The Traumatic Inoculation Process Affects TSPO Radioligand Uptake in Experimental Orthotopic Glioblastoma.

BACKGROUND: The translocator protein (TSPO) has been proven to have great potential as a target for the positron emission tomography (PET) imaging of glioblastoma. However, there is an ongoing debate about the potential various sources of the TSPO PET signal. This work investigates the impact of the inoculation-driven immune response on the PET signal in experimental orthotopic glioblastoma. METHODS: Serial [18F]GE-180 and O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET) PET scans were performed at day 7/8 and day 14/15 after the inoculation of GL261 mouse glioblastoma cells (n = 24) or saline (sham, n = 6) into the right striatum of immunocompetent C57BL/6 mice. An additional n = 25 sham mice underwent [18F]GE-180 PET and/or autoradiography (ARG) at days 7, 14, 21, 28, 35, 50 and 90 in order to monitor potential reactive processes that were solely related to the inoculation procedure. In vivo imaging results were directly compared to tissue-based analyses including ARG and immunohistochemistry. RESULTS: We found that the inoculation process represents an immunogenic event, which significantly contributes to TSPO radioligand uptake. [18F]GE-180 uptake in GL261-bearing mice surpassed [18F]FET uptake both in the extent and the intensity, e.g., mean target-to-background ratio (TBRmean) in PET at day 7/8: 1.22 for [18F]GE-180 vs. 1.04 for [18F]FET, p < 0.001. Sham mice showed increased [18F]GE-180 uptake at the inoculation channel, which, however, continuously decreased over time (e.g., TBRmean in PET: 1.20 at day 7 vs. 1.09 at day 35, p = 0.04). At the inoculation channel, the percentage of TSPO/IBA1 co-staining decreased, whereas TSPO/GFAP (glial fibrillary acidic protein) co-staining increased over time (p < 0.001). CONCLUSION: We identify the inoculation-driven immune response to be a relevant contributor to the PET signal and add a new aspect to consider for planning PET imaging studies in orthotopic glioblastoma models.

Pericytes are protective in experimental pneumococcal meningitis through regulating leukocyte infiltration and blood-brain barrier function.

BACKGROUND: Brain pericytes participate in the regulation of cerebral blood flow and the maintenance of blood-brain barrier integrity. Because of their perivascular localization, their receptor repertoire, and their potential ability to respond to inflammatory and infectious stimuli by producing various cytokines and chemokines, these cells are also thought to play an active role in the immune response to brain infections. This assumption is mainly supported by in vitro studies, investigations in in vivo disease models are largely missing. Here, we analysed the role of brain pericytes in pneumococcal meningitis, in vitro and in vivo in two animal models of pneumococcal meningitis. METHODS: Primary murine and human pericytes were stimulated with increasing concentrations of different serotypes of Streptococcus pneumoniae in the presence or absence of Toll-like receptor inhibitors and their cell viability and cytokine production were monitored. To gain insight into the role of pericytes in brain infection in vivo, we performed studies in a zebrafish embryo model of pneumococcal meningitis in which pericytes were pharmacologically depleted. Furthermore, we analyzed the impact of genetically induced pericyte ablation on disease progression, intracranial complications, and brain inflammation in an adult mouse model of this disease. RESULTS: Both murine and human pericytes reacted to pneumococcal exposure with the release of selected cytokines. This cytokine release is pneumolysin-dependent, TLR-dependent in murine (but not human) pericytes and can be significantly increased by macrophage-derived IL-1b. Pharmacological depletion of pericytes in zebrafish embryos resulted in increased cerebral edema and mortality due to pneumococcal meningitis. Correspondingly, in an adult mouse meningitis model, a more pronounced blood-brain barrier disruption and leukocyte infiltration, resulting in an unfavorable disease course, was observed following genetic pericyte ablation. The degree of leukocyte infiltration positively correlated with an upregulation of chemokine expression in the brains of pericyte-depleted mice. CONCLUSIONS: Our findings show that pericytes play a protective role in pneumococcal meningitis by impeding leukocyte migration and preventing blood-brain barrier breaching. Thus, preserving the integrity of the pericyte population has the potential as a new therapeutic strategy in pneumococcal meningitis.

Interleukin-6-controlled, mesenchymal stem cell-based sodium/iodide symporter gene therapy improves survival of glioblastoma-bearing mice.

New treatment strategies are urgently needed for glioblastoma (GBM)-a tumor resistant to standard-of-care treatment with a high risk of recurrence and extremely poor prognosis. Based on their intrinsic tumor tropism, adoptively applied mesenchymal stem cells (MSCs) can be harnessed to deliver the theranostic sodium/iodide symporter (NIS) deep into the tumor microenvironment. Interleukin-6 (IL-6) is a multifunctional, highly expressed cytokine in the GBM microenvironment including recruited MSCs. MSCs engineered to drive NIS expression in response to IL-6 promoter activation offer the possibility of a new tumor-targeted gene therapy approach of GBM. Therefore, MSCs were stably transfected with an NIS-expressing plasmid controlled by the human IL-6 promoter (IL-6-NIS-MSCs) and systemically applied in mice carrying orthotopic GBM. Enhanced radiotracer uptake by 18F-Tetrafluoroborate-PET/magnetic resonance imaging (MRI) was detected in tumors after IL-6-NIS-MSC application as compared with mice that received wild-type MSCs. Ex vivo analysis of tumors and non-target organs showed tumor-specific NIS protein expression. Subsequent 131I therapy after IL-6-NIS-MSC application resulted in significantly delayed tumor growth assessed by MRI and improved median survival up to 60% of GBM-bearing mice as compared with controls. In conclusion, the application of MSC-mediated NIS gene therapy focusing on IL-6 biology-induced NIS transgene expression represents a promising approach for GBM treatment.

Screening of Chemical Libraries Using Xenopus Embryos and Tadpoles for Phenotypic Drug Discovery.

Phenotypic drug discovery assesses the effect of small molecules on the phenotype of cells, tissues, or whole organisms without a priori knowledge of the target or pathway. Using vertebrate embryos instead of cell-based assays has the advantage that the screening of small molecules occurs in the context of the complex biology and physiology of the whole organism. Fish and amphibians are the only classes of vertebrates with free-living larvae amenable to high-throughput drug screening in multiwell dishes. For both animal classes, particularly zebrafish and Xenopus, husbandry requirements are straightforward, embryos can be obtained in large numbers, and they develop ex utero so their development can be monitored easily with a dissecting microscope. At 350 million years, the evolutionary distance between amphibians and humans is significantly shorter than that between fish and humans, which is estimated at 450 million years. This increases the likelihood that drugs discovered by screening in amphibian embryos will be active in humans. Here, we describe the basic protocol for the medium- to high-throughput screening of chemical libraries using embryos of the African clawed frog Xenopus laevis Bioactive compounds are identified by observing phenotypic changes in whole embryos and tadpoles. In addition to the discovery of compounds with novel bioactivities, the phenotypic screening protocol also allows for the identification of compounds with in vivo toxicity, eliminating early hits that are poor drug candidates. We also highlight important considerations for designing chemical screens, choosing chemical libraries, and performing secondary screens using whole mount in situ hybridization or immunostaining.

Mesenchymal Stem Cell-mediated Image-guided Sodium Iodide Symporter (NIS) Gene Therapy Improves Survival of Glioblastoma-bearing Mice.

PURPOSE: Mesenchymal stem cells (MSC) have emerged as cellular-based vehicles for the delivery of therapeutic genes in cancer therapy based on their inherent tumor-homing capability. As theranostic gene, the sodium iodide symporter (NIS) represents a successful target for noninvasive radionuclide-based imaging and therapy. In this study, we applied genetically engineered MSCs for tumor-targeted NIS gene transfer in experimental glioblastoma (GBM)-a tumor with an extremely poor prognosis. EXPERIMENTAL DESIGN: A syngeneic, immunocompetent GL261 GBM mouse model was established by subcutaneous and orthotopic implantation. Furthermore, a subcutaneous xenograft U87 model was used. Bone marrow-derived MSCs were stably transfected with a NIS-expressing plasmid driven by the constitutively active cytomegalovirus promoter (NIS-MSC). After multiple or single intravenous injection of NIS-MSCs, tumoral iodide uptake was monitored in vivo using 123I-scintigraphy or 124I-PET. Following validation of functional NIS expression, a therapy trial with 131I was performed on the basis of the most optimal application regime as seen by 124I-PET imaging in the orthotopic approach. RESULTS: A robust tumoral NIS-specific radionuclide accumulation was observed after NIS-MSC and radioiodide application by NIS-mediated in vivo imaging. NIS immunofluorescence staining of GBM and non-target tissues showed tumor-selective MSC homing along with NIS expression. Application of therapeutically effective 131I led to significantly delayed tumor growth and prolonged median survival after NIS-MSC treatment as compared with controls. CONCLUSIONS: A strong tumor-selective recruitment of systemically applied MSCs into GBM was found using NIS as reporter gene followed by successful therapeutic application of radioiodide demonstrating the potential use of NIS-based MSCs as therapy vehicles as a new GBM therapy approach.

Dual EGFR- and TfR-targeted gene transfer for sodium iodide symporter gene therapy of glioblastoma.

Sodium iodide symporter (NIS) gene transfer for active accumulation of iodide in tumor cells is a powerful theranostic strategy facilitating both diagnostic and therapeutic application of radioiodide. In glioblastoma (GBM), the blood-brain barrier (BBB) presents an additional delivery barrier for nucleic acid nanoparticles. In the present study, we designed dual-targeted NIS plasmid DNA complexes containing targeting ligands for the transferrin receptor (TfR) and the epidermal growth factor receptor (EGFR), thus providing the potential for active transport across the BBB followed by targeting of tumor cells. In vitro 125I transfection studies confirmed TfR- and EGFR-dependent transfection efficiency and NIS-specific iodide uptake of dual-targeted polyplexes. In vivo gene transfer in mice bearing orthotopic U87 GBM xenografts was assessed at 48 h after intravenous polyplex injection by positron emission tomography (PET) imaging using 18F-labeled tetrafluoroborate (TFB) as tracer. The tumoral 18F-TFB uptake of mice treated with dual-targeted polyplexes (0.56% ± 0.08% ID/mL) was significantly higher compared with mice treated with EGFR-mono-targeted (0.33% ± 0.03% ID/mL) or TfR-mono-targeted (0.27% ± 0.04% ID/mL) polyplexes. In therapy studies, application of 131I induced a superior therapeutic effect of the dual-targeted therapy, demonstrated by a significant delay in tumor growth and prolonged survival.

Deletion of endothelial α-parvin inhibits tumour angiogenesis, reduces tumour growth and induces tumour cell apoptosis.

Alpha-parvin (α-pv), an adaptor protein that mediates integrin-dependent cell-matrix interactions, is essential for endothelial cells migration and proliferation and is a key player in physiological angiogenesis. The role of α-pv in pathological angiogenesis is unknown. Here we demonstrate that endothelial α-pv is required for tumour angiogenesis. Using an inducible knockout approach in which the α-pv gene (Parva) was inactivated specifically in endothelial cells of brain tumour-bearing mice, we show that loss of endothelial α-pv results in reduced vessel density and decreased vascular complexity of the pathological neo-vasculature without affecting the structure of the brain vasculature around tumour. Reduced tumour vascularisation is associated with a significant increase in tumour cell apoptosis and a reduction in tumour volume. Together, our data show for the first time that endothelial α-pv is required for tumour vascularisation and tumour progression in vivo.

PSMA PET Imaging in Glioblastoma: A Preclinical Evaluation and Theranostic Outlook.

BACKGROUND: Prostate specific membrane antigen (PSMA) PET imaging has recently gained attention in glioblastoma (GBM) patients as a potential theranostic target for PSMA radioligand therapy. However, PSMA PET has not yet been established in a murine GBM model. Our goal was to investigate the potential of PSMA PET imaging in the syngeneic GL261 GBM model and to give an outlook regarding the potential of PMSA radioligand therapy in this model. METHODS: We performed an 18F-PSMA-1007 PET study in the orthotopic GL261 model (n=14 GBM, n=7 sham-operated mice) with imaging at day 4, 8, 11, 15, 18 and 22 post implantation. Time-activity-curves (TAC) were extracted from dynamic PET scans (0-120 min p. i.) in a subset of mice (n=4 GBM, n=3 sham-operated mice) to identify the optimal time frame for image analysis, and standardized-uptake-values (SUV) as well as tumor-to-background ratios (TBR) using contralateral normal brain as background were calculated in all mice. Additionally, computed tomography (CT), ex vivo and in vitro 18F-PSMA-1007 autoradiographies (ARG) were performed. RESULTS: TAC analysis of GBM mice revealed a plateau of TBR values after 40 min p. i. Therefore, a 30 min time frame between 40-70 min p. i. was chosen for PET quantification. At day 15 and later, GBM mice showed a discernible PSMA PET signal on the inoculation site, with highest TBRmean in GBM mice at day 18 (7.3 ± 1.3 vs. 1.6 ± 0.3 in shams; p=0.024). Ex vivo ARG confirmed high tracer signal in GBM compared to healthy background (TBRmean 26.9 ± 10.5 vs. 1.6 ± 0.7 in shams at day 18/22 post implantation; p=0.002). However, absolute uptake values in the GL261 tumor remained low (e.g., SUVmean 0.21 ± 0.04 g/ml at day 18) resulting in low ratios compared to dose-relevant organs (e.g., mean tumor-to-kidney ratio 1.5E-2 ± 0.5E-2). CONCLUSIONS: Although 18F-PSMA-1007 PET imaging of GL261 tumor-bearing mice is feasible and resulted in high TBRs, absolute tumoral uptake values remained low and hint to limited applicability of the GL261 model for PSMA-directed therapy studies. Further investigations are warranted to identify suitable models for preclinical evaluation of PSMA-targeted theranostic approaches in GBM.

Selective sodium iodide symporter (NIS) genetherapy of glioblastoma mediatedby EGFR-targeted lipopolyplexes.

Lipo-oligomers, post-functionalized with ligands to enhance targeting, represent promising new vehicles for the tumor-specific delivery of therapeutic genes such as the sodium iodide symporter (NIS). Due to its iodide trapping activity, NIS is a powerful theranostic tool for diagnostic imaging and the application of therapeutic radionuclides. 124I PET imaging allows non-invasive monitoring of the in vivo biodistribution of functional NIS expression, and application of 131I enables cytoreduction. In our experimental design, we used epidermal growth factor receptor (EGFR)-targeted polyplexes (GE11) initially characterized in vitro using 125I uptake assays. Mice bearing an orthotopic glioblastoma were treated subsequently with mono-dibenzocyclooctyne (DBCO)-PEG24-GE11/NIS or bisDBCO-PEG24-GE11/NIS, and 24-48 h later, 124I uptake was assessed by positron emission tomography (PET) imaging. The best-performing polyplex in the imaging studies was then selected for 131I therapy studies. The in vitro studies showed EGFR-dependent and NIS-specific transfection efficiency of the polyplexes. The injection of monoDBCO-PEG24-GE11/NIS polyplexes 48 h before 124I application was characterized to be the optimal regime in the imaging studies and was therefore used for an 131I therapy study, showing a significant decrease in tumor growth and a significant extension of survival in the therapy group. These studies demonstrate the potential of EGFR-targeted polyplex-mediated NIS gene therapy as a new strategy for the therapy of glioblastoma.

APLN/APLNR Signaling Controls Key Pathological Parameters of Glioblastoma.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. GBM-expansion depends on a dense vascular network and, coherently, GBMs are highly angiogenic. However, new intratumoral blood vessels are often aberrant with consequences for blood-flow and vascular barrier function. Hence, the delivery of chemotherapeutics into GBM can be compromised. Furthermore, leaky vessels support edema-formation, which can result in severe neurological deficits. The secreted signaling peptide Apelin (APLN) plays an important role in the formation of GBM blood vessels. Both APLN and the Apelin receptor (APLNR) are upregulated in GBM cells and control tumor cell invasiveness. Here we summarize the current evidence on the role of APLN/APLNR signaling during brain tumor pathology. We show that targeting APLN/APLNR can induce anti-angiogenic effects in GBM and simultaneously blunt GBM cell infiltration. In addition, we discuss how manipulation of APLN/APLNR signaling in GBM leads to the normalization of tumor vessels and thereby supports chemotherapy, reduces edema, and improves anti-tumorigenic immune reactions. Hence, therapeutic targeting of APLN/APLNR signaling offers an interesting option to address different pathological hallmarks of GBM.

Comparing Tumor Cell Invasion and Myeloid Cell Composition in Compatible Primary and Relapsing Glioblastoma.

Glioblastoma (GBM) recurrence after treatment is almost inevitable but addressing this issue with adequate preclinical models has remained challenging. Here, we introduce a GBM mouse model allowing non-invasive and scalable de-bulking of a tumor mass located deeply in the brain, which can be combined with conventional therapeutic approaches. Strong reduction of the GBM volume is achieved after pharmacologically inducing a tumor-specific cell death mechanism. This is followed by GBM re-growth over a predictable timeframe. Pharmacological de-bulking followed by tumor relapse was accomplished with an orthotopic mouse glioma model. Relapsing experimental tumors recapitulated pathological features often observed in recurrent human GBM, like increased invasiveness or altered immune cell composition. Orthotopic implantation of GBM cells originating from biopsies of one patient at initial or follow-up treatment reproduced these findings. Interestingly, relapsing GBM of both models contained a much higher ratio of monocyte-derived macrophages (MDM) versus microglia than primary GBM. This was not altered when combining pharmacological de-bulking with invasive surgery. We interpret that factors released from viable primary GBM cells preferentially attract microglia whereas relapsing tumors preponderantly release chemoattractants for MDM. All in all, this relapse model has the capacity to provide novel insights into clinically highly relevant aspects of GBM treatment.

Cannabidiol converts NF-κB into a tumor suppressor in glioblastoma with defined antioxidative properties.

BACKGROUND: The transcription factor NF-κB drives neoplastic progression of many cancers including primary brain tumors (glioblastoma [GBM]). Precise therapeutic modulation of NF-κB activity can suppress central oncogenic signaling pathways in GBM, but clinically applicable compounds to achieve this goal have remained elusive. METHODS: In a pharmacogenomics study with a panel of transgenic glioma cells, we observed that NF-κB can be converted into a tumor suppressor by the non-psychotropic cannabinoid cannabidiol (CBD). Subsequently, we investigated the anti-tumor effects of CBD, which is used as an anticonvulsive drug (Epidiolex) in pediatric neurology, in a larger set of human primary GBM stem-like cells (hGSC). For this study, we performed pharmacological assays, gene expression profiling, biochemical, and cell-biological experiments. We validated our findings using orthotopic in vivo models and bioinformatics analysis of human GBM datasets. RESULTS: We found that CBD promotes DNA binding of the NF-κB subunit RELA and simultaneously prevents RELA phosphorylation on serine-311, a key residue that permits genetic transactivation. Strikingly, sustained DNA binding by RELA-lacking phospho-serine 311 was found to mediate hGSC cytotoxicity. Widespread sensitivity to CBD was observed in a cohort of hGSC defined by low levels of reactive oxygen species (ROS), while high ROS content in other tumors blocked CBD-induced hGSC death. Consequently, ROS levels served as a predictive biomarker for CBD-sensitive tumors. CONCLUSIONS: This evidence demonstrates how a clinically approved drug can convert NF-κB into a tumor suppressor and suggests a promising repurposing option for GBM therapy.

TAMEP are brain tumor parenchymal cells controlling neoplastic angiogenesis and progression.

Aggressive brain tumors like glioblastoma depend on support by their local environment and subsets of tumor parenchymal cells may promote specific phases of disease progression. We investigated the glioblastoma microenvironment with transgenic lineage-tracing models, intravital imaging, single-cell transcriptomics, immunofluorescence analysis as well as histopathology and characterized a previously unacknowledged population of tumor-associated cells with a myeloid-like expression profile (TAMEP) that transiently appeared during glioblastoma growth. TAMEP of mice and humans were identified with specific markers. Notably, TAMEP did not derive from microglia or peripheral monocytes but were generated by a fraction of CNS-resident, SOX2-positive progenitors. Abrogation of this progenitor cell population, by conditional Sox2-knockout, drastically reduced glioblastoma vascularization and size. Hence, TAMEP emerge as a tumor parenchymal component with a strong impact on glioblastoma progression.

Apelin Controls Angiogenesis-Dependent Glioblastoma Growth.

Glioblastoma (GBM) present with an abundant and aberrant tumor neo-vasculature. While rapid growth of solid tumors depends on the initiation of tumor angiogenesis, GBM also progress by infiltrative growth and vascular co-option. The angiogenic factor apelin (APLN) and its receptor (APLNR) are upregulated in GBM patient samples as compared to normal brain tissue. Here, we studied the role of apelin/APLNR signaling in GBM angiogenesis and growth. By functional analysis of apelin in orthotopic GBM mouse models, we found that apelin/APLNR signaling is required for in vivo tumor angiogenesis. Knockdown of tumor cell-derived APLN massively reduced the tumor vasculature. Additional loss of the apelin signal in endothelial tip cells using the APLN-knockout (KO) mouse led to a further reduction of GBM angiogenesis. Direct infusion of the bioactive peptide apelin-13 rescued the vascular loss-of-function phenotype specifically. In addition, APLN depletion massively reduced angiogenesis-dependent tumor growth. Consequently, survival of GBM-bearing mice was significantly increased when APLN expression was missing in the brain tumor microenvironment. Thus, we suggest that targeting vascular apelin may serve as an alternative strategy for anti-angiogenesis in GBM.

Glioblastoma Exhibits Inter-Individual Heterogeneity of TSPO and LAT1 Expression in Neoplastic and Parenchymal Cells.

Molecular imaging is essential for diagnosis and treatment planning for glioblastoma patients. Positron emission tomography (PET) with tracers for the detection of the solute carrier family 7 member 5 (SLC7A5; also known as the amino acid transporter light chain L system, LAT1) and for the mitochondrial translocator protein (TSPO) is successfully used to provide additional information on tumor volume and prognosis. The current approaches for TSPO-PET and the visualization of tracer ([18F] Fluoroethyltyrosine, FET) uptake by LAT1 (FET-PET) do not yet exploit the full diagnostic potential of these molecular imaging techniques. Therefore, we investigated the expression of TSPO and LAT1 in patient glioblastoma (GBM) samples, as well as in various GBM mouse models representing patient GBMs of different genetic subtypes. By immunohistochemistry, we found that TSPO and LAT1 are upregulated in human GBM samples compared to normal brain tissue. Next, we orthotopically implanted patient-derived GBM cells, as well as genetically engineered murine GBM cells, representing different genetic subtypes of the disease. To determine TSPO and LAT1 expression, we performed immunofluorescence staining. We found that both TSPO and LAT1 expression was increased in tumor regions of the implanted human or murine GBM cells when compared to the neighboring mouse brain tissue. While LAT1 was largely restricted to tumor cells, we found that TSPO was also expressed by microglia, tumor-associated macrophages, endothelial cells, and pericytes. The Cancer Genome Atlas (TCGA)-data analysis corroborates the upregulation of TSPO in a bigger cohort of GBM patient samples compared to tumor-free brain tissue. In addition, AIF1 (the gene encoding for the myeloid cell marker Iba1) was also upregulated in GBM compared to the control. Interestingly, TSPO, as well as AIF1, showed significantly different expression levels depending on the GBM genetic subtype, with the highest expression being exhibited in the mesenchymal subtype. High TSPO and AIF1 expression also correlated with a significant decrease in patient survival compared to low expression. In line with this finding, the expression levels for TSPO and AIF1 were also significantly higher in (isocitrate-dehydrogenase wild-type) IDHWT compared to IDH mutant (IDHMUT) GBM. LAT1 expression, on the other hand, was not different among the individual GBM subtypes. Therefore, we could conclude that FET- and TSPO-PET confer different information on pathological features based on different genetic GBM subtypes and may thus help in planning individualized strategies for brain tumor therapy in the future. A combination of TSPO-PET and FET-PET could be a promising way to visualize tumor-associated myeloid cells and select patients for treatment strategies targeting the myeloid compartment.

Targeting APLN/APLNR Improves Antiangiogenic Efficiency and Blunts Proinvasive Side Effects of VEGFA/VEGFR2 Blockade in Glioblastoma.

Antiangiogenic therapy of glioblastoma (GBM) with bevacizumab, a VEGFA-blocking antibody, may accelerate tumor cell invasion and induce alternative angiogenic pathways. Here we investigate the roles of the proangiogenic apelin receptor APLNR and its cognate ligand apelin in VEGFA/VEGFR2 antiangiogenic therapy against distinct subtypes of GBM. In proneural GBM, apelin levels were downregulated by VEGFA or VEGFR2 blockade. A central role for apelin/APLNR in controlling GBM vascularization was corroborated in a serial implantation model of the angiogenic switch that occurs in human GBM. Apelin and APLNR are broadly expressed in human GBM, and knockdown or knockout of APLN in orthotopic models of proneural or classical GBM subtypes significantly reduced GBM vascularization compared with controls. However, reduction in apelin expression led to accelerated GBM cell invasion. Analysis of stereotactic GBM biopsies from patients as well as from in vitro and in vivo experiments revealed increased dissemination of APLNR-positive tumor cells when apelin levels were reduced. Application of apelin-F13A, a mutant APLNR ligand, blocked tumor angiogenesis and GBM cell invasion. Furthermore, cotargeting VEGFR2 and APLNR synergistically improved survival of mice bearing proneural GBM. In summary, we show that apelin/APLNR signaling controls GBM angiogenesis and invasion and that both pathologic features are blunted by apelin-F13A. We suggest that apelin-F13A can improve the efficiency and reduce the side effects of established antiangiogenic treatments for distinct GBM subtypes. SIGNIFICANCE: Pharmacologic targeting of the APLNR acts synergistically with established antiangiogenic treatments in glioblastoma and blunts therapy resistance to current strategies for antiangiogenesis.See related commentary by Amoozgar et al., p. 2104.

CNS myeloid cells critically regulate heat hyperalgesia.

Activation of non-neuronal microglia is thought to play a causal role in spinal processing of neuropathic pain. To specifically investigate microglia-mediated effects in a model of neuropathic pain and overcome the methodological limitations of previous approaches exploring microglia function upon nerve injury, we selectively ablated resident microglia by intracerebroventricular ganciclovir infusion into male CD11b-HSVTK-transgenic mice, which was followed by a rapid, complete, and persistent (23 weeks) repopulation of the CNS by peripheral myeloid cells. In repopulated mice that underwent sciatic nerve injury, we observed a normal response to mechanical stimuli, but an absence of thermal hypersensitivity ipsilateral to the injured nerve. Furthermore, we found that neuronal expression of calcitonin gene-related peptide (CGRP), which is a marker of neurons essential for heat responses, was diminished in the dorsal horn of the spinal cord in repopulated mice. These findings identify distinct mechanisms for heat and mechanical hypersensitivity and highlight a crucial contribution of CNS myeloid cells in the facilitation of noxious heat.

Decreased demand for olfactory periglomerular cells impacts on neural precursor cell viability in the rostral migratory stream.

The subventricular zone (SVZ) provides a constant supply of new neurons to the olfactory bulb (OB). Different studies have investigated the role of olfactory sensory input to neural precursor cell (NPC) turnover in the SVZ but it was not addressed if a reduced demand specifically for periglomerular neurons impacts on NPC-traits in the rostral migratory stream (RMS). We here report that membrane type-1 matrix metalloproteinase (MT1-MMP) deficient mice have reduced complexity of the nasal turbinates, decreased sensory innervation of the OB, reduced numbers of olfactory glomeruli and reduced OB-size without alterations in SVZ neurogenesis. Large parts of the RMS were fully preserved in MT1-MMP-deficient mice, but we detected an increase in cell death-levels and a decrease in SVZ-derived neuroblasts in the distal RMS, as compared to controls. BrdU-tracking experiments showed that homing of NPCs specifically to the glomerular layer was reduced in MT1-MMP-deficient mice in contrast to controls while numbers of tracked cells remained equal in other OB-layers throughout all experimental groups. Altogether, our data show the demand for olfactory interneurons in the glomerular layer modulates cell turnover in the RMS, but has no impact on subventricular neurogenesis.

Monitoring of Tumor Growth with [(18)F]-FET PET in a Mouse Model of Glioblastoma: SUV Measurements and Volumetric Approaches.

Noninvasive tumor growth monitoring is of particular interest for the evaluation of experimental glioma therapies. This study investigates the potential of positron emission tomography (PET) using O-(2-(18)F-fluoroethyl)-L-tyrosine ([(18)F]-FET) to determine tumor growth in a murine glioblastoma (GBM) model-including estimation of the biological tumor volume (BTV), which has hitherto not been investigated in the pre-clinical context. Fifteen GBM-bearing mice (GL261) and six control mice (shams) were investigated during 5 weeks by PET followed by autoradiographic and histological assessments. [(18)F]-FET PET was quantitated by calculation of maximum and mean standardized uptake values within a universal volume-of-interest (VOI) corrected for healthy background (SUVmax/BG, SUVmean/BG). A partial volume effect correction (PVEC) was applied in comparison to ex vivo autoradiography. BTVs obtained by predefined thresholds for VOI definition (SUV/BG: ≥1.4; ≥1.6; ≥1.8; ≥2.0) were compared to the histologically assessed tumor volume (n = 8). Finally, individual "optimal" thresholds for BTV definition best reflecting the histology were determined. In GBM mice SUVmax/BG and SUVmean/BG clearly increased with time, however at high inter-animal variability. No relevant [(18)F]-FET uptake was observed in shams. PVEC recovered signal loss of SUVmean/BG assessment in relation to autoradiography. BTV as estimated by predefined thresholds strongly differed from the histology volume. Strikingly, the individual "optimal" thresholds for BTV assessment correlated highly with SUVmax/BG (ρ = 0.97, p < 0.001), allowing SUVmax/BG-based calculation of individual thresholds. The method was verified by a subsequent validation study (n = 15, ρ = 0.88, p < 0.01) leading to extensively higher agreement of BTV estimations when compared to histology in contrast to predefined thresholds. [(18)F]-FET PET with standard SUV measurements is feasible for glioma imaging in the GBM mouse model. PVEC is beneficial to improve accuracy of [(18)F]-FET PET SUV quantification. Although SUVmax/BG and SUVmean/BG increase during the disease course, these parameters do not correlate with the respective tumor size. For the first time, we propose a histology-verified method allowing appropriate individual BTV estimation for volumetric in vivo monitoring of tumor growth with [(18)F]-FET PET and show that standardized thresholds from routine clinical practice seem to be inappropriate for BTV estimation in the GBM mouse model.

Wittig derivatization of sesquiterpenoid polygodial leads to cytostatic agents with activity against drug resistant cancer cells and capable of pyrrolylation of primary amines.

Many types of cancer, including glioma, melanoma, non-small cell lung cancer (NSCLC), among others, are resistant to proapoptotic stimuli and thus poorly responsive to current therapies based on the induction of apoptosis in cancer cells. The current investigation describes the synthesis and anticancer evaluation of unique C12-Wittig derivatives of polygodial, a sesquiterpenoid dialdehyde isolated from Persicaria hydropiper (L.) Delabre. These compounds were found to undergo an unprecedented pyrrole formation with primary amines in a chemical model system, a reaction that could be relevant in the biological environment and lead to the pyrrolation of lysine residues in the target proteins. The anticancer evaluation of these compounds revealed their promising activity against cancer cells displaying various forms of drug resistance, including resistance to proapoptotic agents. Mechanistic studies indicated that compared to the parent polygodial, which displays fixative general cytotoxic action against human cells, the C12-Wittig derivatives exerted their antiproliferative action mainly through cytostatic effects explaining their activity against apoptosis-resistant cancer cells. The possibility for an intriguing covalent modification of proteins through a novel pyrrole formation reaction, as well as useful activities against drug resistant cancer cells, make the described polygodial-derived chemical scaffold an interesting new chemotype warranting thorough investigation.