Supplementary Figure 1: Radioiodide uptake of GL261 brain tumors after systemic MSC-mediated NIS reporter gene delivery in comparison to thyroid as endogenous NIS-expressing organ assessed by 124I PET/CT imaging. Results are expressed as mean value ± SEM.Supplementary Figure 2: Ex vivo analysis of non-target organs after NIS-MSC gene delivery. NIS (A) and Neomycin (B; selection marker of NIS-MSCs) mRNA expression was detected by qPCR in liver, spleen, kidney and lung after NIS-MSC application and showed no expression above the background level of untreated tumors (which was arbitrarily set to one; NIS mRNA levels of untreated ΔΔCt = 0.0003 and Neomycin mRNA levels of untreated ΔΔCt = 0.002). Data are represented as mean-fold change ± SEM (*p<0.05).

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.

Abstract The DNA damage response (DDR) and the blood tumor barrier (BTB) restrict chemotherapeutic success for primary brain tumors like glioblastoma (GBM). Coherently, GBM almost invariably relapse with fatal outcome. We here show that interaction of GBM and myeloid cells simultaneously induces chemoresistance on the genetic and the vascular level by activating GP130 receptor signaling, which can be addressed therapeutically. We performed transcriptomic and immunohistochemical screens with human brain material, pharmacological experiments with a humanized organotypic GBM model, proteomics and cell-based assays and observed that nanomolar concentrations of the signaling peptide Humanin promoted TMZ resistance through DDR activation. GBM mouse models recapitulating intratumoral Humanin-release showed accelerated BTB formation. Genetic analysis of pericyte to endothelial cell signaling in transgenic mice revealed increased GP130 activation in Humanin releasing tumors as compared to controls. GP130 blockade attenuated both DDR activity and BTB formation resulting in improved preclinical chemotherapeutic efficacy. Altogether, we describe an overarching mechanism for TMZ resistance and outline a translatable strategy with predictive markers to improve chemotherapy for GBM.

<p>APLNR expression is upregulated in the infiltrating tumor zone in glioblastoma.</p>

AbstractPurpose: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.

Apelin expression is correlated with angiogenesis in human glioblastoma.

Apelin expression is correlated with angiogenesis in human glioblastoma.

<p>APLNR expression is upregulated in the infiltrating tumor zone in glioblastoma.</p>

<p>Glioblastoma cell- and host-derived apelin controls tumor angiogenesis.</p>

Mice with orthotopically implanted glioblastoma were imaged during contrast injection, with the goals of establishing semi-quantitative DCE in this model and to investigate the impact of apelin-controlled tumour angiogenesis. Wild-type mice with control U87 tumours showed higher initial contrast accumulation but also faster washout compared to apelin knockout mice implanted with apelin knockdown tumour cells, consistent with apelin contributing to tumour vascularization. Control and apelin knockout mice had low contrast accumulation with genetically engineered human glioma-initiating cells.

APLNR is expressed in glioblastoma cells and receptor internalization is activated by apelin peptides specifically.

<p>Apelin is expressed in tumor vessels.</p>

Supplementary Text and Methods

APLNR expression is upregulated in the infiltrating tumor zone in glioblastoma.

Apelin is expressed in tumor vessels.

<p>Apelin is expressed in tumor vessels.</p>

Apelin is expressed in tumor vessels.

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.

<p>Anti-invasive role of apelin in orthotopic models of proneural and classical glioblastoma.</p>

APLNR is expressed in glioblastoma cells and receptor internalization is activated by apelin peptides specifically.