Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity.

Humoral immunity relies on the efficient differentiation of memory B cells (MBCs) into antibody-secreting cells (ASCs). T helper (Th) signals upregulate B cell receptor (BCR) signaling by potentiating Src family kinases through increasing CD45 phosphatase activity (CD45 PA). In this study, we show that high CD45 PA in MBCs enhances BCR signaling and is essential for their effective ASC differentiation. Mechanistically, Th signals upregulate CD45 PA through intensifying the surface binding of a CD45 ligand, Galectin-1. CD45 PA works as a sensor of T cell help and defines high-affinity germinal center (GC) plasma cell (PC) precursors characterized by IRF4 expression in vivo. Increasing T cell help in vitro results in an incremental CD45 PA increase and enhances ASC differentiation by facilitating effective induction of the transcription factors IRF4 and BLIMP1. This study connects Th signals with BCR signaling through Galectin-1-dependent regulation of CD45 PA and provides a mechanism for efficient ASC differentiation of MBCs.

Deep sequencing of a recurrent oligodendroglioma and the derived xenografts reveals new insights into the evolution of human oligodendroglioma and candidate driver genes.

We previously reported the establishment of a rare xenograft derived from a recurrent oligodendroglioma with 1p/19q codeletion. Here, we analyzed in detail the exome sequencing datasets from the recurrent oligodendroglioma (WHO grade III, recurrent O2010) and the first-generation xenograft (xenograft1). Somatic SNVs and small InDels (n = 80) with potential effects at the protein level in recurrent O2010 included variants in IDH1 (NM_005896:c.395G>A; p. Arg132His), FUBP1 (NM_003902:c.1307_1310delTAGA; p.Ile436fs), and CIC (NM_015125:c.4421T>G; p.Val1474Gly). All but 2 of these 80 variants were also present in xenograft1, along with 7 new variants. Deep sequencing of the 87 SNVs and InDels in the original tumor (WHO grade III, primary O2005) and in a second-generation xenograft (xenograft2) revealed that only 11 variants, including IDH1 (NM_005896:c.395G>A; p. Arg132His), PSKH1 (NM_006742.2:c.650G>A; p.Arg217Gln), and SNX12 (NM_001256188:c.470G>A; p.Arg157His), along with a variant in the TERT promoter (C250T, NM_198253.2: c.-146G>A), were already present in primary O2005. Allele frequencies of the 11 variants were calculated to assess their potential as putative driver genes. A missense change in NDST4 (NM_022569:c.2392C>G; p.Leu798Val) on 4q exhibited an increasing allele frequency (~ 20%, primary O2005, 80%, recurrent O2010 and 100%, xenograft1), consistent with a selection event. Sequencing of NDST4 in a cohort of 15 oligodendrogliomas, however, revealed no additional cases with potential protein disrupting variants. Our analysis illuminated a tumor evolutionary series of events, which included 1p/19q codeletion, IDH1 R132H, and TERT C250T as early events, followed by loss of function of NDST4 and mutations in FUBP1 and CIC as late events.

Enhanced germinal center reaction by targeting vaccine antigen to major histocompatibility complex class II molecules.

Enhancing the germinal center (GC) reaction is a prime objective in vaccine development. Targeting of antigen to MHCII on APCs has previously been shown to increase antibody responses, but the underlying mechanism has been unclear. We have here investigated the GC reaction after targeting antigen to MHCII in (i) a defined model with T and B cells of known specificity using adjuvant-free vaccine proteins, and (ii) an infectious disease model using a DNA vaccine. MHCII-targeting enhanced presentation of peptide: MHCII on APCs, and increased the numbers of GC B cells, TFH, and plasma cells. Antibodies appeared earlier and levels were increased. BCR of GC B cells and serum antibodies had increased avidity for antigen. The improved responses required cross-linking of BCR and MHCII in either cis or trans. The enhanced GC reaction induced by MHCII-targeting of antigen has clear implications for design of more efficient subunit vaccines.

Brain-resident memory CD8+ T cells induced by congenital CMV infection prevent brain pathology and virus reactivation.

Congenital HCMV infection is a leading infectious cause of long-term neurodevelopmental sequelae. Infection of newborn mice with mouse cytomegalovirus (MCMV) intraperitoneally is a well-established model of congenital human cytomegalovirus infection, which best recapitulates the hematogenous route of virus spread to brain and subsequent pathology. Here, we used this model to investigate the role, dynamics, and phenotype of CD8+ T cells in the brain following infection of newborn mice. We show that CD8+ T cells infiltrate the brain and form a pool of tissue-resident memory T cells (TRM cells) that persist for lifetime. Adoptively transferred virus-specific CD8+ T cells provide protection against primary MCMV infection in newborn mice, reduce brain pathology, and remain in the brain as TRM cells. Brain CD8+ TRM cells were long-lived, slowly proliferating cells able to respond to local challenge infection. Importantly, brain CD8+ TRM cells controlled latent MCMV and their depletion resulted in virus reactivation and enhanced inflammation in brain.

Cathepsin L silencing increases As2O3 toxicity in malignantly transformed pilocytic astrocytoma MPA58 cells by activating caspases 3/7.

Low-grade, pilocytic astrocytomas are treated by resection, but additional therapy is necessary for those tumors with anaplastic features. Arsenic trioxide (As2O3) is emerging as an effective chemotherapeutic agent for treatment of malignant glioblastoma multiforme, where Cathepsin L silencing enables lower, less harmful As2O3 concentrations to achieve the desired cytotoxic effect. Here, we evaluated the effects of As2O3 combined with stable Cathepsin L shRNA silencing on cell viability/metabolic activity, and apoptosis in primary cultures of recurrent malignantly transformed pilocytic astrocytoma (MPA). These cells expressed high Cathepsin L levels, and when grown as monolayers and spheroids, they were more resistant to As2O3 than the U87MG glioblastoma cell line. Caspases 3/7 activity in MPA58 spheroids was not significantly affected by As2O3, possibly due to higher chemoresistance of primary biopsy tissue of less malignant astrocytoma versus the malignant U87MG cell line. However, As2O3 treatment was cytotoxic to MPA spheroids after silencing of Cathepsin L expression. While Cathepsin L silencing only slightly decreased the live/dead cell ratio in As2O3-treated MPA-si spheroids under our experimental conditions, there was an increase in As2O3-mediated apoptosis in MPA-si spheroids, as indicated by elevated caspases 3/7 activity. Therefore, Cathepsin L silencing by gene manipulation can be applied when a more aggressive approach is needed in treatment of pilocytic astrocytomas with anaplastic features.

Engraftment of Human Glioblastoma Cells in Immunocompetent Rats through Acquired Immunosuppression.

Transplantation of glioblastoma patient biopsy spheroids to the brain of T cell-compromised Rowett (nude) rats has been established as a representative animal model for human GBMs, with a tumor take rate close to 100%. In immunocompetent littermates however, primary human GBM tissue is invariably rejected. Here we show that after repeated passaging cycles in nude rats, human GBM spheroids are enabled to grow in the brain of immunocompetent rats. In case of engraftment, xenografts in immunocompetent rats grow progressively and host leukocytes fail to enter the tumor bed, similar to what is seen in nude animals. In contrast, rejection is associated with massive infiltration of the tumor bed by leukocytes, predominantly ED1+ microglia/macrophages, CD4+ T helper cells and CD8+ effector cells, and correlates with elevated serum levels of pro-inflammatory cytokines IL-1α, IL-18 and TNF-α [corrected]. We observed that in nude rat brains, an adaptation to the host occurs after several in vivo passaging cycles, characterized by striking attenuation of microglial infiltration. Furthermore, tumor-derived chemokines that promote leukocyte migration and their entry into the CNS such as CXCL-10 and CXCL-12 are down-regulated, and the levels of TGF-ß2 increase. We propose that through serial in vivo passaging in nude rats, human GBM cells learn to avoid and or/ suppress host immunity. Such adapted GBM cells are in turn able to engraft in immunocompetent rats without signs of an inflammatory response.

Selected Aspects in the Pathogenesis of Autoimmune Diseases.

Autoimmune processes can be found in physiological circumstances. However, they are quenched with properly functioning regulatory mechanisms and do not evolve into full-blown autoimmune diseases. Once developed, autoimmune diseases are characterized by signature clinical features, accompanied by sustained cellular and/or humoral immunological abnormalities. Genetic, environmental, and hormonal defects, as well as a quantitative and qualitative impairment of immunoregulatory functions, have been shown in parallel to the relative dominance of proinflammatory Th17 cells in many of these diseases. In this review we focus on the derailed balance between regulatory and Th17 cells in the pathogenesis of autoimmune diseases. Additionally, we depict a cytokine imbalance, which gives rise to a biased T-cell homeostasis. The assessment of Th17/Treg-cell ratio and the simultaneous quantitation of cytokines, may give a useful diagnostic tool in autoimmune diseases. We also depict the multifaceted role of dendritic cells, serving as antigen presenting cells, contributing to the development of the pathognomonic cytokine signature and promote cellular and humoral autoimmune responses. Finally we describe the function and role of extracellular vesicles in particular autoimmune diseases. Targeting these key players of disease progression in patients with autoimmune diseases by immunomodulating therapy may be beneficial in future therapeutic strategies.

Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids.

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.

In vivo bioluminescence imaging validation of a human biopsy-derived orthotopic mouse model of glioblastoma multiforme.

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.

A novel, diffusely infiltrative xenograft model of human anaplastic oligodendroglioma with mutations in FUBP1, CIC, and IDH1.

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.

EGFR wild-type amplification and activation promote invasion and development of glioblastoma independent of angiogenesis.

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.

In vivo animal models for studying brain metastasis: value and limitations.

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.

In vivo models of primary brain tumors: pitfalls and perspectives.

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.

Cellular host responses to gliomas.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: Human glioma cell lines and glioma spheroid orthotopic implants were used in rodents. In both models, the xenografts recruited large numbers of host nestin-expressing cells, which formed a 'network' with glioma. The host nestin-expressing cells appeared to originate in the subventricular zone ipsilateral to the tumor, and were clearly distinguishable from pericytes that expressed smooth muscle actin. These distinct cell populations established close physical contact in a 'pair-wise' manner and migrated together to the deeper layers of tumor satellites and gave rise to tumor vasculature. The GBM biopsy xenografts displayed two different phenotypes: (a) low-generation tumors (first in vivo passage in rats) were highly invasive and non-angiogenic, and host nestin-positive cells that infiltrated into these tumors displayed astrocytic or elongated bipolar morphology; (b) high-generation xenografts (fifth passage) had pronounced cellularity, were angiogenic with 'glomerulus-like' microvascular proliferations that contained host nestin-positive cells. Stromal cell-derived factor-1 and its receptor CXCR4 were highly expressed in and around glioma xenografts, suggesting their role in glioma progression and invasion. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate a robust migration of nestin-expressing host cells to glioma, which together with pericytes give rise to tumor vasculature. Mapping the cellular composition of glioma microenvironment and deciphering the complex 'crosstalk' between tumor and host may ultimately aid the development of novel anti-glioma therapies.

The regulation of cysteine cathepsins and cystatins in human gliomas.

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.

NUMB does not impair growth and differentiation status of experimental gliomas.

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.

A reproducible brain tumour model established from human glioblastoma biopsies.

BACKGROUND: 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. METHODS: In this work, we collected data on growth kinetics from a material of 29 biopsies xenografted in nude rats, and characterised this model with an emphasis on neuropathological and radiological features. RESULTS: The tumour take rate for xenografted GBM biopsies were 96% and remained close to 100% at subsequent passages in vivo, whereas only one of four lower grade tumours engrafted. Average time from transplantation to the onset of symptoms was 125 days +/- 11.5 SEM. Histologically, the primary xenografts recapitulated the invasive features of the parent tumours while endothelial cell proliferations and necrosis were mostly absent. After 4-5 in vivo passages, the tumours became more vascular with necrotic areas, but also appeared more circumscribed. MRI typically revealed changes related to tumour growth, several months prior to the onset of symptoms. CONCLUSIONS: In vivo passaging of patient GBM biopsies produced tumours representative of the patient tumours, with high take rates and a reproducible disease course. The model provides combinations of angiogenic and invasive phenotypes and represents a good alternative to in vitro propagated cell lines for dissecting mechanisms of brain tumour progression.

Remission of invasive, cancer stem-like glioblastoma xenografts using lentiviral vector-mediated suicide gene therapy.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: We used a rodent xenograft model that recapitulates the invasive and angiogenic features of human glioblastoma to analyze the transduction pattern and therapeutic efficacy of lentiviral pseudotyped vectors. Both, lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) and vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors very efficiently transduced human glioblastoma cells in vitro and in vivo. In contrast, pseudotyped gammaretroviral vectors, similar to those evaluated for clinical therapy of glioblastoma, showed inefficient gene transfer in vitro and in vivo. Both pseudotyped lentiviral vectors transduced cancer stem-like cells characterized by their CD133-, nestin- and SOX2-expression, the ability to form spheroids in neural stem cell medium and to express astrocytic and neuronal differentiation markers under serum conditions. In a therapeutic approach using the suicide gene herpes simplex virus thymidine kinase (HSV-1-tk) fused to eGFP, both lentiviral vectors mediated a complete remission of solid tumors as seen on MRI resulting in a highly significant survival benefit (p<0.001) compared to control groups. In all recurrent tumors, surviving eGFP-positive tumor cells were found, advocating prodrug application for several cycles to even enhance and prolong the therapeutic effect. CONCLUSIONS/SIGNIFICANCE: In conclusion, lentiviral pseudotyped vectors are promising candidates for gene therapy of glioma in patients. The inefficient gene delivery by gammaretroviral vectors is in line with the results obtained in clinical therapy for GBM and thus confirms the high reproducibility of the invasive glioma animal model for translational research.