Orthotopic Glioblastoma Model Research Articles

A novel technique of serial biopsy in mouse brain tumour models

Biopsy is often used to investigate brain tumour-specific abnormalities so that treatments can be appropriately tailored. Dacomitinib (PF-00299804) is a tyrosine kinase inhibitor (TKI), which is predicted to only be effective in cancers where the targets of this drug (EGFR, ERBB2, ERBB4) are abnormally active. Here we describe a method by which serial biopsy can be used to validate response to dacomitinib treatment in vivo using a mouse glioblastoma model. In order to determine the feasibility of conducting serial brain biopsies in mouse models with minimal morbidity, and if successful, investigate whether this can facilitate evaluation of chemotherapeutic response, an orthotopic model of glioblastoma was used. Immunodeficient mice received cortical implants of the human glioblastoma cell line, U87MG, modified to express the constitutively-active EGFR mutant, EGFRvIII, GFP and luciferase. Tumour growth was monitored using bioluminescence imaging. Upon attainment of a moderate tumour size, free-hand biopsy was performed on a subgroup of animals. Animal monitoring using a neurological severity score (NSS) showed that all mice survived the procedure with minimal perioperative morbidity and recovered to similar levels as controls over a period of five days. The technique was used to evaluate dacomitinib-mediated inhibition of EGFRvIII two hours after drug administration. We show that serial tissue samples can be obtained, that the samples retain histological features of the tumour, and are of sufficient quality to determine response to treatment. This approach represents a significant advance in murine brain surgery that may be applicable to other brain tumour models. Importantly, the methodology has the potential to accelerate the preclinical in vivo drug screening process.

Synthesis of a novel non‐diuretic, brain‐penetrating, ethacrynic acid analog and demonstration of its potent efficacy in orthotopic glioblastoma (GBM) models

The incidence of pediatric and adult brain tumors has continued to rise in the USA and the alkylating agent temozolomide has remained the sole choice in the chemotherapy of these lethal malignancies. Therefore, there is a very urgent need to design new, more effective brain‐penetrating drugs that can significantly advance the survival brain tumor patients. We are highly interested in exploiting the elevated oxidative stress present in gliomas and have synthesized a hydrophobic, non‐diuretic analog of ethacrynic acid (EA) called KSS72 by removing the carboxylate side chain. KSS72 was selectively cytotoxic to cancer cells and pharmacokinetics following intravenous or oral administrations in mice showed its excellent penetrance through the blood‐brain‐barrier, accumulating at levels equivalent to (TMZ) in the brain. In vitro assays measuring protein carbonyl content, GSH content, ROS generation, GSTpi enzyme activity and others showed that KSS72 triggers a redox imbalance by inhibiting GSTpi and by lowering the antioxidant (GSH) and reducing equivalent (NADPH) levels, leading to a significant elevation of ROS levels. We also showed the upregulation of endoplasmic reticulum (ER) stress‐responsive proteins, activation of MAPK, autophagy and apoptotic pathways by KSS72 in several cell lines. In view of its excellent brain penetrance and multiple cytocidal events mediated by ROS, we tested the antitumor efficacy of KSS72 in human GBM cell lines and intracranial GBM xenografts. SF‐188 human GBM cells expressing firefly luciferase were injected into the brain just left of bregma for the development of orthotopic GBM in nude mice. These mice were given 25 mg/kg/day of KSS72 intraperitoneally for 2 weeks. The animals when imaged by IVIS after luciferin injections showed a complete lack of intracranial tumors in all KSS72 administered animals. H&E staining of mouse brain sections confirmed the total elimination of GBM by KSS72. KSS72 did not exert any toxicity on host tissues and serum ALT and AST levels were not altered. In summary, we conclude that KSS72 acting through multiple pathways of oxidative stress is a hugely promising non‐toxic anti‐glioma drug with potential to enter clinical trials.Support or Funding InformationCPRIT grant RP130266 to KSS.

Human mesenchymal stromal cells inhibit tumor growth in orthotopic glioblastoma xenografts

BackgroundMesenchymal stem/stromal cells (MSCs) represent an attractive tool for cell-based cancer therapy mainly because of their ability to migrate to tumors and to release bioactive molecules. However, the impact of MSCs on tumor growth has not been fully established. We previously demonstrated that murine MSCs show a strong tropism towards glioblastoma (GBM) brain xenografts and that these cells are able to uptake and release the chemotherapeutic drug paclitaxel (PTX), maintaining their tropism towards the tumor. Here, we address the therapy-relevant issue of using MSCs from human donors (hMSCs) for local or systemic administration in orthotopic GBM models, including xenografts of patient-derived glioma stem cells (GSCs).MethodsU87MG or GSC1 cells expressing the green fluorescent protein (GFP) were grafted onto the striatum of immunosuppressed rats. Adipose hMSCs (Ad-hMSCs), fluorescently labeled with the mCherry protein, were inoculated adjacent to or into the tumor. In rats bearing U87MG xenografts, systemic injections of Ad-hMSCs or bone marrow (BM)-hMSCs were done via the femoral vein or carotid artery. In each experiment, either PTX-loaded or unloaded hMSCs were used. To characterize the effects of hMSCs on tumor growth, we analyzed survival, tumor volume, tumor cell proliferation, and microvascular density.ResultsOverall, the AD-hMSCs showed remarkable tropism towards the tumor. Intracerebral injection of Ad-hMSCs significantly improved the survival of rats with U87MG xenografts. This effect was associated with a reduction in tumor growth, tumor cell proliferation, and microvascular density. In GSC1 xenografts, intratumoral injection of Ad-hMSCs depleted the tumor cell population and induced migration of resident microglial cells. Overall, PTX loading did not significantly enhance the antitumor potential of hMSCs. Systemically injected Ad- and BM-hMSCs homed to tumor xenografts. The efficiency of hMSC homing ranged between 0.02 and 0.5% of the injected cells, depending both on the route of cell injection and on the source from which the hMSCs were derived. Importantly, systemically injected PTX-loaded hMSCs that homed to the xenograft induced cytotoxic damage to the surrounding tumor cells.ConclusionshMSCs have a therapeutic potential in GBM brain xenografts which is also expressed against the GSC population. In this context, PTX loading of hMSCs seems to play a minor role.

Effects of the lysosomal destabilizing drug siramesine on glioblastoma in vitro and in vivo

BackgroundGlioblastoma is the most frequent and most malignant brain tumor with the patients having a median survival of only 14.6 months. Although glioblastoma patients are treated with surgery, radiation and chemotherapy recurrence is inevitable. A stem-like population of radio- and chemoresistant brain tumor-initiating cells combined with the invasive properties of the tumors is believed to be critical for treatment resistance. In the present study, the aim was to investigate the effect of a novel therapeutic strategy using the lysosomotropic detergent siramesine on glioblastomas.MethodsStandard glioma cell lines and patient-derived spheroids cultures with tumor-initiating stem-like cells were used to investigate effects of siramesine on proliferation and cell death. Responsible mechanisms were investigated by inhibitors of caspases and cathepsins. Effects of siramesine on migrating tumor cells were investigated by a flat surface migration assay and by implanting spheroids into organotypic rat brain slice cultures followed by confocal time-lapse imaging. Finally the effect of siramesine was investigated in an orthotopic mouse glioblastoma model. Results obtained in vitro and in vivo were confirmed by immunohistochemical staining of histological sections of spheroids, spheroids in brain slice cultures and tumors in mice brains.ResultsThe results showed that siramesine killed standard glioma cell lines in vitro, and loss of acridine orange staining suggested a compromised lysosomal membrane. Co-treatment of the cell lines with inhibitors of caspases and cathepsins suggested differential involvement in cell death. Siramesine caused tumor cell death and reduced secondary spheroid formation of patient-derived spheroid cultures. In the flat surface migration model siramesine caused tumor cell death and inhibited tumor cell migration. This could not be reproduced in the organotypic three dimensional spheroid-brain slice culture model or in the mice xenograft model.ConclusionsIn conclusion the in vitro results obtained with tumor cells and spheroids suggest a potential of lysosomal destabilizing drugs in killing glioblastoma cells, but siramesine was without effect in the organotypic spheroid-brain slice culture model and the in vivo xenograft model.

Preclinical therapeutic efficacy of a novel blood-brain barrier-penetrant dual PI3K/mTOR inhibitor with preferential response in PI3K/PTEN mutant glioma.

Glioblastoma (GBM) is an ideal candidate disease for signal transduction targeted therapy because the majority of these tumors harbor genetic alterations that result in aberrant activation of growth factor signaling pathways. Loss of heterozygosity of chromosome 10, mutations in the tumor suppressor gene PTEN, and PI3K mutations are molecular hallmarks of GBM and indicate poor prognostic outcomes in many cancers. Consequently, inhibiting the PI3K pathway may provide therapeutic benefit in these cancers. PI3K inhibitors generally block proliferation rather than induce apoptosis. To restore the sensitivity of GBM to apoptosis induction, targeted agents have been combined with conventional therapy. However, the molecular heterogeneity and infiltrative nature of GBM make it resistant to traditional single agent therapy. Our objectives were to test a dual PI3K/mTOR inhibitor that may cross the blood–brain barrier (BBB) and provide the rationale for using this inhibitor in combination regimens to chemotherapy-induced synergism in GBM. Here we report the preclinical potential of a novel, orally bioavailable PI3K/mTOR dual inhibitor, DS7423 (hereafter DS), in in-vitro and in-vivo studies. DS was tested in mice, and DS plasma and brain concentrations were determined. DS crossed the BBB and led to potent suppression of PI3K pathway biomarkers in the brain. The physiologically relevant concentration of DS was tested in 9 glioma cell lines and 22 glioma-initiating cell (GIC) lines. DS inhibited the growth of glioma tumor cell lines and GICs at mean 50% inhibitory concentration values of less than 250 nmol/L. We found that PI3K mutations and PTEN alterations were associated with cellular response to DS treatment; with preferential inhibition of cell growth in PI3KCA-mutant and PTEN altered cell lines. DS showed efficacy and survival benefit in the U87 and GSC11 orthotopic models of GBM. Furthermore, administration of DS enhanced the antitumor efficacy of temozolomide against GBM in U87 glioma models, which shows that PI3K/mTOR inhibitors may enhance alkylating agent-mediated cytotoxicity, providing a novel regimen for the treatment of GBM. Our present findings establish that DS can specifically be used in patients who have PI3K pathway activation and/or loss of PTEN function. Further studies are warranted to determine the potential of DS for glioma treatment.

Enhanced expression of Vastatin inhibits angiogenesis and prolongs survival in murine orthotopic glioblastoma model

BackgroundAntiangiogenic therapies are considered promising for the treatment of glioblastoma (GB). The non-collagenous C-terminal globular NC1 domain of type VIII collagen a1 chain, Vastatin, is an endogenous antiangiogenic polypeptide. Sustained enhanced expression of Vastatin was shown to inhibit tumour growth and metastasis in murine hepatocellular carcinoma models. In this study, we further explored the efficacy of Vastatin in the treatment of GB xenografts.MethodTreatment of Vastatin was carried out using a nanopolymer gene vector PEI600-CyD-Folate (H1). Antiangiogenic effect of Vastatin was tested in vitro by using co-culture system and conditioned medium. An orthotopic GB murine model was established to examine the in vivo therapeutic effect of Vastatin alone treatment and its combination with temozolomide.ResultsVastatin gene transfection mediated by H1 could target tumour cells specifically and suppress the proliferation of microvessel endothelial cells (MECs) through a paracrine inhibition manner. Enhancing Vastatin expression by intracerebral injection of H1-Vastatin significantly prolonged animal survival from 48 to 75 days in GB murine model, which was comparable to the effect of Endostatin, the most studied endogenous antiangiogenic polypeptide. The diminished presence of CD34 positive cells in the GB xenografts suggested that Vastatin induced significant antiangiogenesis. Moreover, a synergistic effect in extending survival was detected when H1-Vastatin was administered with temozolomide (TMZ) in GB chemoresistant murine models.ConclusionOur results suggest, for the first time, that Vastatin is an antiangiogenic polypeptide with significant potential therapeutic benefit for GB. H1-Vastatin gene therapy may have important implications in re-sensitizing recurrent GB to standard chemotherapeutic agents.

Intracranial AAV-IFN-β gene therapy eliminates invasive xenograft glioblastoma and improves survival in orthotopic syngeneic murine model.

The highly invasive property of glioblastoma (GBM) cells and genetic heterogeneity are largely responsible for tumor recurrence after the current standard‐of‐care treatment and thus a direct cause of death. Previously, we have shown that intracranial interferon‐beta (IFN‐β) gene therapy by locally administered adeno‐associated viral vectors (AAV) successfully treats noninvasive orthotopic glioblastoma models. Here, we extend these findings by testing this approach in invasive human GBM xenograft and syngeneic mouse models. First, we show that a single intracranial injection of AAV encoding human IFN‐β eliminates invasive human GBM8 tumors and promotes long‐term survival. Next, we screened five AAV‐IFN‐β vectors with different promoters to drive safe expression of mouse IFN‐β in the brain in the context of syngeneic GL261 tumors. Two AAV‐IFN‐β vectors were excluded due to safety concerns, but therapeutic studies with the other three vectors showed extensive tumor cell death, activation of microglia surrounding the tumors, and a 56% increase in median survival of the animals treated with AAV/P2‐Int‐mIFN‐β vector. We also assessed the therapeutic effect of combining AAV‐IFN‐β therapy with temozolomide (TMZ). As TMZ affects DNA replication, an event that is crucial for second‐strand DNA synthesis of single‐stranded AAV vectors before active transcription, we tested two TMZ treatment regimens. Treatment with TMZ prior to AAV‐IFN‐β abrogated any benefit from the latter, while the reverse order of treatment doubled the median survival compared to controls. These studies demonstrate the therapeutic potential of intracranial AAV‐IFN‐β therapy in a highly migratory GBM model as well as in a syngeneic mouse model and that combination with TMZ is likely to enhance its antitumor potency.

Locoregional Confinement and Major Clinical Benefit of 188Re-Loaded CXCR4-Targeted Nanocarriers in an Orthotopic Human to Mouse Model of Glioblastoma.

Purpose: Gold standard beam radiation for glioblastoma (GBM) treatment is challenged by resistance phenomena occurring in cellular populations well prepared to survive or to repair damage caused by radiation. Among signals that have been linked with radio-resistance, the SDF1/CXCR4 axis, associated with cancer stem-like cell, may be an opportune target. To avoid the problem of systemic toxicity and blood-brain barrier crossing, the relevance and efficacy of an original system of local brain internal radiation therapy combining a radiopharmaceutical with an immuno-nanoparticle was investigated.Experiment design: The nanocarrier combined lipophilic thiobenzoate complexes of rhenium-188 loaded in the core of a lipid nanocapsule (LNC188Re) with a function-blocking antibody, 12G5 directed at the CXCR4, on its surface. The efficiency of 12G5-LNC188Re was investigated in an orthotopic and xenogenic GBM model of CXCR4-positive U87MG cells implanted in the striatum of Scid mice.Results: We demonstrated that 12G5-LNC188Re single infusion treatment by convection-enhanced delivery resulted in a major clinical improvement in median survival that was accompanied by locoregional effects on tumor development including hypovascularization and stimulation of the recruitment of bone marrow derived CD11b- or CD68-positive cells as confirmed by immunohistochemistry analysis. Interestingly, thorough analysis by spectral imaging in a chimeric U87MG GBM model containing CXCR4-positive/red fluorescent protein (RFP)-positive- and CXCR4-negative/RFP-negative-GBM cells revealed greater confinement of DiD-labeled 12G5-LNCs than control IgG2a-LNCs in RFP compartments.Main conclusion: These findings on locoregional impact and targeting of disseminated cancer cells in tumor margins suggest that intracerebral active targeting of nanocarriers loaded with radiopharmaceuticals may have considerable benefits in clinical applications.

Alisertib demonstrates significant antitumor activity in bevacizumab resistant, patient derived orthotopic models of glioblastoma

Aurora A kinase (AURKA), a member of the serine/threonine kinase family, plays a critical role in cell division, and it is widely overexpressed in a variety of tumors including glioblastoma (GBM). Alisertib (MLN8237) is an orally administered selective AURKA inhibitor with potent antiproliferative activity, currently undergoing clinical testing in different tumor types. In vitro evaluation of alisertib against the primary GBM lines, GBM6, GBM10, GBM12 and GBM39 showed significant antitumor activity with IC50s ranging between 30 and 95nM. Orthotopic xenografts of GBM10 and the bevacizumab resistant lines GBM6 and GBM39 were established by implantating 3 × 105 cells in the caudate nucleus of nude mice; animals were randomized to treatment with either alisertib 30mg/kg/day or vehicle. In all three models, treatment with alisertib resulted in a statistically significant prolongation of survival (p < 0.0001). In addition, alisertib administration in these mice decreased phosphorylated aurora-A, induced mitotic arrest and significantly decreased histone H3 phosphorylation in tumors. In conclusion, alisertib displays significant antitumor activity against primary GBM lines and xenografts, including patient derived GBM lines resistant to bevacizumab; these data support clinical translation in GBM.

Culture on 3D Chitosan‐Hyaluronic Acid Scaffolds Enhances Stem Cell Marker Expression and Drug Resistance in Human Glioblastoma Cancer Stem Cells

The lack of in vitro models that support the growth of glioblastoma (GBM) stem cells (GSCs) that underlie clinical aggressiveness hinders developing new, effective therapies for GBM. While orthotopic patient-derived xenograft models of GBM best reflect in vivo tumor behavior, establishing xenografts is a time consuming, costly, and frequently unsuccessful endeavor. To address these limitations, a 3D porous scaffold composed of chitosan and hyaluronic acid (CHA) is synthesized. Growth and expression of the cancer stem cell (CSC) phenotype of the GSC GBM6 taken directly from fresh xenogratfs grown on scaffolds or as adherent monolayers is compared. While 2D adherent cultures grow as monolayers of flat epitheliod cells, GBM6 cells proliferate within pores of CHA scaffolds as clusters of self-adherent ovoid cells. Growth on scaffolds is accompanied by greater expression of genes that mediate epithelial-mesenchymal transition and maintain a primitive, undifferentiated phenotype, hallmarks of CSCs. Scaffold-grown cells also display higher expression of genes that promote resistance to hypoxia-induced oxidative stress. In accord, scaffold-grown cells show markedly greater resistance to clinically utilized alkylating agents compared to adherent cells. These findings suggest that our CHA scaffolds better mimic in vivo biological and clinical behavior and provide insights for developing novel individualized treatments.

STMC-30. TRAFFICKING AND EFFECT ON SURVIVAL OF BEVACIZUMAB IN GLIOBLASTOMA

Most patients with recurrent glioblastoma (GBM) are treated with bevacizumab, a humanized monoclonal antibody (mAb) that binds VEGF-A and inhibits its binding to VEGFR. Approximately 30% of GBM patients are non-responsive to bevacizumab and the underlying mechanism for the lack of response is unknown. It has been assumed that bevacizumab solely targets circulating VEGF-A. We hypothesized that bevacizumab and human IgGs in general gain access to the perivascular niche that contains cancer stem cells (CSCs) in GBM. We found that bevacizumab gains access to the perivascular tumor area through leaky blood vessels and was internalized by tumor cells in an orthotopic established xenograft mouse model of GBM. In vitro, CSCs (CD133+) from GBM rapidly internalized bevacizumab into membrane protrusions that contained actin and internalization was significantly inhibited by a macropinocytosis inhibitor (EIPA), suggesting CSCs internalize bevacizumab via macropinocytosis. Furthermore, bevacizumab or human IgG were largely detected in the Rab4+ “fast” recycling compartment at 5 min, and in the LAMP1+ compartment (late endosome/lysosome) at 3 hr in the CSCs. We observed similar trafficking patterns for bevacizumab in our in vivo model. CSCs from GBM do not express the neonatal Fc receptor, the canonical pathway for recycling of IgG. Bevacizumab induces autophagy in CSCs due to VEGF deprivation and this mechanism is blocked with the addition of growth factors. Taken together, our data show that in GBM, bevacizumab gains access to the perivascular tumor space, is macropinocytosed by CSCs where it is trafficked to a recycling compartment or to the late endosome/lysosome, and that it induces autophagy promoting survival of these tumor cells. These data suggest that alterations in endocytosis or recycling in the CSCs could impact the fate of therapeutic IgGs like bevacizumab and ultimately influence a patients’ response to GBM therapy.

Highlights from the Literature

Tumors with high glycolytic or hypoxic properties must produce sufficient levels of acetyl-CoA to maintain cellular survival and proliferation under adverse nutrient conditions. Acetyl-CoA can be generated by oxidation of glucose, glutamine, or fatty acids. There are principal differences between the metabolism of normal, well-fed cells and tumor cells, especially under nutrientdeprived conditions. Whereas the former consume glucose/ pyruvate/acetyl-CoA-derived citrate to synthesize acetyl-CoA, which is then used for fatty acid and cholesterol synthesis, generation of nucleotides and amino acids, and histone acetylation, the latter divert the main metabolite of glucose into aerobic glycolysis from pyruvate-derived mitochondrial acetylCoA to lactate. The “bioenergetic substrate gap” develops when under oxygen-limiting conditions the ability of a cell to make acetyl-CoA is impaired, and the question arises how sufficient amounts of citrate via ATP citrate lyase are produced. In the brain, glial cells are capable of oxidizing acetate. In an extremely elegant study, integrating observations from clinical experiments, ex vivo data, and preclinical mouse and cell culture studies, Mashimo and Pichumani et al from the Bachoo and Maher laboratories showed that primary and metastatic tumors growing in the brain have the capacity to oxidize acetate while simultaneously oxidizing glucose, providing an explanation for the yet unknown carbon source for the generation of the critical metabolite acetyl-CoA. Previously, C-nuclear magnetic resonance analysis showed that upon infusion of C-glucose during surgery for primary or metastatic brain tumors, ,50% of carbon in the acetyl-CoA pool was derived from glucose. The principal hypothesis by Mashimo and Pichumani et al is that glioma cells retain the acetatemetabolizing ability of the “healthy” glial cells. For metastases, it had been speculated that the microenvironment provokes a change in cells from non-acetate-metabolizing organs, like lung and breast, to allow them to metabolize acetate. In their work, relevant human orthotopic tumor mouse models of glioblastoma and brain metastases with remarkable preservation of the hallmark histopathological and molecular features (ER-/PR-negative/Her2-positive breast cancer, EGFR/ALK/KRAS wild-type non-small cell lung cancer, VHL-nil clear cell renal carcinoma, or BRAF melanoma) were used in C-NMR tissue examinations with very clear co-infusion experiments of C-glucose and C-acetate. Each of the metastatic cell lines oxidized glucose but showed a relevant bioenergetics carbon substrate gap. In co-infusion experiments with C-glucose and C-acetate, which produce distinct labeling patterns in the citric acid cycle intermediates, in six glioblastoma human orthotopic lines derived from patients prior to treatment and one line derived from the progression of one initial line a considerable, tumor-specific and variable consumption of acetate was demonstrated. The finding that the human tumors oxidize acetate was validated in the clinical setting by infusing C-acetate during glioblastoma and brain metastases operations. In contrast, glutamine is taken up by primary and metastatic brain tumors but not metabolized in the citric acid cycle in vivo. In a study published back-to-back with the Mashimo and Pichumani et al paper, Comerford and colleagues demonstrate that the nucleocytosolic acetyl-CoA synthetase enzyme (ACSS2) is expressed and has a critical role in hepatocellular carcinoma, gliomas, and breast and lung cancer for acetate utilization. Immunoreactivity to ACSS2 is correlated with glioma WHO grade (II-IV) and reduced survival in grade II/III gliomas. ACSS2 expression was also correlated with accumulation of mutation in diverse genetic mouse glioma models. Reintroduction of ACSS2 in ACSS2-knockout mouse embryonic fibroblasts enabled these cells to increase acetate incorporation into glutamate. The authors conclude that despite extensive molecular alterations in glioblastoma the ability to oxidize acetate, which is typical for glial cells, is at least maintained. The unexpected finding that metastases to the CNS can metabolize acetate may be discussed as an adaptation to the brain microenvironment or rather a more general property of tumor cells. ACSS2 upregulation is a prerequisite for the tumor cell to convert actetate to acetyl-CoA. Therefore, ACSS2 might be a vulnerability specific to the tumor metabolism. Further, the studies summarized provide nice examples of the relevance of in vivo models for research in cancer metabolism and also the strength of clinical cross-validation.

Inhibitory Kinetics and Mechanism of Flavonoids Extracted from Cotinus coggygria Scop. Against Glioblastoma Cancer

ABSTRACTThis proposal seeks to study the potential therapeutic modality of chemoprevention and anticancer effects and mechanisms of the flavonoids from Cotinus coggygria Scop. on glioblastoma cancer. In the current study, the total flavonoids (TFs) isolated from Cotinus coggygria Scop. var. cinerea Engl. (Cotinus coggygria Scop.) and the major flavonoids of Cotinus coggygria Scop. (CCFs) were identified, and the inhibitory kinetics of TF and CCF on glioblastoma cell lines were calculated. We also investigated whether TF or CCF regulated the apoptotic mechanism in cellular models of glio-blastoma cells. Finally, we evaluated whether treatment with TF or CCF suppressed tumor growth and inhibited migration in orthotopic mouse models of glioblastoma in vivo. In this study, the CCFs were identified as rutin, myricetin, and fisetin. TF and CCF remarkably inhibited cell proliferation and downregulated the PI3K/Akt and ERK signaling pathway in glioblastoma cell lines. Furthermore, the mitochondrial caspase-dependent cascade was regulated by TF and myricetin. In addition, TF and myricetin exhibited significant antitumor effects on glioblastoma in vivo. Taken together, these results suggest that phytochemical and biological data provide evidence for the active components in Cotinus coggygria, and that the TFs are responsible for the anticancer effects on glioblastoma cell growth via induction of apoptosis. In addition, the representative compound myricetin could provide a clinically relevant therapeutic opportunity. Therefore, our data strongly suggest that myricetin-deprived CCF can serve as a potent chemopreventive herbal medicine.

P05.04 Morphine pretreatment improves the therapeutic efficacy of doxorubicin in an orthotopic model of human glioblastoma

AbstractIntroduction:Morphine is the most frequently employed analgesic in pain therapy and its use is well documented during antineoplastic agents administration. In addition, morphine is able to alter the neuronal and glial microenvironment, leading to the stroke of the Blood-Brain Barrier (BBB) and enhancing the access of molecules normally unable to cross it, as Doxorubicin (Dox).We have developed a xenograft mice model of human glioblastoma in order to verify the effectiveness of Dox alone or in association with morphine.Materials and Methods:Foxn1 mice were injected with U87MG-luc cells in the left lobe of the brain and treated with Dox (5 mg/kg and 2.5 mg/kg, weekly) with or without morphine (10 mg/kg, weekly).Intracranial tumor growth was quantified by Bioluminescence Imaging (BLI).Results:The data showed that only Dox 5mg/kg determined a significant regression of BLI in a xenograft mouse model of brain glioma while the lower dose of 2.5 mg/kg was not effective.The average BLI for Dox 5mg/kg was 5-fold lower than that measured for Dox 2.5mg/kg (P=0.0238) and morphine 10 mg/kg (P=0.0098) and 8 fold lower than vehicle (P=0.0012).However, when administered with morphine to brain xenograft in nude mice, Dox 2.5mg/kg showed a decreasing radiance values corresponding to decreasing tumor growth. The average BLI for Dox 2.5 mg/kg plus morphine was 5-fold lower than Dox 2.5mg/kg alone (P=0.0053) and 8-fold lower than vehicle (P=0.0004).Even more interesting, the average BLI for Dox 2.5mg/kg plus morphine was lower than Dox 5mg/kg during the whole experiment, indicating the effectiveness of combined administration of morphine and low doses of Dox as proper dosage to treat poor prognosis brain tumors.In addition, a weekly schedule (up to 5 weeks) with a combination of 10 mg/kg morphine and 2.5 mg/kg Dox did not cause a body weight loss of animals; rather, it induced a body weight gain of +7,98% as a clear signal of minimal toxicity.Conclusions:The present findings show that molecules as morphine are able to interfere with molecules normally unable to cross the BBB. This mechanism could be used for new approaches in therapy of refractory CNS tumors as glioblastoma.

Targeting hexokinase 2 enhances response to radio-chemotherapy in glioblastoma

First-line cancer therapies such as alkylating agents and radiation have limited survival benefits for Glioblastoma (GBM) patients. Current research strongly supports the notion that inhibition of aberrant tumor metabolism holds promise as a therapeutic strategy when used in combination with radiation and chemotherapy. Hexokinase 2 (HK2) has been shown to be a key driver of altered metabolism in GBM, and presents an attractive therapeutic target. To date, no study has fully assessed the therapeutic value of targeting HK2 as a mechanism to sensitize cells to standard therapy, namely in the form of radiation and temozolomide (TMZ). Using cell lines and primary cultures of GBM, we showed that inducible knockdown of HK2 altered tumor metabolism, which could not be recapitulated by HK1 or HK3 loss. HK2 loss diminished both in vivo tumor vasculature as well as growth within orthotopic intracranial xenograft models of GBMs, and the survival benefit was additive with radiation and TMZ. Radio-sensitization following inhibition of HK2 was mediated by increased DNA damage, and could be rescued through constitutive activation of ERK signaling. This study supports HK2 as a potentially effective therapeutic target in GBM.

Dactolisib (NVP-BEZ235) toxicity in murine brain tumour models.

BackgroundGlioblastomas (GBMs) are highly malignant brain tumours with a poor prognosis, and current cytotoxic regimens provide only a limited survival benefit. The PI3K/Akt/mTOR pathway has been an attractive target for therapy due to its high activation in GBMs as well as other cancers. The dual pan-PI3K/mTOR kinase inhibitor dactolisib (NVP-BEZ235) is an anti-neoplastic compound currently under investigation. However, little is known about its efficacy in human GBMs. We aimed at evaluating the efficacy of dactolisib in human glioblastoma cells, as well as in murine models carrying human GBM xenografts.MethodsTo assess the effect of dactolisib in vitro, MTS assay, manual cell count, BrdU incorporation and Annexin V staining experiments were used to observe growth and apoptosis. Furthermore, Akt phosphorylation (S473), a downstream target of PI3K, was explored by western blotting. Animal studies utilizing orthotopic xenograft models of glioblastoma were performed in nude rats and NOD/SCID mice to monitor survival benefit or inhibition of tumor growth.ResultsWe found that dactolisib in vitro shows excellent dose dependent anti-growth properties and increase in apoptosis. Moreover, dose dependent inhibition of Akt phosphorylation (S473), a downstream effect of PI3K, was observed by western blotting. However, in two independent animal studies utilizing nude rats and NOD/SCID mice in orthotopic xenograft models of glioblastoma, we observed no survival benefit or inhibition of tumour growth. Severe side effects were observed, such as elevated levels of blood glucose and the liver enzyme alanine transaminase (ALT), in addition to diarrhoea, hair loss (alopecia), skin rash and accumulation of saliva in the oral cavity.ConclusionTaken together, our results suggest that despite the anti-neoplastic efficacy of dactolisib in glioma treatment in vitro, its utility in vivo is questionable due to toxicity.

Abstract 628: A method to examine spatial relationships between tumor cells and vasculature using a mouse orthotopic PDX glioblastoma model

Abstract Introduction: Glioblastoma progression requires angiogenesis and/or vascular co-option. A major pathway for its infiltration is the perivascular space. We present herein a method to visualize glioblastoma cells with respect to tumor-associated vasculature within and beyond the tumor mass. Experimental procedures: A neurosphere cell line, HF3253, developed from cultures of a human glioblastoma biopsy sample was labeled with PKH26, a red fluorescent dye, using a commercially available kit (Sigma-Aldrich). The labeled cells were implanted into the right striatum in immunocompromised nude mice (n = 5) and allowed to grow for 2-4 weeks. Terminally (one mouse at 2, two at 3 and the remaining two at 4 weeks), the mice were intravenously injected with fluorescent isothiocyanate (FITC) dextran of 70 kDa molecular weight. The brains were removed after 5 min of dextran circulation, immersion fixed in 10% neutral buffered formalin for 2 days and then sectioned into 100 μm thick coronal slices using a vibratome. Slices were stained with the nuclear counterstain 4’,6-diamidino-2-phenylindole (DAPI) to visualize native mouse brain cells at 405 nm in an Olympus FV1200 laser scanning confocal microscope. Relative distributions of the dextran and PKH26 fluorochromes were also examined using the same microscope. FITC-dextran (green fluorochrome)-labeled microvessels and PKH26-labeled tumor cells (red fluorochrome) were excited by a laser beam at 488 and 568 nm, respectively, and emissions were detected with a photomultiplier tube through 522 and 585 nm emission filters, respectively. Results: The normal vasculature on the contralateral side appeared well perfused, filled with the green dextran fluorescence. Intricate, fine networks of microvessels were present. The ipsilateral hemisphere showed bright red spots of tumor cells in the striatum in closely distributed groups among the blue DAPI-stained mouse cells. Compared to contralateral side, the vasculature associated with such groups was dilated and irregular, with fewer interconnections. Interestingly, these irregular networks were also observed away from the main tumor cell mass in 3- and 4-week-old tumors, suggesting probable future infiltrative pathways. Red-labeled single cells and groups of tumor cells could be seen along vascular branches penetrating brain tissue adjacent to the tumor. The strong fluorescence of the tumor cell tracer PKH26 persisted over the 4 week experimental period, suggesting its utility in imaging tumor infiltration in this model. Conclusions: This technique generated high resolution images of tumor cell distribution and associated microvasculature in an orthotopic glioblastoma model. With further quantification, it can help in establishing infiltrative patterns of different PDX tumor models, and may also be useful to measure discrete responses to therapies that affect tumor cells and/or vasculature. Citation Format: Tavarekere N. Nagaraja, Kelly Keenan, Susan Irtenkauf, Laura Hasselbach, Swayamprava Panda, Glauber Cabral, James R. Ewing, Tom Mikkelsen, Ana deCarvalho. A method to examine spatial relationships between tumor cells and vasculature using a mouse orthotopic PDX glioblastoma model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 628.

Abstract 2525: Sema3C promotes glioma stem cell survival and radioresistance

Abstract Introduction: Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs) are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the non-stem tumor cell (NSTC) population is not well defined. Methods/Materials: GSCs were enriched from patient specimens and functionally validated. We used a candidate approach to screen for secreted factors that were preferentially used by GSCs but not NSTCs. Expression was assessed by Western blot, and confirmed by immunohistochemistry and immunofluorescence in vitro and in human and mouse GBM tissues. Ligand-receptor interactions were confirmed by co-immunoprecipitation. Rac activity was assessed by Rac-GTP pulldown. Co-culture epistasis experiments were performed using knockdown and recombinant protein approaches. Glioma stem cell marker analysis (CD133, Sox2, Olig2), cell viability (ATP-dependent assays), apoptosis (TUNEL, PI/AnnexinV, cleaved caspase 3/7), self-renewal (tumorsphere formation and limiting dilution assay) and invasion (transwell migration assay) were performed. GSCs were separated in PlexinD1 hi and lo populations by FACS. Radiation (3 Gy) was delivered by Cs-137 irradiator. GSCs were used to establish orthotopic xenograft models of glioblastoma and tumor growth and animal survival were assessed. Results: We have found that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/NF-κB signaling in an autocrine and paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs) or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype in vivo. Furthermore, GSCs that expressed high levels of Plexin receptors were more resistant to radiation, and Sema3C and Plexin receptors were upregulated after irradiation. Together, these data support that Sema3C signaling contributes to radiation resistance. Conclusions: Our study supports targeting Sema3C to break this GSC-specific autocrine/paracrine loop to improve glioblastoma treatment, potentially with a high therapeutic index. Citation Format: Jianghong Man, Jocelyn Shoemake, Qiulian Wu, Jennifer Yu. Sema3C promotes glioma stem cell survival and radioresistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2525.

Abstract 3276: The tissue and cellular destination of therapeutic IgGs in glioblastoma

Abstract Most patients with recurrent glioblastoma (GBM) are treated with bevacizumab, a humanized monoclonal antibody (mAb) that binds VEGF-A and inhibits its binding to VEGFR. Approximately 30% of GBM patients are non-responsive to bevacizumab and the underlying mechanism for the lack of response is not known. It has been assumed that bevacizumab solely targets circulating VEGF-A in blood. We hypothesized that bevacizumab and human IgGs in general gain access to the perivascular niche that contains cancer stem cells (CSCs) in GBM. We found that bevacizumab gains access to the perivascular tumor area through leaky blood vessels and was internalized by tumor cells in an orthotopic xenograft mouse model of GBM. In vitro, CSCs (CD133+) from GBM rapidly internalized either bevacizumab or human IgG into membrane protrusions that contained actin and internalization was significantly inhibited by a macropinocytosis inhibitor (EIPA), suggesting CSCs internalize bevacizumab or human IgG via macropinocytosis. Furthermore, bevacizumab or human IgG was largely detected in the Rab4+ “fast” recycling compartment at 5 min, and both were largely detected in the LAMP1+ compartment (late endosome/lysosome) at 3 hr in the CSCs. CSCs (CD133+) from GBM do not express the neonatal Fc receptor, the canonical pathway for recycling of IgG. Administration of bevacizumab to an orthotopic xenograft mouse model of established GBM showed that bevacizumab was partially co-localized with Rab4+ or with LAMP1+ in perivascular tumor cells, consistent with our in vitro findings. Taken together, our data show that in GBM, humanized IgG, including bevacizumab, gains access to the perivascular tumor space and is then macropinocytosed by CSCs and trafficked to a recycling compartment or to the late endosome/lysosome. These data suggest that alterations in endocytosis or recycling in the CSCs could impact the fate of therapeutic IgGs like bevacizumab and ultimately influence a patients’ response to GBM therapy. Citation Format: Gaelle Muller-Greven, Cathleen Carlin, Steven Toms, Manmeet Ahluwalia, Markus Bredel, Justin Lathia, Jeremy Rich, Petra Hamerlik, Candece L. Gladson. The tissue and cellular destination of therapeutic IgGs in glioblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3276.

Abstract LB-346: Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages

Abstract OBJECTIVE: Here we aimed to overcome resistance to anti-VEGF therapy in glioblastoma (GBM), the most common and aggressive adult primary brain tumor. INTRODUCTION: Current standard of care, including chemo-radiation, confers modest overall survival benefits of less than 1.5 years in patients. GBMs are highly dependent on angiogenesis, and anti-VEGF therapy is a promising approach to prolong survival. While anti-VEGF therapy prolongs progression-free survival in GBM, patients rapidly become refractory and overall survival is not increased. We previously demonstrated that angiopoietin-2 (Ang-2) may confer resistance to anti-VEGF receptor (VEGFR) treatment, as ectopic overexpression of Ang-2 compromises the benefits of VEGFR inhibition in murine GBM. Additionally, circulating Ang-2 levels in GBM patients rebound after an initial decrease following administration of cediranib, a pan-VEGFR tyrosine kinase inhibitor, suggesting Ang-2 may mediate resistance to anti-VEGF pathway inhibition. EXPERIMENTAL DESIGN: In two orthotopic models of GBM, Gl261 (murine) and U87 (human), we used MRI and Optical Frequency Domain Imaging (OFDI) to measure whether dual inhibition of Ang-2 and VEGFR reduces tumor burden and improves survival. We examined morphological changes in the tumor vasculature by immunohistochemistry, and phenotypical changes in tumor associated macrophages (TAMs) by flow cytometry. RESULTS: Dual therapy with cediranib (a pan-VEGFRs inhibitor) and MEDI3617 (an Ang-2 neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth. In U87 tumors, combined treatment increased tumor necrosis. Dual therapy enhanced morphological normalization of vessels and led to changes along the M1/M2 spectrum of CD45+CD11b+F4/80+ TAMs. We analyzed the functional role of TAMs by inhibiting TAM recruitment with an anti-colony stimulating factor-1 antibody (anti-CSF-1) in combination with dual therapy. Depletion of TAMs compromised the survival benefit of anti-Ang-2/VEGFR therapy, suggesting that TAMs mediate the survival benefits of dual therapy. CONCLUSION: Here we successfully demonstrated that dual inhibition of Ang-2 and VEGFRs prolongs survival in preclinical GBM models by reducing the viable tumor burden, improving vascular normalization, and reshaping the tumor immune-microenvironment mediated by TAMs. This combination may represent a potential therapeutic strategy to overcome the limitations of anti-VEGF monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells. Citation Format: Zohreh Amoozgar, Teresa E. Peterson, Nathaniel D. Kirkpatrick, Yuhui Huang, Christian T. Farrar, Koen A. Marijt, Jonas Kloepper, Meenal Datta, Giorgio Seano, Keehoon Jung, Walid S. Kamoun, Trupti Vardam, Matija Snuderl, Jermaine Goveia, Sampurna Chatterjee, Ana Batista, Alona Muzikansky, Ching Ching Leow, Lei Xu, Tracy T. Batchelor, Dan G. Duda, Dai Fukumura, Jain K. Jain. Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-346.