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Malignant gliomas, the largest group of primary intracerebrial tumors, are one of the most-difficult-to-cure cancers. The outcome has not significantly improved in recent years, despite considerable advances in our understanding of the molecular pathogenesis and improvement of surgical techniques, radio- and chemo-therapy. For glioblastoma multiforme (GBM), the most malignant form of glioma, the median survival time is approximately 15 months after diagnosis. Although complete remission of experimental GBM on MRI has been reported by using a lentiviral vector based suicide gene therapy approach1, recurrence of tumors at distant sites is common which is mainly caused by invasive glioma cells that escape treatment. Thus, a better distribution of the suicide gene is needed in order to target and efficiently kill the infiltrative glioma cells to prolong recurrence-free time span and improve the therapeutic effect. By introducing EGFR, a gene that has been reported to promote invasion of glioma cells2 into our LCMV-pseudotyped lentiviral vector system, we want to enhance the distribution of the suicide gene HSV-TK. This might lead to a more efficient killing of glioma cells in both tumor core and invasive areas.

Highlights • Microglia are proneurogenic in a model of prion disease. • Blocking the expansion of microglia prevents the aberrant differentiation of newborn neurons. • TGFβ dominates the proneurogenic actions of microglia during chronic neurodegeneration., Neurogenesis is altered in neurodegenerative disorders, partly regulated by inflammatory factors. We have investigated whether microglia, the innate immune brain cells, regulate hippocampal neurogenesis in neurodegeneration. Using the ME7 model of prion disease we applied gain- or loss-of CSF1R function, as means to stimulate or inhibit microglial proliferation, respectively, to dissect the contribution of these cells to neurogenesis. We found that increased hippocampal neurogenesis correlates with the expansion of the microglia population. The selective inhibition of microglial proliferation caused a reduction in neurogenesis and a restoration of normal neuronal differentiation, supporting a pro-neurogenic role for microglia. Using a gene screening strategy, we identified TGFβ as a molecule controlling the microglial pro-neurogenic response in chronic neurodegeneration, supported by loss-of-function mechanistic experiments. By the selective targeting of microglial proliferation we have been able to uncover a pro-neurogenic role for microglia in chronic neurodegeneration, suggesting promising therapeutic targets to normalise the neurogenic niche during neurodegeneration.

Cancer-cell heterogeneity dramatically influences treatment success, but escapes detection by classical histology. Mass-spectrometric imaging (MSI) represents a powerful method for visualizing the spatial distribution of proteins in tissue sections. Herein we asked whether MSI also facilitates detection of tumor heterogeneity. We first transduced the human neuroendocrine-carcinoma BON cell line following the red-green-blue (RGB) marking principle. RGB marking allows for specific color-coding of individual clones. Mice transplanted with RGB-marked BON cells developed liver tumors. We identified 16 primary tumors clearly distinguishable by histology and fluorescence imaging, but also based on a common tumor-specific signal pattern detected by MSI. Importantly, this pattern was clearly confined to tumor tissue while was absent from surrounding liver tissue. At the same time, we observed protein signals differentially present in a few or even single tumors. Since these signals were independent of RGB marking, they apparently reflected unique intrinsic protein-signal patterns of individual tumors. Thus, our data propose MSI as a tool for identifying divergent tissue by 'fingerprints' of protein signals, allowing not only for differentiation of tumor from healthy tissue but also detection of tumor heterogeneity. In conclusion, by visualizing tumor heterogeneity, MSI ideally complements microscopy-based methods. This might help to better understand tumor biology and develop future treatment strategies.

Identifying and tracking cells in the brain is a challenging task, requiring advanced molecular biology techniques and precise anatomical information. We have applied the principle of RGB marking to the long-term marking and tracking of progenitor and mature cells in the adult brain. Briefly, multicolor RGB marking is based on the simultaneous, lentiviral vector-mediated expression of three genes encoding fluorescent proteins in the three basic colors (red, green, and blue). Here, we show the application of RGB marking to the stable multicolor marking of adult granule cells in the hippocampus. This technique provides each individual cell with a characteristic hue, which facilitates their anatomical identification and spatial tracking. Multicolor RBG marking of mature neurons can provide an effective approach to dissect the function of individual cells, as it allows combination with functional techniques, facilitating the understanding of complex neuronal populations such as that of the dentate gyrus.

Malignant gliomas, the largest group of primary intracerebral tumours, are one of the most difficult-to-cure cancers. For glioblastoma, the most malignant form of glioma, the median survival time is generally less than one year. Lentiviral vector mediated suicide gene therapy has been reported to be a potential therapeutic option for glioma and has been validated in clinically relevant animal models. At this end, specific targeting of tumor cells sparing normal brain tissue is a significant concern. Although several studies may suggest that HSV-Tk mediated suicide gene therapy is not likely to be toxic for normal brain cells, it has never been investigated which effect lentiviral vector mediated HSV-Tk suicide gene therapy exerts on the normal brain in a non-tumor setting. We addressed this important question by delivering the suicide gene HSV-Tk. 007 to the brain of healthy, immunocompetent rats using lentiviral vectors pseudotyped with VSV-G. First, we studied behavior patterns and assessed any potential neurological symptoms in treatment and the control groups, during prodrug treatment with Ganciclovir. We did not observe any significant changes in their physical and behavioral patterns. Then, we carried detailed histological/immunohistochemical analyses of the brains to analyze potential differences in the number of transduced brain cells between control and treatment groups. There was no significant difference in numbers of neurons, astrocytes and oligodendrocyte progenitor cells among the groups, indicating successful survival of these cells after pro-drug treatment. Furthermore, potential inflammation and apoptosis of brain cells were also investigated. No significant infiltration of immune cells was detected across the groups. We could not detect any sign of apoptosis or necrosis across all groups. In conclusion, our study which suggests that lentiviral suicide gene therapy mediated by HSV-Tk. 007 is not toxic for normal brain cells.View Large Image | Download PowerPoint Slide

Human adenoviruses are non-enveloped DNA viruses causing various infections; their pathogenicity varies dependent on virus species and type. Although acute infections can sometimes take severe courses, they are rarely fatal in immune-competent individuals. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are hyperacute and highly contagious infections of the eye caused by human adenovirus types within species D. Currently there is no causal treatment available to counteract these diseases effectively. The E2B region of the adenovirus genome encodes for the viral DNA polymerase, which is required for adenoviral DNA replication. Here we propose novel model systems to test this viral key factor, DNA polymerase, as a putative target for the development of efficient antiviral therapy based on RNA interference. Using our model cell lines we found that different small interfering RNAs mediate significant suppression (up to 90%) of expression levels of viral DNA polymerase upon transfection. Moreover, permanent expression of short hairpin RNA based on the most effective small interfering RNA led to a highly significant, more than tenfold reduction in replication for different human group D adenoviruses involved in ocular infections.

Given the increasing importance of immunotherapeutic treatment strategies in neuro-oncology, the investigation of immune escape mechanisms, which lead to treatment resistance, is crucial. Therefore, studying tumor heterogeneity to understand the clonal dynamics and emergence of immune escape variants during cancer immunoediting can yield valuable insights into the adaptive processes of treatment resistance. We applied a novel multicolor labeling strategy, termed optical barcoding, which makes use of flow-cytometric read-out to compare in-vivo clonality in an immunocompetent versus -deficient environment. The optically barcoded syngeneic GL261 glioma was administered to wildtype and immunodeficient C57BL/6 pfp-/- rag2-/- mice, and tumor growth was monitored. We also analyzed the phenotype of tumor-infiltrating lymphocytes and the gene-expression signatures of explanted tumors using RNAseq. Ex-vivo analysis of individual tumor clones demonstrated that tumors grown in immunocompetent C57BL/6 mice were composed of a significantly different clonal architecture compared to pfp-/- rag2-/- C57BL/6. Whereas pfp-/- rag2-/- mice displayed a homogeneous clonal distribution in all tumors, immunoedited tumors in wildtype mice exhibited reduced clonality with emergence of immune escape clones, independent of proliferation rates. Beyond the expected prolonged survival of wildtype mice, immunoediting was further associated with differences in macroscopic growth patterns, vascularity and invasiveness. Tissue staining, time-course microarray analyses and flow-cytometric phenotyping of tumor-infiltrating immune cells highlighted the exemplary role of PD-L1 as mechanism to adapt to interferon-ɣ and infiltration of T-cells. Furthermore, RNAseq profiling of GL261 tumors grown in wt vs immunodeficient mice allowed the identification of an interferon-ɣ-mediated T-cell-shaped gene expression signature and new pathways potentially involved in the immune escape of glioma. Taken together, using optical barcoding we have demonstrated that the process of cancer immunoediting during tumor evolution not only shapes the gene expression profile and growth pattern, but also has a profound impact on the intratumoral heterogeneity of glioma in the syngeneic GL261 model.

Most of the genomic and epigenetic alterations in GBM have been described thanks to the effort of the Tumor Cancer Genome Atlas and others, highlighting the highly heterogeneous and complex genomic landscape of GBM. Our understanding of how such heterogeneity is maintained is however still limited, and tumor growth driven by micro-environmental selective pressure follows a clonal-evolution model that leads to the emergence of new subpopulation and regional heterogeneity. We established GBM-patient derived cell lines (PDCL) and mouse xenografts from tissue resections and followed the growth progression of multiple clones in vivo by using optical barcoding to track and quantify the clonal evolution. We isolated single cells out of GBM-PDCL and tagged them with specific combinations of two out of six different fluorescent proteins, thus generating 21 optically barcoded (OBC) clones. These clones were then grown either separately or as a mix in vitro and in vivo after intracranial injection. Using this innovative technique, we were able to track the fate of individual clones and analyzed their growth dynamics in different microenvironments. We discovered that fast growing clones in vitro are not necessarily the ones that are responsible for most of the tumor growth in vivo. Interestingly, the clones that do not have the capacity to overgrow other clones when injected in the brain as a mix, are still tumorigenic when injected alone. Moreover, we discovered that distinct clones were more resistant to radiotherapy than others, opening the possibility to identify specific clonal subpopulations in the tumor that are resistant to treatment and will probably develop into the major resistant clone when the tumor will recur. Understanding how tumor heterogeneity is shaped by the microenvironment and how the different clonal population react to treatment will help predicting drug treatment efficiency and potential resistance.

In neuroscience it is a technical challenge to identify and follow the temporal and spatial distribution of cells as they differentiate. We hypothesised that RGB marking, the tagging of individual cells with unique hues resulting from simultaneous expression of the three basic colours red, green and blue, provides a convenient toolbox for the study of the CNS anatomy at the single-cell level. Using γ-retroviral and lentiviral vector sets we describe for the first time the in-vivo multicolour RGB marking of neurons in the adult brain. RGB marking also enabled us to track the spatial and temporal fate of neural stem cells in the adult brain. The application of different viral envelopes and promoters provided a useful approach to track the generation of neurons vs. glial cells at the neurogenic niche, allowing the identification of the prominent generation of new astrocytes to the striatum. Multicolour RGB marking could serve as a universal and reproducible method to study and manipulate the CNS at the single-cell level, in both health and disease.

PURPOSE To analyze the neuroprotective effect of intravitreally grafted neural stem (NS) cells genetically modified to secrete ciliary neurotrophic factor (CNTF) on intraorbitally lesioned retinal ganglion cells (RGCs) in adult mice. METHODS Adherently cultivated NS cells were genetically modified to express a secretable variant of mouse CNTF together with the fluorescent reporter protein Venus. Clonal CNTF-secreting NS cell lines were established using fluorescence activated cell sorting, and intravitreally grafted into adult mice 1 day after an intraorbital crush of the optic nerve. Brn-3a-positive RGCs were counted in flat-mounted retinas at different postlesion intervals to evaluate the neuroprotective effect of the CNTF-secreting NS cells on the axotomized RGCs. Anterograde axonal tracing experiments were performed to analyze the regrowth of the injured RGC axons in CNTF-treated retinas. RESULTS Intravitreally grafted NS cells preferentially differentiated into astrocytes that survived in the host eyes, stably expressed CNTF, and significantly attenuated the loss of the axotomized RGCs over a period of at least 4 months, the latest postlesion time point analyzed. Depending on the postlesion interval analyzed, the number of RGCs in eyes with grafted CNTF-secreting NS cells was 2.8-fold to 6.4-fold higher than in eyes with grafted control NS cells. The CNTF-secreting NS cells additionally induced long-distance regrowth of the lesioned RGC axons. CONCLUSIONS Genetically modified clonal NS cell lines may serve as a useful tool for preclinical studies aimed at evaluating the therapeutic potential of a sustained cell-based intravitreal administration of neuroprotective factors in mouse models of glaucoma.

TAL-effector nucleases (TALENs) are attractive tools for sequence-specific genome modifications, but their delivery still remains problematic. It is well known that the presence of multiple sequence repeats in TALEN genes hampers the use of lentiviral vectors. We report that lentiviral vectors readily package full-length vector mRNAs encoding TALENs, but recombination during reverse transcription prevents successful delivery. We reasoned that preventing reverse transcription of lentiviral-vector RNA would allow transfer of TALENs as mRNA. We demonstrate that lentiviral particles containing genetically inactivated reverse transcriptase (RT) mediated efficient transduction of cultured cells and supported transient transgene expression. For proof-of-principle, we transferred CCR5- and TCR-specific TALEN pairs for efficient targeted genome editing and abrogated expression for each of the receptor proteins in different cell lines. Combining the high specificity of TALENs with efficient lentiviral gene delivery should advance genome editing in vitro and potentially in vivo, and RT-deficient lentiviral vectors may be useful for transient expression of various other genes-of-interest.

Bone homeostasis critically relies on the RANKL-RANK-OPG axis which can be targeted by the fully human monoclonal antibody denosumab in conditions with increased bone resporption such as bone metastases. The binding site and therefore the molecular mechanism by which this antibody inhibits RANKL has not been characterized so far. Here, we used random peptide phage display library screenings to identify the denosumab epitope on RANKL. Alignments of phage derived peptide sequences with RANKL suggested that this antibody recognized a linear epitope between position T233 and Y241. Mutational analysis confirmed the core residues as critical for this interaction. The spatial localization of this epitope on a 3-dimensional model of RANKL showed that it overlapped with the major binding sites of OPG and RANK on RANKL. We conclude that denosumab inhibits RANKL by both functional and molecular mimicry of the natural decoy receptor OPG.

BACKGROUND: Neural stem cells (NSCs) have an inherent brain tumor tropism that can be exploited for targeted delivery of therapeutic genes to invasive gliomas. We have recently introduced the concept of intranasal application of NSCs as a noninvasive and direct alternative delivery method for glioma-targeting NSCs. Here, we demonstrate that the repeated non-invasive intranasal administration of tumor-targeting NSC is able to deliver a novel suicide gene (TK.007) to intracerebrally growing human glioblastoma xenografts. METHODS: Murine NSC were genetically modified to stably express a novel, codon-optimized HSVtk(A168H) mutant (TK.007). The biological activity of the NSC-mediated TK.007/ganciclovir (GCV) system was assessed in cell survival and bystander assays using various human glioma cell lines. Therapeutic effects of intratumoral and intranasal NSC-TK007 applications alone and the sequential combinations of both was tested using an intracranial U87 human glioblastoma model in nude mice. All animals received 50 mg/kg GCV i.p. for five consecutive days. In all experiments two control groups received either NaCl instead of GCV or NSC containing the control vector instead of NSC-TK.007. Therapeutic efficiency was determined by assessment of tumor size by 7T MR-imaging and by establishment of Kaplan-Meier survival curves. RESULTS: Cell survival and bystander assays confirmed the GCV catalytic activity of NSC-expressed TK007 in a GCV dose dependent manner and a significant bystander effect at 12.5% of cells. Tumor targeted migration was demonstrated by intravital imaging of luciferase-expressing NSC. In glioma-bearing mice a single intratumoral application of NSC-TK007 followed by systemic prodrug application of GCV lead to a significant tumor growth inhibition of 72% versus the control groups at day 22 and translated in a prolonged survival time. Mice recieving a cyclic intranasal application of NSC-TK.007 alone displayed a signifcant longer survival than the controls. This effect was significantly enhanced when cyclic intranasal NSC-TK.007 therapy was preceded by a single intratumoral application of NSC-TK007 and led to a significant tumor growth inhibition of >95% and improved survival time. CONCLUSIONS: Our data demonstrates that the NSC-mediated TK.007/GCV therapy, which is based on the clinically most widely used suicide system to date, is safe, nontoxic, and effective in glioblastoma-bearing mice. Most importantly, our findings establish that the intranasal application of therapeutically-modified NSC is a safe and non-invasive application method which allows a chronic treatment during the disease course. SECONDARY CATEGORY: Clinical Neuro-Oncology.

Increased neurogenesis has been reported in neurodegenerative disease, but its significance is unclear. In a mouse model of prion disease, Gomez-Nicola et al. detect increased neurogenesis in the dentate gyrus that partially counteracts neuronal loss. Targeting neurogenesis may have therapeutic potential., The study of neurogenesis during chronic neurodegeneration is crucial in order to understand the intrinsic repair mechanisms of the brain, and key to designing therapeutic strategies. In this study, using an experimental model of progressive chronic neurodegeneration, murine prion disease, we define the temporal dynamics of the generation, maturation and integration of new neurons in the hippocampal dentate gyrus, using dual pulse-chase, multicolour γ-retroviral tracing, transmission electron microscopy and patch-clamp. We found increased neurogenesis during the progression of prion disease, which partially counteracts the effects of chronic neurodegeneration, as evidenced by blocking neurogenesis with cytosine arabinoside, and helps to preserve the hippocampal function. Evidence obtained from human post-mortem samples, of both variant Creutzfeldt-Jakob disease and Alzheimer’s disease patients, also suggests increased neurogenic activity. These results open a new avenue into the exploration of the effects and regulation of neurogenesis during chronic neurodegeneration, and offer a new model to reproduce the changes observed in human neurodegenerative diseases.

Multiple myeloma is a mostly incurable malignancy characterized by the expansion of a malignant plasma cell (PC) clone in the human bone marrow (BM). Myeloma cells closely interact with the BM stroma, which secretes soluble factors that foster myeloma progression and therapy resistance. Growth arrest-specific gene 6 (Gas6) is produced by BM-derived stroma cells and can promote malignancy. However, the role of Gas6 and its receptors Axl, Tyro3 and Mer (TAM receptors) in myeloma is unknown. We therefore investigated their expression in myeloma cell lines and in the BM of myeloma patients and healthy donors. Gas6 showed increased expression in sorted BMPCs of myeloma patients compared with healthy controls. The fraction of Mer(+) BMPCs was increased in myeloma patients in comparison with healthy controls whereas Axl and Tyro3 were not expressed by BMPCs in the majority of patients. Downregulation of Gas6 and Mer inhibited the proliferation of different myeloma cell lines, whereas knocking down Axl or Tyro3 had no effect. Inhibition of the Gas6 receptor Mer or therapeutic targeting of Gas6 by warfarin reduced myeloma burden and improved survival in a systemic model of myeloma. Thus, the Gas6-Mer axis represents a novel candidate for therapeutic intervention in this incurable malignancy.

RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies.

Chronic hepatitis B virus (HBV) infection has been associated with alterations in lipid metabolism. Moreover, the Na+-taurocholate cotransporting polypeptide (NTCP), responsible for bile acid (BA) uptake into hepatocytes, was identified as the functional cellular receptor mediating HBV entry. The aim of the study was to determine whether HBV alters the liver metabolic profile by employing HBV-infected and uninfected human liver chimeric mice. Humanized urokinase plasminogen activator/severe combined immunodeficiency mice were used to establish chronic HBV infection. Gene expression profiles were determined by real-time polymerase chain reaction using primers specifically recognizing transcripts of either human or murine origin. Liver biopsy samples obtained from HBV-chronic individuals were used to validate changes determined in mice. Besides modest changes in lipid metabolism, HBV-infected mice displayed a significant enhancement of human cholesterol 7α-hydroxylase (human [h]CYP7A1; median 12-fold induction; P0.0001), the rate-limiting enzyme promoting the conversion of cholesterol to BAs, and of genes involved in transcriptional regulation, biosynthesis, and uptake of cholesterol (human sterol-regulatory element-binding protein 2, human 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and human low-density lipoprotein receptor), compared to uninfected controls. Significant hCYP7A1 induction and reduction of human small heterodimer partner, the corepressor of hCYP7A1 transcription, was also confirmed in liver biopsies from HBV-infected patients. Notably, administration of Myrcludex-B, an entry inhibitor derived from the pre-S1 domain of the HBV envelope, provoked a comparable murine CYP7A1 induction in uninfected mice, thus designating the pre-S1 domain as the viral component triggering such metabolic alterations.Binding of HBV to NTCP limits its function, thus promoting compensatory BA synthesis and cholesterol provision. The intimate link determined between HBV and liver metabolism underlines the importance to exploit further metabolic pathways, as well as possible NTCP-related viral-drug interactions.

Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC) patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181). However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic., Bundesministerium für Wissenschaft und Forschung (TOMCAT, grant number 01EZ0824; http://www.bmbf.de/), Landesexzellenzinitiative Hamburg (Nanotechnology in Medicine – NAME)

A continuous intraocular delivery of neurotrophic factors (NFs) is being explored as a strategy to rescue photoreceptor cells and visual functions in degenerative retinal disorders that are currently untreatable. To establish a cell-based intraocular delivery system for a sustained administration of NFs to the dystrophic mouse retina, we used a polycistronic lentiviral vector to genetically modify adherently cultivated murine neural stem (NS) cells. The vector concurrently encoded a gene of interest, a reporter gene, and a resistance gene and thus facilitated the selection, cloning, and in vivo tracking of the modified cells. To evaluate whether modified NS cells permit delivery of functionally relevant quantities of NFs to the dystrophic mouse retina, we expressed a secretable variant of ciliary neurotrophic factor (CNTF) in NS cells and grafted the cells into the vitreous space of Pde6brd1 and Pde6brd10 mice, two animal models of retinitis pigmentosa. In both mouse lines, grafted cells attached to the retina and lens, where they differentiated into astrocytes and some neurons. Adverse effects of the transplanted cells on the morphology of host retinas were not observed. Importantly, the CNTF-secreting NS cells significantly attenuated photoreceptor degeneration in both mutant mouse lines. The neuroprotective effect was significantly more pronounced when clonally derived NS cell lines selected for high expression levels of CNTF were grafted into Pde6brd1 mice. Intravitreal transplantations of modified NS cells may thus represent a useful method for preclinical studies aimed at evaluating the therapeutic potential of a cell-based intraocular delivery of NFs in mouse models of photoreceptor degeneration.

Acute myeloid leukemia (AML) represents a clonal disease of hematopoietic progenitors characterized by acquired heterogenous genetic changes that alter normal mechanisms of proliferation, self-renewal, and differentiation.(1) Although 40% to 45% of patients younger than 65 years of age can be cured with current therapies, only 10% of older patients reach long-term survival.(1) Because only very few novel AML drugs were approved in the past 2 decades, there is an urgent need to identify novel targets and therapeutic strategies to treat underserved AML patients. We report here that Axl, a member of the Tyro3, Axl, Mer receptor tyrosine kinase family,(2-4) represents an independent prognostic marker and therapeutic target in AML. AML cells induce expression and secretion of the Axl ligand growth arrest-specific gene 6 (Gas6) by bone marrow-derived stromal cells (BMDSCs). Gas6 in turn mediates proliferation, survival, and chemoresistance of Axl-expressing AML cells. This Gas6-Axl paracrine axis between AML cells and BMDSCs establishes a chemoprotective tumor cell niche that can be abrogated by Axl-targeting approaches. Axl inhibition is active in FLT3-mutated and FLT3 wild-type AML, improves clinically relevant end points, and its efficacy depends on presence of Gas6 and Axl. Axl inhibition alone or in combination with chemotherapy might represent a novel therapeutic avenue for AML.

Several cell adhesion molecules (CAMs) including selectins, integrins, cadherins and immunoglobulin-like CAMs are involved in leukocyte adhesion especially at sites of inflammation. In cancer cells, these CAMs have been associated with the growth and metastatic behavior in several malignant entities. In this study adhesion of LAN 1 and SK-N-SH neuroblastoma cells to selectins, hyaluronan and endothelial cells were determined under flow conditions. Furthermore cells were injected subcutaneously into wildtype and selectin deficient scid mice and their growth and metastatic behavior were analyzed. Under shear stress SK-N-SH cells firmly adhered to E-selectin-Fc-fusion protein, hyaluronan and endothelial cells, while LAN 1 cells showed less or hardly any adhesive events by comparison. In the SK-N-SH xenograft model metastasis formation was slightly dependent on the expression of selectins, while LAN 1 cells developed metastases completely independent of selectin expression. The different adhesive and metastatic properties of LAN 1 and SK-N-SH cells are reflected by a different expression profile of several CAMs. The results indicate that endothelial selectins are not essential for metastasis formation of human LAN 1 and SK-N-SH cells. However, other CAMs namely CD44, N-cadherin, NCAM and integrins were upregulated or downregulated, respectively, in SK-N-SH and LAN 1 cells and are potential adhesion molecules involved in the metastatic cascade of these cells.

e23010Background: E-selectin-mediated adhesion of circulating tumor cells (CTCs) to vascular endothelial cells (ECs) initiates CTC extravasation at a distant site and is hence a critical step of th...

Background Despite progress in the development of combined antiretroviral therapies (cART), HIV infection remains a significant challenge for human health. Current problems of cART include multi-drug-resistant virus variants, long-term toxicity and enormous treatment costs. Therefore, the identification of novel effective drugs is urgently needed. Methods We developed a straightforward screening approach for simultaneously evaluating the sensitivity of multiple HIV gag-pol mutants to antiviral drugs in one assay. Our technique is based on multi-colour lentiviral self-inactivating (SIN) LeGO vector technology. Results We demonstrated the successful use of this approach for screening compounds against up to four HIV gag-pol variants (wild-type and three mutants) simultaneously. Importantly, the technique was adapted to Biosafety Level 1 conditions by utilising ecotropic pseudotypes. This allowed upscaling to a large-scale screening protocol exploited by pharmaceutical companies in a successful proof-of-concept experiment. Conclusions The technology developed here facilitates fast screening for anti-HIV activity of individual agents from large compound libraries. Although drugs targeting gag-pol variants were used here, our approach permits screening compounds that target several different, key cellular and viral functions of the HIV life-cycle. The modular principle of the method also allows the easy exchange of various mutations in HIV sequences. In conclusion, the methodology presented here provides a valuable new approach for the identification of novel anti-HIV drugs.

3549 Background: Acquired resistance to epidermal growth factor receptor (EGFR) targeted antibodies represents a clinical challenge in the treatment of gastrointestinal tumors such as metastatic colorectal cancer, but its molecular mechanisms are incompletely understood. Methods: We scanned KRAS exon 2/3/4, NRAS exon 2/3/4 and the overlapping epitopes of the EGFR antibodies cetuximab and panitumumab for mutations in pre- and post-treatment tumor tissue of 21 patients with gastrointestinal cancer treated with chemotherapy +/- EGFR antibodies by targeted next-generation sequencing (“tumor tissue” cohort). Binding, signaling and drug sensitivity studies were performed in Ba/F3 cells stably transduced with wt and mutant EGFR. Results were validated in circulating tumor DNA (ctDNA) of an independent “liquid biopsy” cohort of 27 patients. Results: We describe a novel EGFR exon 12 mutation acquired in tumors of 1 out of 3 patients treated with panitumumab in the “tumor tissue” cohort. This mutation introduces a ...

Introduction: •Malignant gliomas, the largest group of primary intracerebral tumours, are one of the most difficult-to-cure cancers. The outcome has not improved significantly in the past years, despite considerable advances in our understanding of the molecular pathogenesis and the improvement of surgical techniques, radio- and chemotherapy. For glioblastoma multiforme (GBM), the most malignant form of glioma, the median survival time is generally less than one year.•Invasive glioma cells escape current therapies and are initiating recurrent tumors. Although complete remission of experimental GBM on MRI was observed, recurrent tumors came up which were frequently observed at different sites compared to the primary tumor 1. Thus invasive cells migrated to thes areas and initiated recurrent tumors.•The distribution of the therapeutic gene needs to be enhanced in order to target the invasive glioma cells and thereby improve the therapeutic effect and prolong the recurrence-free time window.By introducing EGFR, a gene that enhances invasion2, in addition to the suicide gene HSV-tk into our vector system, we want to improve the distribution of the therapeutic gene and also track invasive tumor cells.Key findings: •Expression of EGFR initiates an invasive program in transduced glioblastoma cells in vitro.•EGFR mediated infiltration of glioblastoma cells is retained in vivo in a rat xenograft model. [figure3 a &b]•The bicistronic lentiviral vector containing HSV-TK and EGFR [figure4] shows satisfactory cytotoxic profile in vitro. [figure5a & b] View Large Image | Download PowerPoint SlideConclusion: Expression of EGFR can enhance infiltrative nature of glioblastoma cells both in vitro and in vivo. We are currently taking preparations for an in vivo therapeutic study of our bicistronic lentiviral construct containing a recombinant HSV-TK and EGFR in a clinically relevant GBM rat model.

We have recently introduced the concept of intranasal application of NSCs as a noninvasive and direct alternative delivery method for glioma-targeting NSCs. Here, we demonstrate that the repeated non-invasive intranasal administration of tumor-targeting NSC is able to deliver a suicide gene (TK.007) to intracerebrally growing human glioblastoma xenografts. Murine NSC were genetically modified to stably express a novel, codon-optimized HSVtk(A168H) mutant (TK.007). The biological activity of the NSC-mediated TK.007/ganciclovir (GCV) system was assessed in cell survival and bystander assays using various human glioma cell lines. Therapeutic effects of intratumoral and intranasal NSC-TK007 applications alone and the sequential combinations of both was tested using an intracranial U87 human glioblastoma model in nude mice. Therapeutic efficiency was determined by assessment of tumor size by 7T MR-imaging and by establishment of Kaplan-Meier survival curves. Tumor targeted migration was demonstrated by intravital imaging of luciferase-expressing NSC. Cell survival and bystander assays confirmed the GCV catalytic activity of NSC-expressed TK007 in a GCV dose dependent manner and a significant bystander effect at 12.5% of cells. Tumor targeted migration was mediated by intracerebral gradients of SDF-1. In glioma-bearing mice a single intratumoral application of NSC-TK007 followed by systemic prodrug application of GCV lead to a significant tumor growth inhibition of 72% versus the control groups at day 22 and translated in a prolonged survival time. Mice recieving a cyclic intranasal application of NSC-TK.007 alone displayed a signifcant longer survival than the controls. This effect was significantly enhanced when cyclic intranasal NSC-TK.007 therapy was preceded by a single intratumoral application of NSC-TK007 and led to a significant tumor growth inhibition of >95% and significantly improved survival time. Our findings establish that the intranasal application of therapeutically-modified NSC is a safe and non-invasive application method which allows a chronic treatment during the disease course.

Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3(r)CDDP(1000) in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases.

7027 Background: Axl, the receptor for Growth Arrest-specific protein 6 (Gas6) plays a role in AML pathobiology (Blood Suppl. Nov 2011; 118: 940). Here, we investigated whether Axl represents a therapeutic target in AML. Methods: Gas6 levels were measured by ELISA and immunohistochemistry. Axl expression was detected by flow cytometry. Co-cultures of (murine) BM stroma cells (primary, OP9, S17) with Mv4-11 and OCI-AML5 cell lines were performed. Results: We found (i) higher expression of Axl in AML BM compared to healthy BM donors (66.20 ± 10.87 vs. 0.65 ± 0.10 %; n=8/6; p+CD38- AML stem cells compared to healthy CD34+CD38- BM stem cells (58.43 ± 4.63 % vs. 6.00 ± 2.01 %; n=7/6; p+leukemia cells forms a chemoprotective niche for leukemia cells. In line with these findings Axl blockade chemosensitizes Mv4-11 cells for treatment with doxorubicine in vivo. Conclusions: Axl represents a therapeutic target in AML and Axl inhibition by BGB324 holds potential to treat chemosensitive and chemoresistant AML.

Retroviral gene marking has been used successfully in preclinical and clinical transplantation settings. Highly sensitive techniques for vector insertion-site determination, such as linear amplification-mediated polymerase chain reaction (LAM-PCR) in conjunction with next-generation sequencing, have been introduced to assess the composition of gene-marked hematopoiesis at a single-cell level. Here we used these novel techniques for directly comparing clonal reconstitution kinetics in mice transplanted with bone-marrow-derived stem cells genetically marked with either a standard, spleen focus-forming virus long terminal repeat (LTR)-driven γ-retroviral, or a lentiviral self-inactivating vector containing an identical but internal spleen focus-forming virus-derived enhancer/promoter. We observed that the use of the lentiviral self-inactivating vector for gene marking was associated with a broader repertoire of differently marked hematopoietic clones. More importantly, we found a significantly higher probability of insertions in growth-promoting, clonal-dominance-associated genes in the spleen focus-forming virus LTR-driven γ-retroviral vector at later time points of analysis. Based on our data, we suggest that the combined use of LAM-PCR and next-generation sequencing represents a potent tool for the analysis of clonal reconstitution kinetics in the context of gene marking with integrated vectors. At the same time, our findings prove that the use of multiple restriction enzymes for LAM-PCR is indispensable to detect most or ideally all individual stem cell clones contributing to hematopoiesis. We have also found that techniques such as quantitative PCR can be helpful to retrospectively analyze reconstitution kinetics for individual hematopoietic stem cell clones. Finally, our results confirm the notion that marking with lentiviral self-inactivating vectors is associated with a lower risk of genotoxicity as compared with γ-retroviral LTR vectors.