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Boron neutron capture therapy (BNCT) is an encouraging therapeutic modality for cancer treatment. Prostate-specific membrane antigen (PSMA) is a cell membrane protein that is abundantly overexpressed in prostate cancer and can be targeted with radioligand therapies to stimulate clinical responses in patients. In principle, a spatially targeted neutron beam together with specifically targeted PSMA ligands could enable prostate cancer-targeted BNCT. Thus, we developed and tested PSMA-targeted poly(lactide- co -glycolide)-block-poly(ethylene glycol) (PLGA- b -PEG) nanoparticles (NPs) loaded with carborane and tethered to the radiometal chelator deferoxamine B (DFB) for simultaneous positron emission tomography (PET) imaging and selective delivery of boron to prostate cancer. Monomeric PLGA- b -PEGs were covalently functionalized with either DFB or the PSMA ligand ACUPA. Different nanoparticle formulations were generated by nanoemulsification of the corresponding unmodified and DFB- or ACUPA-modified monomers in varying percent fractions. The nanoparticles were efficiently labeled with 89 Zr and were subjected to in vitro and in vivo evaluation. The optimized DFB(25)ACUPA(75) NPs exhibited strong in vitro binding to PSMA in direct binding and competition radioligand binding assays in PSMA(+) PC3-Pip cells. [ 89 Zr]DFB(25) NPs and [ 89 Zr]DFB(25)ACUPA(75) NPs were injected to mice with bilateral PSMA(-) PC3-Flu and PSMA(+) PC3-Pip dual xenografts. The NPs demonstrated twofold superior accumulation in PC3-Pip tumors to that of PC3-Flu tumors with a tumor/blood ratio of 25; however, no substantial effect of the ACUPA ligands was detected. Moreover, fast release of carborane from the NPs was observed, resulting in a low boron delivery to tumors in vivo . In summary, these data demonstrate the synthesis, characterization, and initial biological assessment of PSMA-targeted, carborane-loaded PLGA- b -PEG nanoparticles and establish the foundation for future efforts to enable their best use in vivo .

The fate of nanocarrier materials at the cellular level constitutes a critical checkpoint in the development of effective nanomedicines, determining whether tissue level accumulation results in therapeutic benefit. The cytotoxicity and cell internalization of ∼18 nm 3-helix micelle (3HM) loaded with doxorubicin (DOX) were analyzed in patient-derived glioblastoma (GBM) cells in vitro . The half-maximal inhibitory concentration (IC 50 ) of 3HM-DOX increased to 6.2 μg/mL from <0.5 μg/mL for free DOX in patient-derived GBM6 cells, to 15.0 μg/mL from 6.5 μg/mL in U87MG cells, and to 21.5 μg/mL from ∼0.5 μg/mL in LN229 cells. Modeling analysis of previous 3HM biodistribution results predicts that these cytotoxic concentrations are achievable with intravenous injection in rodent GBM models. 3HM-DOX formulations were internalized intact and underwent intracellular trafficking distinct from free DOX. 3HM was quantified to have an internalization half-life of 12.6 h in GBM6 cells, significantly longer than that reported for some liposome and polymer systems. 3HM was found to traffic through active endocytic processes, with clathrin-mediated endocytosis being the most involved of the pathways studied. Inhibition studies suggest substantial involvement of receptor recognition in 3HM uptake. As the 3HM surface is PEG-ylated with no targeting functionalities, protein corona-cell surface interactions, such as the apolipoprotein-low-density lipoprotein receptor, are expected to initiate internalization. The present work gives insights into the cytotoxicity, pharmacodynamics, and cellular interactions of 3HM and 3HM-DOX relevant for ongoing preclinical studies. This work also contributes to efforts to develop predictive mathematical models tracking the accumulation and biodistribution kinetics at a systemic level.

The treatment of glioblastoma (GBM) remains challenging in part due to the presence of stem-like tumor-propagating cells that are resistant to standard therapies consisting of radiation and temozolomide. Among the novel and targeted agents under evaluation for the treatment of GBM are BRAF/MAPK inhibitors, but their effects on tumor-propagating cells are unclear. Here, we characterized the behaviors of CD133(+) tumor-propagating cells isolated from primary GBM cell lines. We show that CD133(+) cells exhibited decreased sensitivity to the antiproliferative effects of BRAF/MAPK inhibition compared to CD133(-) cells. Furthermore, CD133(+) cells exhibited an extended G2-M phase and increased polarized asymmetric cell divisions. At the molecular level, we observed that polo-like kinase (PLK) 1 activity was elevated in CD133(+) cells, prompting our investigation of BRAF/PLK1 combination treatment effects in an orthotopic GBM xenograft model. Combined inhibition of BRAF and PLK1 resulted in significantly greater antiproliferative and proapoptotic effects beyond those achieved by monotherapy (P < 0.05). We propose that PLK1 activity controls a polarity checkpoint and compensates for BRAF/MAPK inhibition in CD133(+) cells, suggesting the need for concurrent PLK1 inhibition to improve antitumor activity against a therapy-resistant cell compartment., (©2015 American Association for Cancer Research.)

This report describes results from our analysis of the activity and biodistribution of a novel pan-ERBB inhibitor, NT113, when used in treating mice with intracranial glioblastoma (GBM) xenografts. Approaches used in this investigation include: bioluminescence imaging (BLI) for monitoring intracranial tumor growth and response to therapy; determination of survival benefit from treatment; analysis of tumor IHC reactivity for indication of treatment effect on proliferation and apoptotic response; Western blot analysis for determination of effects of treatment on ERBB and ERBB signaling mediator activation; and high-performance liquid chromatography for determination of NT113 concentration in tissue extracts from animals receiving oral administration of inhibitor. Our results show that NT113 is active against GBM xenografts in which wild-type EGFR or EGFRvIII is highly expressed. In experiments including lapatinib and/or erlotinib, NT113 treatment was associated with the most substantial improvement in survival, as well as the most substantial tumor growth inhibition, as indicated by BLI and IHC results. Western blot analysis results indicated that NT113 has inhibitory activity, both in vivo and in vitro, on ERBB family member phosphorylation, as well as on the phosphorylation of downstream signaling mediator Akt. Results from the analysis of animal tissues revealed significantly higher NT113 normal brain-to-plasma and intracranial tumor-to-plasma ratios for NT113, relative to erlotinib, indicating superior NT113 partitioning to intracranial tissue compartments. These data provide a strong rationale for the clinical investigation of NT113, a novel ERBB inhibitor, in treating patients with GBM., (©2014 American Association for Cancer Research.)

Purpose: Cidofovir (CDV) is an U.S. Food and Drug Administration (FDA)-approved nucleoside antiviral agent used to treat severe human cytomegalovirus (HCMV) infection. Until now, no clear therapeutic effects of CDV have been reported outside of the setting of viral infection, including a potential role for CDV as an antineoplastic agent for the treatment of brain tumors., Experimental Design: We investigated the cytotoxicity of CDV against the glioblastoma cells, U87MG and primary SF7796, both in vitro and in vivo, using an intracranial xenograft model. Standard techniques for cell culturing, immunohistochemistry, Western blotting, and real-time PCR were employed. The survival of athymic mice (n = 8-10 per group) bearing glioblastoma tumors, treated with CDV alone or in combination with radiation, was analyzed by the Kaplan-Meier method and evaluated with a two-sided log-rank test., Results: CDV possesses potent antineoplastic activity against HCMV-infected glioblastoma cells. This activity is associated with the inhibition of HCMV gene expression and with activation of cellular apoptosis. Surprisingly, we also determined that CDV induces glioblastoma cell death in the absence of HCMV infection. CDV is incorporated into tumor cell DNA, which promotes double-stranded DNA breaks and induces apoptosis. In the setting of ionizing radiotherapy, the standard of care for glioblastoma in humans, CDV augments radiation-induced DNA damage and, further, promotes tumor cell death. Combination therapy with CDV and radiotherapy significantly extended the survival of mice bearing intracranial glioblastoma tumors., Conclusion: We have identified a novel antiglioma property of the FDA-approved drug CDV, which heightens the cytotoxic effect of radiotherapy, the standard of care therapy for glioblastoma., (©2013 AACR.)

Purpose: The aim of this study was to identify conserved pharmacodynamic and potential predictive biomarkers of response to anti-VEGF therapy using gene expression profiling in preclinical tumor models and in patients., Experimental Design: Surrogate markers of VEGF inhibition [VEGF-dependent genes or VEGF-dependent vasculature (VDV)] were identified by profiling gene expression changes induced in response to VEGF blockade in preclinical tumor models and in human biopsies from patients treated with anti-VEGF monoclonal antibodies. The potential value of VDV genes as candidate predictive biomarkers was tested by correlating high or low VDV gene expression levels in pretreatment clinical samples with the subsequent clinical efficacy of bevacizumab (anti-VEGF)-containing therapy., Results: We show that VDV genes, including direct and more distal VEGF downstream endothelial targets, enable detection of VEGF signaling inhibition in mouse tumor models and human tumor biopsies. Retrospective analyses of clinical trial data indicate that patients with higher VDV expression in pretreatment tumor samples exhibited improved clinical outcome when treated with bevacizumab-containing therapies., Conclusions: In this work, we identified surrogate markers (VDV genes) for in vivo VEGF signaling in tumors and showed clinical data supporting a correlation between pretreatment VEGF bioactivity and the subsequent efficacy of anti-VEGF therapy. We propose that VDV genes are candidate biomarkers with the potential to aid the selection of novel indications as well as patients likely to respond to anti-VEGF therapy. The data presented here define a diagnostic biomarker hypothesis based on translational research that warrants further evaluation in additional retrospective and prospective trials., (©2013 AACR.)

The prognosis for diffuse infiltrating pontine gliomas (DIPG) remains extremely poor, with the majority of patients surviving less than 2 years. Here, we have adapted standard xenograft techniques to study glioma growth in the mouse brainstem, and have utilized the mouse model for studying a relevant therapeutic for treating DIPGs. bioluminescence imaging monitoring revealed a progressive increase in signal following the injection of either of two tumor cell types into the brainstem. Mice with orthotopic GS2 tumors, and receiving a single 100 mg/kg dose of temozolomide showed a lengthy period of decreased tumor luminescence, with substantially increased survival relative to untreated mice (P < 0.001). A small molecule inhibitor that targets cdk4/6 was used to test AM-38 brainstem xenograft response to treatment. Drug treatment resulted in delayed tumor growth, and significantly extended survival. Our results demonstrate the feasibility of using an orthotopic brainstem tumor model in athymic mice, and for application to testing therapeutic agents in treating DIPG.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in humans. Because the phosphatidylinositol-3-kinase (PI3K) signaling pathway is activated in more than 88% of GBM, new drugs which target this pathway, such as the mTOR inhibitor Everolimus, are currently in clinical trials. Early tumor response to molecularly targeted treatments remains challenging to assess non-invasively, because it is often associated with tumor stasis or slower tumor growth. Innovative neuroimaging methods are therefore critically needed to provide metabolic or functional information that is indicative of targeted therapeutic action at early time points during the course of treatment. In this study, we demonstrated for the first time that hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) can be used on a clinical MR system to monitor early metabolic response of orthotopic GBM tumors to Everolimus treatment through measurement of the HP lactate-to-pyruvate ratios. The study was performed on a highly invasive non-enhancing orthotopic GBM tumor model in rats (GS-2 tumors), which replicates many fundamental features of human GBM tumors. Seven days after initiation of treatment there was a significant drop in the HP lactate-to-pyruvate ratio from the tumor tissue in treated animals relative to day 0 (67%±27% decrease). In the control group, no significant changes in the HP lactate-to-pyruvate ratios were observed. Importantly, at the 7 day time point, conventional MR imaging (MRI) was unable to detect a significant difference in tumor size between control and treated groups. Inhibition of tumor growth by conventional MRI was observed from day 15 of treatment. This implies that the decrease in the HP lactate-to-pyruvate ratio could be detected before any treatment-induced inhibition of tumor growth. Using immunohistochemical staining to further examine tumor response to treatment, we found that the decrease in the HP lactate-to-pyruvate ratio was associated with a drop in expression of lactate dehydrogenase, the enzyme that catalyzes pyruvate to lactate conversion. Also evident was decreased staining for carbonic anhydrase IX (CA-IX), an indicator of hypoxia-inducible factor 1α (HIF-1α) activity, which, in turn, regulates expression of lactate dehydrogenase. To our knowledge, this study is the first report of the use of HP 13C MRSI at a clinical field strength to monitor GBM response to molecularly targeted treatments. It highlights the potential of HP lactate-to-pyruvate ratio as an early biomarker of response, thereby supporting further investigation of this non-invasive imaging approach for eventual clinical application., (Published by Elsevier Inc.)

Telomerase is a ribonucleoprotein complex that elongates telomeric DNA and appears to play an important role in cellular immortalization of cancers. Because telomerase is expressed in the vast majority of malignant gliomas but not in normal brain tissues, it is a logical target for gliomaspecific therapy. The telomerase inhibitor GRN163, a 13-mer oligonucleotide N3'-->P5' thio-phosphoramidate (Geron Corporation, Menlo Park, Calif.), is complementary to the template region of the human telomerase RNA subunit hTR. When athymic mice bearing U-251 MG human brain tumor xenografts in their flanks were treated intratumorally with GRN163, a significant growth delay in tumor size was observed (P < 0.01 in all groups) as compared to the tumor size in mice receiving a mismatched oligonucleotide or the carrier alone. We also investigated biodistribution of the drug in vivo in an intracerebral rat brain-tumor model. Fluorescein-labeled GRN163 was loaded into an osmotic minipump and infused directly into U-251 MG brain tumors over 7 days. Examination of the brains revealed that GRN163 was present in tumor cells at all time points studied. When GRN163 was infused into intracerebral U-251 MG tumors shortly after their implantation, it prevented their establishment and growth. Lastly, when rats with larger intracerebral tumors were treated with the inhibitor, GRN163 increased animal survival times. Our results demonstrate that the antitelomerase agent GRN163 inhibits growth of glioblastoma in vivo, exhibits favorable intracerebral tumor uptake properties, and prevents the growth of intracerebral tumors. These findings support further development of this compound as a potential anticancer agent.

BackgroundLiposomal drug packaging is well established as an effective means for increasing drug half-life, sustaining drug activity, and increasing drug efficacy, whether administered locally or distally to the site of disease. However, information regarding the relative effectiveness of peripheral (distal) versus local administration of liposomal therapeutics is limited. This issue is of importance with respect to the treatment of central nervous system cancer, for which the blood-brain barrier presents a significant challenge in achieving sufficient drug concentration in tumors to provide treatment benefit for patients.MethodsWe compared the anti-tumor activity and efficacy of a nanoliposomal formulation of irinotecan when delivered peripherally by vascular route with intratumoral administration by convection-enhanced delivery (CED) for treating intracranial glioblastoma xenografts in athymic mice.ResultsOur results show significantly greater anti-tumor activity and survival benefit from CED of nanoliposomal irinotecan. In 2 of 3 efficacy experiments, there were animal subjects that experienced apparent cure of tumor from local administration of therapy, as indicated by a lack of detectable intracranial tumor through bioluminescence imaging and histopathologic analysis. Results from investigating the effectiveness of combination therapy with nanoliposomal irinotecan plus radiation revealed that CED administration of irinotecan plus radiation conferred greater survival benefit than did irinotecan or radiation monotherapy and also when compared with radiation plus vascularly administered irinotecan.ConclusionsOur results indicate that liposomal formulation plus direct intratumoral administration of therapeutic are important for maximizing the anti-tumor effects of irinotecan and support clinical trial evaluation of this therapeutic plus route of administration combination.

Despite the use of radiation and chemotherapy, the prognosis for children with diffuse brainstem gliomas is extremely poor. There is a need for relevant brainstem tumor models that can be used to test new therapeutic agents and delivery systems in pre-clinical studies. We report the development of a brainstem-tumor model in rats and the application of bioluminescence imaging (BLI) for monitoring tumor growth and response to therapy as part of this model. Luciferase-modified human glioblastoma cells from five different tumor cell sources (either cell lines or serially-passaged xenografts) were implanted into the pontine tegmentum of athymic rats using an implantable guide-screw system. Tumor growth was monitored by BLI and tumor volume was calculated by three-dimensional measurements from serial histopathologic sections. To evaluate if this model would allow detection of therapeutic response, rats bearing brainstem U-87 MG or GS2 glioblastoma xenografts were treated with the DNA methylating agent temozolomide (TMZ). For each of the tumor cell sources tested, BLI monitoring revealed progressive tumor growth in all animals, and symptoms caused by tumor burden were evident 26-29 days after implantation of U-87 MG, U-251 MG, GBM6, and GBM14 cells, and 37-47 days after implantation of GS2 cells. Histopathologic analysis revealed tumor growth within the pons in all rats and BLI correlated quantitatively with tumor volume. Variable infiltration was evident among the different tumors, with GS2 tumor cells exhibiting the greatest degree of infiltration. TMZ treatment groups were included for experiments involving U-87 MG and GS2 cells, and in each case TMZ delayed tumor growth, as indicated by BLI monitoring, and significantly extended survival of animal subjects. Our results demonstrate the development of a brainstem tumor model in athymic rats, in which tumor growth and response to therapy can be accurately monitored by BLI. This model is well suited for pre-clinical testing of therapeutics that are being considered for treatment of patients with brainstem tumors.

Abstract: A retro-convection enhanced delivery (R-CED) method has been developed to improve the entry of intravenously administered therapeutics within solid brain tumors. R-CED uses an osmotic gradient to withdraw brain interstitial fluid (ISF) in a controlled manner via an implanted microdialysis catheter. Withdrawal of ISF increases the local tissue specific gravity in normal brain and increases twofold the extravasation of intravenous Evans blue (EB) albumin in normal brain and in an orthotopic 9L tumor. R-CED also increases the extravasation of 70 nm fluorescent liposomes fivefold in the 9L tumor. Thus the transmembrane osmotic gradient induces movement of substances in the blood into the tissue parenchyma. Following probe removal, the magnitude of the R-CED effect on EB-albumin extravasation decreases to control values within 1.5 h in normal brain; however, the effect persists beyond 6 h in the tumor. There was no evidence of histologic damage to the neurons at either 6 h or 2 weeks after R-CED. These studies establish the feasibility of applying R-CED to increase the distribution of systemically administered drugs in both the normal tissue–tumor margin as well as in the central tumor core, holding forth the possibility of improved antitumor drug efficacy. [Copyright &y& Elsevier]