Congenital human cytomegalovirus infection is a leading infectious cause of long-term neurodevelopmental sequelae, including mental retardation and hearing defects. Strict species specificity of cytomegaloviruses has restricted the scope of studies of cytomegalovirus infection in animal models. To investigate the pathogenesis of congenital human cytomegalovirus infection, we developed a mouse cytomegalovirus model that recapitulates the major characteristics of central nervous system infection in human infants, including the route of neuroinvasion and neuropathological findings. Following intraperitoneal inoculation of newborn animals with mouse cytomegalovirus, the virus disseminates to the central nervous system during high-level viremia and replicates in the brain parenchyma, resulting in a focal but widespread, non-necrotizing encephalitis. Central nervous system infection is coupled with the recruitment of resident and peripheral immune cells as well as the expression of a large number of pro-inflammatory cytokines. Although infiltration of cellular constituents of the innate immune response characterizes the early immune response in the central nervous system, resolution of productive infection requires virus-specific CD8(+) T cells. Perinatal mouse cytomegalovirus infection results in profoundly altered postnatal development of the mouse central nervous system and long-term motor and sensory disabilities. Based on an enhanced understanding of the pathogenesis of this infection, prospects for novel intervention strategies aimed to improve the outcome of congenital human cytomegalovirus infection are proposed.Cellular & Molecular Immunology advance online publication, 21 July 2014; doi:10.1038/cmi.2014.51.
The enantiomeric separation ability of the newly prepared chiral stationary phases containing acridino-18-crown-6 ether selectors was studied by high-performance liquid chromatography (HPLC). The chiral stationary phases separated the enantiomers of selected protonated primary aralkylamines efficiently. The best results were found for the separation of the mixtures of enantiomers of NO2 -PEA.
Glioblastoma multiforme (GBM), the most aggressive brain malignancy, is characterized by extensive cellular proliferation, angiogenesis, and single-cell infiltration into the brain. We have previously shown that a xenograft model based on serial xenotransplantation of human biopsy spheroids in immunodeficient rodents maintains the genotype and phenotype of the original patient tumor. The present work further extends this model for optical assessment of tumor engraftment and growth using bioluminescence imaging (BLI). A method for successful lentiviral transduction of the firefly luciferase gene into multicellular spheroids was developed and implemented to generate optically active patient tumor cells. Luciferase-expressing spheroids were injected into the brains of immunodeficient mice. BLI photon counts and tumor volumes from magnetic resonance imaging (MRI) were correlated. Luciferase-expressing tumors recapitulated the histopathologic hallmarks of human GBMs and showed proliferation rates and microvessel density counts similar to those of wild-type xenografts. Moreover, we detected widespread invasion of luciferase-positive tumor cells in the mouse brains. Herein we describe a novel optically active model of GBM that closely mimics human pathology with respect to invasion, angiogenesis, and proliferation indices. The model may thus be routinely used for the assessment of novel anti-GBM therapeutic approaches implementing well-established and cost-effective optical imaging strategies.
Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As2O3) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.
Oligodendroglioma poses a biological conundrum for malignant adult human gliomas: it is a tumor type that is universally incurable for patients, and yet, only a few of the human tumors have been established as cell populations in vitro or as intracranial xenografts in vivo. Their survival, thus, may emerge only within a specific environmental context. To determine the fate of human oligodendroglioma in an experimental model, we studied the development of an anaplastic tumor after intracranial implantation into enhanced green fluorescent protein (eGFP) positive NOD/SCID mice. Remarkably after nearly nine months, the tumor not only engrafted, but it also retained classic histological and genetic features of human oligodendroglioma, in particular cells with a clear cytoplasm, showing an infiltrative growth pattern, and harboring mutations of IDH1 (R132H) and of the tumor suppressor genes, FUBP1 and CIC. The xenografts were highly invasive, exhibiting a distinct migration and growth pattern around neurons, especially in the hippocampus, and following white matter tracts of the corpus callosum with tumor cells accumulating around established vasculature. Although tumors exhibited a high growth fraction in vivo, neither cells from the original patient tumor nor the xenograft exhibited significant growth in vitro over a six-month period. This glioma xenograft is the first to display a pure oligodendroglioma histology and expression of R132H. The unexpected property, that the cells fail to grow in vitro even after passage through the mouse, allows us to uniquely investigate the relationship of this oligodendroglioma with the in vivo microenvironment.
Angiogenesis is regarded as a hallmark of cancer progression and it has been postulated that solid tumor growth depends on angiogenesis. At present, however, it is clear that tumor cell invasion can occur without angiogenesis, a phenomenon that is particularly evident by the infiltrative growth of malignant brain tumors, such as glioblastomas (GBMs). In these tumors, amplification or overexpression of wild-type (wt) or truncated and constitutively activated epidermal growth factor receptor (EGFR) are regarded as important events in GBM development, where the complex downstream signaling events have been implicated in tumor cell invasion, angiogenesis and proliferation. Here, we show that amplification and in particular activation of wild-type EGFR represents an underlying mechanism for non-angiogenic, invasive tumor growth. Using a clinically relevant human GBM xenograft model, we show that tumor cells with EGFR gene amplification and activation diffusely infiltrate normal brain tissue independent of angiogenesis and that transient inhibition of EGFR activity by cetuximab inhibits the invasive tumor growth. Moreover, stable, long-term expression of a dominant-negative EGFR leads to a mesenchymal to epithelial-like transition and induction of angiogenic tumor growth. Analysis of human GBM biopsies confirmed that EGFR activation correlated with invasive/non-angiogenic tumor growth. In conclusion, our results indicate that activation of wild-type EGFR promotes invasion and glioblastoma development independent of angiogenesis, whereas loss of its activity results in angiogenic tumor growth.
Brain metastasis is associated with a particular poor prognosis. Novel insight into the brain metastatic process is therefore warranted. Several preclinical models of brain tumor metastasis have been developed during the last 60 years, and they have in part revealed some of the mechanisms underlying the metastatic process. This review discusses mechanisms of brain metastasis with a key focus of the development of animal model systems. This includes the use of rodent, syngeneic brain metastasis models (spontaneous, chemically induced and genetically engineered models) and human xenotransplantation models (ectopic inoculation and orthotopic models). Current information indicates that none of these fully reflect tumor development seen in patients with metastatic disease. The various model systems used, however, have provided important insight into specific mechanisms of the metastatic process related to the brain. By combining the knowledge obtained from animal models, new important information on the molecular mechanisms behind metastasis will be obtained, leading to the future development of new therapeutic strategies.
The cell fate determinant NUMB orchestrates asymmetric cell division in flies and mammals and has lately been suggested to have a tumor suppressor function in breast and lung cancer. Here, we studied NUMB in the context of malignant gliomas. We used ectopic expression of NUMB in order to inhibit proliferation and induce differentiation in glioma cells by alteration of Notch, Hedgehog and p53 signaling. We found that NUMB is consistently expressed in glioma biopsies with predominance of NUMB2/4 isoforms as determined by isoform-specific real-time PCR and Western blotting. Upon lentiviral overexpression, in vitro proliferation rate and the grade of differentiation as assessed by morphology and expression of neural and glial markers remained unchanged. Orthotopic xenografts of NUMB-transduced human U87 glioma cells could be established in nude rats without impairing engraftment or causing significant changes in morphology based on magnetic resonance imaging (MRI). The previously reported alteration of Hedgehog and p53 signaling by NUMB could not be recapitulated in glioma cells. We thus show that in experimental gliomas, NUMB overexpression most likely does not exert a tumor suppressor function such as seen in epithelial cancers.
Establishing clinically relevant animal models of glioblastoma multiforme (GBM) remains a challenge, and many commonly used cell line-based models do not recapitulate the invasive growth patterns of patient GBMs. Previously, we have reported the formation of highly invasive tumour xenografts in nude rats from human GBMs. However, implementing tumour models based on primary tissue requires that these models can be sufficiently standardised with consistently high take rates.
Glioblastoma is the most frequent and most malignant primary brain tumor with a poor prognosis. The translation of therapeutic strategies for glioblastoma from the experimental phase into the clinic has been limited by insufficient animal models, which lack important features of human tumors. Lentiviral gene therapy is an attractive therapeutic option for human glioblastoma, which we validated in a clinically relevant animal model.
Animal modeling for primary brain tumors has undergone constant development over the last 60 years, and significant improvements have been made recently with the establishment of highly invasive glioblastoma models. In this review we discuss the advantages and pitfalls of model development, focusing on chemically induced models, various xenogeneic grafts of human cell lines, including stem cell-like cell lines and biopsy spheroids. We then discuss the development of numerous genetically engineered models available to study mechanisms of tumor initiation and progression. At present it is clear that none of the current animal models fully reflects human gliomas. Yet, the various model systems have provided important insight into specific mechanisms of tumor development. In particular, it is anticipated that a combined comprehensive knowledge of the various models currently available will provide important new knowledge on target identification and the validation and development of new therapeutic strategies.
Glioblastoma multiforme (GBM) is the most aggressive type of malignant primary brain tumors in adults. Molecular and genetic analysis has advanced our understanding of glioma biology, however mapping the cellular composition of the tumor microenvironment is crucial for understanding the pathology of this dreaded brain cancer. In this study we identified major cell populations attracted by glioma using orthotopic rodent models of human glioma xenografts. Marker-specific, anatomical and morphological analyses revealed a robust influx of host cells into the main tumor bed and tumor satellites.
Cysteine cathepsins play an important role in shaping the highly infiltrative growth pattern of human gliomas. We have previously demonstrated that the activity of cysteine cathepsins is elevated in invasive glioblastoma (GBM) cells in vitro, in part due to attenuation of their endogenous inhibitors, the cystatins. To investigate this relationship in vivo, we established U87-MG xenografts in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID)-enhanced green fluorescent protein (eGFP) mice. Here, tumor growth correlated with an elevated enzymatic activity of CatB both in the tumor core and at the periphery, whereas CatS and CatL levels were higher at the xenograft edge compared to the core. Reversely, StefB expression was detected in the tumor core, but it was generally absent in the tumor periphery, suggesting that down-regulation of this inhibitor correlates with in vivo invasion. In human GBM samples, all cathepsins were elevated at the tumor periphery compared to brain parenchyma. CatB was also typically associated with angiogenic endothelia and necrotic areas. StefB was mainly detected in the tumor core, whereas CysC and StefA were evenly distributed, reflecting the observations in the xenografts. However, at the mRNA level, no differences in cathepsins and cystatins were observed between the tumor center and the periphery in both human biopsies and xenografts. Interestingly, in human tumors, cathepsin and stefin transcript levels correlated with CD68 and CXCR4 levels, but not with epidermal growth factor receptor (EGFR). Moreover, we reveal for the first time that an elevated StefA mRNA level is a highly significant prognostic factor for patient survival.
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