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Highlights • Growth factor signaling causes sustained nuclear ERK1/2 activation. • The SCR and BCR/ABL inhibitor dasatinib blocks ERK1/2 and represses cell invasion. • EGF-stimulated cells may escape dasatinib inhibition of invasion through mesenchymal to amoeboid transition. • Combined inhibition of SRC and Rho-kinase signaling is necessary to completely block EGF-induced invasion., Aberrantly activated kinase signaling pathways drive invasion and dissemination in medulloblastoma (MB). A majority of tumor-promoting kinase signaling pathways feed into the mitogen-activated protein kinase (MAPK) extracellular regulated kinase (ERK1/2) pathway. The activation status of ERK1/2 during invasion of MB cells is not known and its implication in invasion control unclear. We established a synthetic kinase activation relocation sensor (SKARS) for the MAPK ERK1/2 pathway in MB cells for real-time measuring of drug response. We used 3D invasion assays and organotypic cerebellum slice culture to test drug effects in a physiologically relevant tissue environment. We found that hepatocyte growth factor (HGF), epidermal growth factor (EGF), or basic fibroblast growth factor (bFGF) caused rapid nuclear ERK1/2 activation in MB cells, which persisted for several hours. Concomitant treatment with the BCR/ABL kinase inhibitor dasatinib completely repressed nuclear ERK1/2 activity induced by HGF and EGF but not by bFGF. Increased nuclear ERK1/2 activity correlated positively with speed of invasion. Dasatinib blocked ERK-associated invasion in the majority of cells, but we also observed fast-invading cells with low ERK1/2 activity. These ERK1/2-low, fast-moving cells displayed a rounded morphology, while ERK-high fast-moving cells displayed a mesenchymal morphology. Dasatinib effectively blocked EGF-induced proliferation while it only moderately repressed tissue invasion, indicating that a subset of cells may evade invasion repression by dasatinib through non-mesenchymal motility. Thus, growth factor-induced nuclear activation of ERK1/2 is associated with mesenchymal motility and proliferation in MB cells and can be blocked with the BCR/ABL kinase inhibitor dasatinib.

Local infiltration and distal dissemination of tumor cells hamper efficacy of current treatments against central nervous system (CNS) tumors and greatly influence mortality and therapy-induced long-term morbidity in survivors. A number of in vitro and ex vivo assay systems have been established to better understand the infiltration and metastatic processes, to search for molecules that specifically block tumor cell infiltration and metastatic dissemination and to pre-clinically evaluate their efficaciousness. These systems allow analytical testing of tumor cell viability and motile and invasive capabilities in simplified and well-controlled environments. However, the urgent need for novel anti-metastatic therapies has provided an incentive for the further development of not only classical in vitro methods but also of novel, physiologically more relevant assay systems including organotypic brain slice culture. In this review, using publicly available peer-reviewed primary research and review articles, we provide an overview of a selection of in vitro and ex vivo techniques widely used to study growth and dissemination of primary metastatic brain tumors. Furthermore, we discuss how our steadily increasing knowledge of tumor biology and the tumor microenvironment could be integrated to improve current research methods for metastatic brain tumors. We believe that such rationally improved methods will ultimately increase our understanding of the biology of brain tumors and facilitate the development of more efficacious anti-metastatic treatments.

Medulloblastoma (MB) is a paediatric cancer of the cerebellum that can develop cerebellar and leptomeningeal metastases. Local brain tissue infiltration, the underlying cause of metastasis and relapse, remains unexplored. We developed a novel approach to investigate tissue infiltration of MB using organotypic cerebellum slice culture (OCSC). We show that cellular and structural components of cerebellar tissue in OCSCs are maintained for up to 30 days ex vivo, and that OCSCs foster tumour growth and cell proliferation. Using cell-based models of sonic hedgehog (SHH) and group 3 (G3) MB, we quantified tumour growth and infiltration and determined the morphological characteristics of the infiltrating cells. We observed basal levels of dissemination occurring in both subgroups with cells migrating either individually or collectively as clusters. Collective cerebellar tissue infiltration of SHH MB cells was further enhanced by EGF but not HGF, demonstrating differential tumour cell responses to microenvironmental cues. We found G3 cells to be hyper proliferative and observed aggressive tumour expansion even in the absence of exogenous growth factors. Our study thus provides unprecedented insights into brain tissue infiltration of SHH and G3 MB cells and reveals the cellular basis of the tumour progressing functions of EGF in SHH MB.

Local tissue infiltration of Medulloblastoma (MB) tumor cells precedes metastatic disease but little is still known about intrinsic regulation of migration and invasion in these cells. We found that MAP4K4, a pro-migratory Ser/Thr kinase, is overexpressed in 30% of primary MB tumors and that increased expression is particularly associated with the frequently metastatic SHH β subtype. MAP4K4 is a driver of migration and invasion downstream of c-Met, which is transcriptionally up-regulated in SHH MB. Consistently, depletion of MAP4K4 in MB tumor cells restricts HGF-driven matrix invasion in vitro and brain tissue infiltration ex vivo. We show that these pro-migratory functions of MAP4K4 involve the activation of the integrin β-1 adhesion receptor and are associated with increased endocytic uptake. The consequent enhanced recycling of c-Met caused by MAP4K4 results in the accumulation of activated c-Met in cytosolic vesicles, which is required for sustained signaling and downstream pathway activation. The parallel increase of c-Met and MAP4K4 expression in SHH MB could predict an increased potential of these tumors to infiltrate brain tissue and cause metastatic disease. Molecular targeting of the underlying accelerated endocytosis and receptor recycling could represent a novel approach to block pro-migratory effector functions of MAP4K4 in metastatic cancers.

The microenvironment shapes cell behavior and determines metastatic outcomes of tumors. We addressed how microenvironmental cues control tumor cell invasion in pediatric medulloblastoma (MB). We show that bFGF promotes MB tumor cell invasion through FGF receptor (FGFR) in vitro and that blockade of FGFR represses brain tissue infiltration in vivo. TGF-β regulates pro-migratory bFGF function in a context-dependent manner. Under low bFGF, the non-canonical TGF-β pathway causes ROCK activation and cortical translocation of ERK1/2, which antagonizes FGFR signaling by inactivating FGFR substrate 2 (FRS2), and promotes a contractile, non-motile phenotype. Under high bFGF, negative-feedback regulation of FRS2 by bFGF-induced ERK1/2 causes repression of the FGFR pathway. Under these conditions, TGF-β counters inactivation of FRS2 and restores pro-migratory signaling. These findings pinpoint coincidence detection of bFGF and TGF-β signaling by FRS2 as a mechanism that controls tumor cell invasion. Thus, targeting FRS2 represents an emerging strategy to abrogate aberrant FGFR signaling.

Medulloblastoma (MB) is a paediatric cancer of the cerebellum that can develop cerebellar and leptomeningeal metastases. Local brain tissue infiltration, the underlying cause of metastasis and relapse, remains unexplored. We developed a novel approach to investigate tissue infiltration of MB using organotypic cerebellum slice culture (OCSC). We show that cellular and structural components of cerebellar tissue in OCSCs are maintained for up to 30 days ex vivo, and that OCSCs foster tumour growth and cell proliferation. Using cell-based models of sonic hedgehog (SHH) and group 3 (G3) MB, we quantified tumour growth and infiltration and determined the morphological characteristics of the infiltrating cells. We observed basal levels of dissemination occurring in both subgroups with cells migrating either individually or collectively as clusters. Collective cerebellar tissue infiltration of SHH MB cells was further enhanced by EGF but not HGF, demonstrating differential tumour cell responses to microenvironmental cues. We found G3 cells to be hyper proliferative and observed aggressive tumour expansion even in the absence of exogenous growth factors. Our study thus provides unprecedented insights into brain tissue infiltration of SHH and G3 MB cells and reveals the cellular basis of the tumour progressing functions of EGF in SHH MB.

AIMS As 4-day-old mice of the severe spinal muscular atrophy (SMA) model (dying at 5-8 days) display pronounced neuromuscular changes in the diaphragm but not the soleus muscle, we wanted to gain more insight into the relationship between muscle development and the emergence of pathological changes and additionally to analyse intercostal muscles which are affected in human SMA. METHODS Structures of muscle fibres and neuromuscular junctions (NMJs) of the diaphragm, intercostal and calf muscles of prenatal (E21) and postnatal (P0 and P4) healthy and SMA mice were analysed by light and transmission electron microscopy. NMJ innervation was studied by whole mount immunofluorescence in diaphragms of P4 mice. RESULTS During this period, the investigated muscles still show a significant neck-to-tail developmental gradient. The diaphragm and calf muscles are most and least advanced, respectively, with respect to muscle fibre fusion and differentiation. The number and depth of subsynaptic folds increases, and perisynaptic Schwann cells (PSCs) acquire a basal lamina on their outer surface. Subsynaptic folds are connected to an extensive network of tubules and beaded caveolae, reminiscent of the T system in adult muscle. Interestingly, intercostal muscles from P4 SMA mice show weaker pathological involvement (that is, vacuolization of PSCs and perineurial cells) than those previously described by us for the diaphragm, whereas calf muscles show no pathological changes. CONCLUSION SMA-related alterations appear to occur only when the muscles have reached a certain developmental maturity. Moreover, glial cells, in particular PSCs, play an important role in SMA pathogenesis.

Spinal muscular atrophy (SMA) is characterized by motoneuron loss and muscle weakness. However, the structural and functional deficits that lead to the impairment of the neuromuscular system remain poorly defined. By electron microscopy, we previously found that neuromuscular junctions (NMJs) and muscle fibres of the diaphragm are among the earliest affected structures in the severe mouse SMA model. Because of certain anatomical features, i.e. its thinness and its innervation from the cervical segments of the spinal cord, the diaphragm is particularly suitable to characterize both central and peripheral events. Here we show by immunohistochemistry that, at postnatal day 3, the cervical motoneurons of SMA mice receive less stimulatory synaptic inputs. Moreover, their mitochondria become less elongated which might represent an early stage of degeneration. The NMJs of the diaphragm of SMA mice show a loss of synaptic vesicles and active zones. Moreover, the partly innervated endplates lack S100 positive perisynaptic Schwann cells (PSCs). We also demonstrate the feasibility of comparing the proteomic composition between diaphragm regions enriched and poor in NMJs. By this approach we have identified two proteins that are significantly upregulated only in the NMJ-specific regions of SMA mice. These are apoptosis inducing factor 1 (AIFM1), a mitochondrial flavoprotein that initiates apoptosis in a caspase-independent pathway, and four and a half Lim domain protein 1 (FHL1), a regulator of skeletal muscle mass that has been implicated in several myopathies.

How pediatric medulloblastoma (MB), a metastatic tumor of the cerebellum, infiltrates the brain tissue prior to recurrence or distant spreading is poorly understood. We hypothesized that growth factors released from the tumor microenvironment trigger tumor cell migration and brain tissue infiltration. Using a medium-throughput in vitro 3D cell migration screen and ex vivo organotypic cerebellum slice culture, we identified and validated novel potential drug targets for preventing brain tissue infiltration and growth of MB. We found that the FGFR1 ligand bFGF, which we detected in the tumor microenvironment of clinical samples and in organotypic cultures, promoted tumor cell infiltration through the adaptor protein FRS2. Interestingly, FRS2 function was antagonized by TGFβ, which induced a contractile, non-motile cell phenotype through ROCK activation and direct FRS2 repression. In the absence of TGFβ, however, bFGF-FRS2 function attenuated ROCK and enabled motility and invasiveness through Rac-PAK and ERK1/2. In contrast, at high concentrations of bFGF as observed in certain areas of MB tumor tissue, TGFβ rescued tumor cell motility impeded by a FGFR1-dependent negative feed-back and triggered invasiveness. Using organotypic cerebellum slice culture, we demonstrated that pharmacological inhibition of FGFR or genetic ablation of FRS2 prevented infiltration and proliferation of MB cells. Using a computer-assisted molecular design approach, we are currently exploring low molecular weight compounds that might selectively block FRS2 function. Together, we describe an antagonistic crosstalk between bFGF and TGFβ signaling pathways in MB that tunes the motile and invasive properties of the tumor cells for brain infiltration under varying environmental conditions. Furthermore, we identified the FRS2 adaptor protein as a novel and promising drug target in this process.

As 4-day-old mice of the severe spinal muscular atrophy (SMA) model (dying at 5-8 days) display pronounced neuromuscular changes in the diaphragm but not the soleus muscle, we wanted to gain more insight into the relationship between muscle development and the emergence of pathological changes and additionally to analyse intercostal muscles which are affected in human SMA.Structures of muscle fibres and neuromuscular junctions (NMJs) of the diaphragm, intercostal and calf muscles of prenatal (E21) and postnatal (P0 and P4) healthy and SMA mice were analysed by light and transmission electron microscopy. NMJ innervation was studied by whole mount immunofluorescence in diaphragms of P4 mice.During this period, the investigated muscles still show a significant neck-to-tail developmental gradient. The diaphragm and calf muscles are most and least advanced, respectively, with respect to muscle fibre fusion and differentiation. The number and depth of subsynaptic folds increases, and perisynaptic Schwann cells (PSCs) acquire a basal lamina on their outer surface. Subsynaptic folds are connected to an extensive network of tubules and beaded caveolae, reminiscent of the T system in adult muscle. Interestingly, intercostal muscles from P4 SMA mice show weaker pathological involvement (that is, vacuolization of PSCs and perineurial cells) than those previously described by us for the diaphragm, whereas calf muscles show no pathological changes.SMA-related alterations appear to occur only when the muscles have reached a certain developmental maturity. Moreover, glial cells, in particular PSCs, play an important role in SMA pathogenesis.