial cell galectin expression in response to combined chemoradiation. Methodology The effects of TMZ, ionizing radiation, or combined chemoradiation on galectin protein secretion and expression were assessed in U87 orthotopically grown GBM tumors in mice, as well as in vitro in U87 human glioma cells and human umbilical vein endothelial cells (HUVECs). Results We found that combination chemoradiation increased galectin-1 and galectin-3 protein expression in U87 glioma cells. In response to radiation alone, U87 cells secreted significant levels of galectin-1 and galectin-3 into the microenvironment. HUVEC co-culture increased U87 galectin-1 and galectin-3 protein expression 14 - 20% following chemoradiation, and conferred a radioprotective benefit to U87 glioma cells. In vivo, radiation alone and combination chemoradiation significantly increased tumor galectin-1 expression in an orthotopic murine model of GBM. Conclusions Glioma cell galectin expression increased following combined chemoradiation, both in vitro and in vivo. The presence of endothelial cells further increased glioma cell galectin expression and survival, suggesting that crosstalk between tumor and endothelial cells in response to standard chemoradiation may be an important factor in mediating glioma recurrence, potentially via galectin upregulation.

AI-11 * c-MET-MEDIATED ENDOTHELIAL-MESENCHYMAL TRANSITION DRIVES GLIOBLASTOMA PROGRESSION AND THERAPEUTIC RESISTANCE

Excessive and abnormal vasculature characterizes the microenvironment that fuels the progression of malignant tumors including glioblastoma multiforme (GBM), the grade IV glioma. GBM is among the most lethal human malignancies, distinguished by extensive microvascular hyperplasia due to intravessel endothelial cell (EC) proliferation with unknown etiology. Here, we show that c-Met phosphorylation induces endothelial-mesenchymal transition (Endo-MT), contributing significantly to vascular abnormality, GBM progression, and therapeutic resistance. Utilizing murine orthotropic GBM models induced by transplantation of GL26 glioma cells and RCAS/tv-a-mediated somatic gene transfer of platelet-derived growth factor (PDGF) in Ink-4a/Arf−/-PTEN−/- mice, we reveal robust Endo-MT in tumor vasculature, characterized by expression of the fibroblast-specific marker FSP-1 in 60% of CD31+ or CD105+ ECs, and the co-expression level correlates with the aggressiveness of glioma in humans. Cell fate analysis by lineage tracing Tie2+ ECs shows over half of FSP-1+ fibroblasts are of EC origin in the mouse GBM models. Tumor-conditioned medium induces in vitro EC acquisition of mesenchymal gene signature including FSP-1 and N-cadherin expression, confirming the microenvironment-dependent Endo-MT. Furthermore, proteomic analysis identifies a critical role of c-Met in Endo-MT, which is required for the increased EC proliferation and migration and vessel hyperpermeability in the GBM microenvironment. EC-specific c-Met phosphorylation induces MMP-14 expression, leading to Endo-MT and vascular abnormality. Finally, pharmacological inhibition of c-Met phosphorylation normalizes blood vessels, reduces vascular density and Endo-MT, blocks tumor progression, and sensitizes tumor to temozolomide treatment; all of which improves overall survival in the GBM-bearing mice. These findings reveal a novel mechanism controlling aberrant vascularization and GBM progression, and suggest that targeting c-Met phosphorylation and Endo-MT may offer selective and efficient therapeutic strategies for the treatment-resistant GBM, and possibly other malignant tumors.

IT-31 * HARNESSING THE IMMUNOLOGIC CAPACITY OF THE LIVER AGAINST GLIOBLASTOMA MULTIFORME

BACKGROUND: Glioblastoma multiforme (GBM) remains invariably associated with poor patient outcomes thus necessitating the development of more targeted therapeutics. To circumvent the challenges associated with cellular therapeutics, we have utilized clinical-grade nanoparticle (NP) vaccine formulations that are known to traffic through the liver. Since the liver is a profoundly important lymphoid organ enriched with innate immune cells that must function in the dualistic role of tolerance when constitutively engaged by gastrointestinal ligands and heighted inflammatory responses when engaged by viral elements, it represents a profound immunological niche for re-directing the immune system in the presence of specific ‘danger’ signals. Therefore, using tumor derived RNA from intracranial gliomas, imbued with the appropriate immunomodulatory RNAs packaged into an off the shelf NP vaccine formulation, we aim to harness the liver's immunologic potential against GBM. METHODS: We sought to assess if vaccination with autologously derived total tumor RNA encapsulated in lipophilic NPs could transfect liver antigen-presenting cells (APCs) and induce anti-tumor immunity in pre-clinical immunocompetent murine GBM models. RESULTS: We screened clinically available nanoliposomal formulations, identified a suitable NP courier for delivery of encapsulated RNA to liver APCs, and demonstrated effective anti-tumor activity against murine GBMs. These NPs were shown to preferentially transfect hepatic cells and APCs therein generating a preponderance of antigen specific T cell immunity against tumor antigens. The percentage of antigen specific T cells was greater from harvested livers compared with harvested secondary lymphoid organs. Moreover, we demonstrated the capacity to significantly enhance the immunity and anti-tumor efficacy of this platform through incorporation of immunomodulatory RNAs encoding for cytokines such as GM-CSF. CONCLUSION: RNA-NPs represent a novel therapeutic platform for inducing potent nontoxic immunity against gliomas by harnessing the immunologic potential of the liver, thus providing a more effective and specific therapy critical in improving clinical outcomes for patients with GBM.

Abstract 2663: SPECT/CT analysis of distribution of intracranially infused interleukin-13 Pseudomonas exotoxin, a targeted agent for interleukin-13 receptor expressing brain tumors.

Abstract Interleukin-13 Pseudomonas exotoxin (IL-13PE), a powerful targeted agent for IL-13 receptor α2 (IL-13Rα2) - positive tumors is being tested for safety and effectiveness in glioblastoma multiforme (GBM) clinical trials. However, distribution of IL-13PE after intra-cranial convection-enhanced delivery (CED) has not been determined. To study this, we radiolabeled IL-13PE with 125I and determined its binding and biological activity in vitro and distribution in a murine model of GBM. 125IL-13PE maintained its specific binding activity to IL-13Rα2 positive U251 glioblastoma tumor cells but not to T98G cells, as these cells did not express IL-13Rα2. 125IL-13PE was as cytotoxic to glioblastoma cells as non-radiolabeled IL-13PE, which killed U251 tumor cells in a concentration dependent manner but not T98G cells. Both binding and cytotoxic activities were blocked by a 100-fold excess of IL-13, which indicated that IL-13PE binding was specific. Athymic nude mice with intracranially implanted U251 tumors were given stereotactic intratumoral injections of 125I-IL-13PE by either a bolus injection or a 3-day-CED. Animals were imaged by micro-SPECT/CT at various time points. SPECT/CT imaging revealed retention of 125I-IL-13PE in U251 tumors. CED showed significantly higher volumes of distribution and maintained detectable drug levels for longer periods of time compared to bolus injection. SPECT/CT images of whole brains corroborated with the autoradiography results performed on sectioned brain. These results indicate that IL-13PE can be successfully radiolabled with 125I without the loss of binding and cytotoxic activity and that 125I-IL-13PE administered by CED showed higher volumes of distribution than the bolus route. Thus, future clinical studies with 125I-IL-13PE or other agents should consider the CED mode of delivery for better distribution in intracranial environments. Volume of distribution of 125I-IL-13PE after bolus or CED administration into mouse brain Injection parameters SPECT Time Vi (μl)* Vd (mm3)** Vd:Vi ratio Bolus - day 1 10 minutes 5 10.2 ± 0.4 204.7 ± 7.0 Bolus - day 7 10 minutes 5 0.1 ± 0.02 2.0 ± 0.4 CED - day 1 3 days 100 120.4 ± 21.8 120.4 ± 21.6 CED - day 7 3 days 100 81.7 ± 6.9 81.7 ± 6.7 *Vi: Volume of injection **Vd: Volume of distribution Citation Format: Akiko Suzuki, Pamela Leland, Peter L. Choyke, Tomio Inoue, Bharat H. Joshi, Raj K. Puri. SPECT/CT analysis of distribution of intracranially infused interleukin-13 Pseudomonas exotoxin, a targeted agent for interleukin-13 receptor expressing brain tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2663. doi:10.1158/1538-7445.AM2013-2663

Abstract LB-503: Pharmacological inhibition of microglia leads to increased survival in a murine model of glioblastoma multiforme in conjunction with ionizing radiation

Abstract Glioblastoma multiforme (GBM) is the most malignant and lethal form of astrocytoma. GBM patients have a median survival time of approximately one year and therefore it is essential to identify novel targets and effective therapies. There is mounting evidence that microglia (specialized brain-resident macrophages) play a significant role in the development and progression of GBM tumors. We have established a microglia-glioma cell co-culture system consisting of normal microglia from C57Bl/6J mice and syngeneic GL261 glioma cells. We have demonstrated that microglia strongly stimulate the invasive behavior of GL261 cells in this system. Our preliminary data also indicate that the microglia significantly stimulate GL261 cell proliferation and resistance to ionizing radiation (IR) in these conditions. In a search for small molecule inhibitors that block the stimulatory effects of microglia on glioma cells, we have examined CNI-1493, a drug that is known to selectively interfere with macrophage function. We found that CNI-1493 potently inhibits microglia-stimulated GL261 invasion. We also examined the effect of intracranially administered CNI-1493 on orthotopic GL261 tumors in the presence or absence of fractionated whole brain irradiation (10 Gy total). We found that although CNI-1493 only has a marginal benefit in the absence of IR, it strongly stimulates animal survival in combination with IR: whereas IR treatment increases survival by 10 days, IR in combination with CNI-1493 treatment extends survival beyond 40 days. In conclusion, our observations support the notion that pharmacological targeting of microglia may be beneficial as adjuvant therapy in conjunction with ionizing radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-503. doi:1538-7445.AM2012-LB-503

Abstract 5474: Optimization of a microdialysis method for evaluation of CP-868,596 penetration in glioblastoma murine models using a sensitive liquid chromatography mass spectrometry (LC-MS/MS) method

Abstract Focal amplification and gene rearrangement of the platelet derived growth factor receptor alpha (PDGFRA) gene, resulting in constitutively active signaling, has been identified in a subset of cases of glioblastoma multiforme (GBM). Thus, inhibiting PDGFR signaling represents a valid treatment strategy for these tumors. CP-868,596, a potent inhibitor of the PDGFR kinase, is currently undergoing preclinical testing for the treatment of GBM. CP-868,596 inhibits the intracellular ATP-binding site of PDGFR, thereby attenuating downstream signaling events. To characterize CP-868,596 penetration in murine high grade glioma, we used a cerebral microdialysis approach. This approach would allow us to measure unbound CP-868,596 concentrations in the tumor extracellular fluid (ECF) using microdialysis under steady-state conditions. To accurately measure the potentially low concentrations of CP-868,596 in the dialysates, we developed and validated a highly sensitive LC-MS/MS method for analysis of CP-868,596. The parent drug CP-868,596 and its deuterated form as an internal standard (ISTD) were separated on a Pursuit 3µ C18 column (50 × 2.0 mm) with mobile phase consisting of 85% acetonitrile and 15% 10mM HCOONH4. The ion transitions at m/z 444.4→301.1 for CP-868,596 and at m/z 448.2→374.2 for the ISTD were selected. Samples were processed using liquid-liquid extraction (LLE) with t-butyl methyl ether. The extracted samples were dried by nitrogen gas and reconstituted with 40% acetonitrile. 10 µLs were injected for analysis. Both within-day and between-day precision (%CV) were ≤ 7% and accuracy ranged from 93.7% to 102.5%. Next, we optimized the microdialysis conditions for CP-868,596. Initial in vitro microdialysis experiments using artificial cerebrospinal-fluid (aCSF) as a perfusate demonstrated low CP-868,596 recovery (8.24 ± 1.26, mean ± SD). Addition of 10% hydroxyl-propyl beta cyclodextrin to the perfusate increased recovery to 91.2% ± 4.6%. Several approaches to estimate probe recovery were tested including retrodialysis and Zero-Flow Rate (ZFR) methods. The ZFR method estimated stock concentrations with > 85% accuracy; hence, this method will be used to estimate recovery under in vivo steady-state conditions. To achieve steady-state plasma concentrations, CP-868,596-loaded miniosmotic pump (Alzet model 2001D) was s.c. implanted in wild-type FVB mice (n=6). Analysis showed that steady-state plasma levels of CP-868,596 were maintained for 30 hours (516.8 ± 92.9 ng/ml, mean ± SD). We predict that our sensitive analytical method (LLOQ 0.1 ng/mL) will allow us to measure the low CP-868,596 concentrations expected during in vivo cerebral microdialysis experiments. Studies measuring CP-868,596 tumor concentrations using the cerebral microdialysis approach in several murine GBM models are ongoing. 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 5474. doi:10.1158/1538-7445.AM2011-5474

MicroPET/CT Imaging of an Orthotopic Model of Human Glioblastoma Multiforme and Evaluation of Pulsed Low-Dose Irradiation

Glioblastoma multiforme (GBM) is an aggressive tumor that typically causes death due to local progression. To assess a novel low-dose radiotherapy regimen for treating GBM, we developed an orthotopic murine model of human GBM and evaluated in vivo treatment efficacy using micro-positron-emission tomography/computed tomography (microPET/CT) tumor imaging. Orthotopic GBM xenografts were established in nude mice and treated with standard 2-Gy fractionation or 10 0.2-Gy pulses with 3-min interpulse intervals, for 7 consecutive days, for a total dose of 14 Gy. Tumor growth was quantified weekly using the Flex Triumph (GE Healthcare/Gamma Medica-Ideas, Waukesha, WI) combined PET-single-photon emission CT (SPECT)-CT imaging system and necropsy histopathology. Normal tissue damage was assessed by counting dead neural cells in tissue sections from irradiated fields. Tumor engraftment efficiency for U87MG cells was 86%. Implanting 0.5 × 10(6) cells produced a 50- to 70-mm(3) tumor in 10 to 14 days. A significant correlation was seen between CT-derived tumor volume and histopathology-measured volume (p = 0.018). The low-dose 0.2-Gy pulsed regimen produced a significantly longer tumor growth delay than standard 2-Gy fractionation (p = 0.045). Less normal neuronal cell death was observed after the pulsed delivery method (p = 0.004). This study successfully demonstrated the feasibility of in vivo brain tumor imaging and longitudinal assessment of tumor growth and treatment response with microPET/CT. Pulsed radiation treatment was more efficacious than the standard fractionated treatment and was associated with less normal tissue damage.

EGFR Gene Overexpression Retained in an Invasive Xenograft Model by Solid Orthotopic Transplantation of Human Glioblastoma Multiforme Into Nude Mice

Orthotopic xenograft animal model from human glioblastoma multiforme (GBM) cell lines often do not recapitulate an extremely important aspect of invasive growth and epidermal growth factor receptor (EGFR) gene overexpression of human GBM. We developed an orthotopic xenograft model by solid transplantation of human GBM into the brain of nude mouse. The orthotopic xenografts sharing the same histopathological features with their original human GBMs were highly invasive and retained the overexpression of EGFR gene. The murine orthotopic GBM models constitute a valuable in vivo system for preclinical studies to test novel therapies for human GBM.

Yes and PI3K Bind CD95 to Signal Invasion of Glioblastoma

Invasion of surrounding brain tissue by isolated tumor cells represents one of the main obstacles to a curative therapy of glioblastoma multiforme. Here we unravel a mechanism regulating glioma infiltration. Tumor interaction with the surrounding brain tissue induces CD95 Ligand expression. Binding of CD95 Ligand to CD95 on glioblastoma cells recruits the Src family member Yes and the p85 subunit of phosphatidylinositol 3-kinase to CD95, which signal invasion via the glycogen synthase kinase 3-beta pathway and subsequent expression of matrix metalloproteinases. In a murine syngeneic model of intracranial GBM, neutralization of CD95 activity dramatically reduced the number of invading cells. Our results uncover CD95 as an activator of PI3K and, most importantly, as a crucial trigger of basal invasion of glioblastoma in vivo.

Combination chemotherapy with 13-cis-retinoic acid and celecoxib in the treatment of glioblastoma multiforme

In a phase II clinical trial, we sought to determine if combining celecoxib with 13-cis-retinoic acid (13-cRA, Accutane) was efficacious in the treatment of recurrent (progressive) glioblastoma multiforme (GBM). In parallel, we also sought to determine to what extent the outcomes from this clinical trial correlated with the findings from studies utilizing two murine intracerebral GBM models, U87MG and U251HF, to determine the predictive value of these murine models. In the clinical trial, 25 patients were studied at recurrence. Stable disease, which occurred in 44% of the patients, was the best response. The median progression-free survival (PFS) was 8 weeks, with a PFS at 6 months of only 19%. For the patients with stable disease, the median PFS was 24 weeks. The toxicity profile was unremarkable. The modest effect on PFS seen in this study agreed with the recent findings of another study, which showed a 19% PFS at 6 months in patients treated with 13-cRA alone. Thus, the combination of 13-cRA with celecoxib is not more effective than 13-cRA in the treatment of progressive GBM. In the murine model study, we found that long-term dosing with 13-cRA or celecoxib alone or in combination did not increase survival in animals with U87MG tumors but modestly increased survival in animals with U251HF tumors. There was no evidence of synergism between the two drugs. From this, we concluded that the animal studies generally predicted that the two agents would have only a modest effect alone and no additive effect when given in combination to patients.