Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Sonic Hedgehog (SHH) signaling drives a minority of MB, correlating with desmoplastic pathology and favorable outcome. The majority, however, arises independently of SHH and displays classic or large cell anaplastic (LCA) pathology and poor prognosis. To identify common signaling abnormalities, we profiled mRNA, demonstrating misexpression of MYCN in the majority of human MB and negligible expression in normal cerebella. We clarified a role in pathogenesis by targeting MYCN (and luciferase) to cerebella of transgenic mice. MYCN-driven MB showed either classic or LCA pathologies, with Shh signaling activated in~5% of tumors, demonstrating that MYCN can drive MB independently of Shh. MB arose at high penetrance, consistent with a role for MYCN in initiation. Tumor burden correlated with bioluminescence, with rare metastatic spread to the leptomeninges, suggesting roles for MYCN in both progression and metastasis. Transient pharmacological down-regulation of MYCN led to both clearance and senescence of tumor cells, and improved survival. Targeted expression of MYCN thus contributes to initiation, progression, and maintenance of MB, suggesting a central role for MYCN in pathogenesis.

Brain hemorrhage is a severe complication of both neoplastic and nonneoplastic brain disease. Mice deficient in the alpha(v)beta8 integrin display defective brain vessel formation resulting in hemorrhage and perinatal death, but the mechanism of brain hemorrhage is unknown. Because the alpha(v)beta8 integrin is expressed by astrocytes and not expressed by endothelium, paracrine interactions between astrocytes and endothelial cells could contribute to the maintenance of brain vessel integrity. We have investigated the mechanisms underlying astrocytic-endothelial paracrine signaling and have found that integrin-mediated activation of transforming growth factor (TGF)-beta by astrocytes influences endothelial cell function. Thus, we identified the integrin alpha(v)beta8 in human perivascular glial cell processes surrounding developing blood vessels. Human astrocytic alpha(v)beta8 was a major cell surface receptor for latent TGF-beta, and alpha(v)beta8-dependent activation of TGF-beta was the major mechanism of TGF-beta activation in primary cultures of astrocytes or freshly dissociated fetal brain cells. This activation of TGF-beta was sufficient to inhibit endothelial migration in fibrin gels and to alter expression of genes affecting proteolytic and angiogenic pathways. Taken together, our data suggest that astrocytic alpha(v)beta8 acts as a central regulator of brain vessel homeostasis through regulation of TGF-beta activation and expression of TGF-beta-responsive genes that promote vessel differentiation and stabilization, most notably plasminogen activator inhibitor-1 and thrombospondin-1.

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Like many tumors, malignant mesothelioma exhibits significant chemoresistance and resistance to apoptosis in vivo that is not seen in current in vitro models. To study the mechanisms of this multicellular resistance, biologically relevant in vitro models are necessary. Therefore, we characterized and tested human mesothelioma tissue grown in vitro as tumor fragment spheroids. After 5-10 d in culture, fragments from each of 15 human mesothelioma tumors rounded into spheroids. The tumor fragment spheroids maintained multiple characteristics of the original tumors for up to 3 mo including the presence of viable mesothelioma cells, macrophages, and a collagenrich stroma. In 14-d-old spheroids, mesothelioma cells showed the same proliferation rate and expression of a death receptor, DR5, as in the original tumor. To determine responses to treatment, we treated tumor fragment spheroids grown from three separate tumors with agents, TNF-related apoptosis-inducing ligand (TRAIL) plus cycloheximide, that induced near total apoptosis in three human mesothelioma cell lines (M28, REN, MS-1) grown as monolayers (94 Ϯ 6% apoptosis; mean Ϯ SEM). Compared with mesothelioma cells in monolayers, mesothelioma cells in the spheroids were resistant to TRAIL plus cycloheximide (32 Ϯ 4% apoptosis; mean Ϯ SEM). Apoptotic resistance of mesothelioma cells was significantly reduced by inhibiting either the PI3K/Akt pathway with LY294002 (47 Ϯ 6% apoptosis) or the mTOR pathway with rapamycin (50 Ϯ 17% apoptosis). We conclude that human mesothelioma can be maintained in vitro in a biologically relevant model that exhibits apoptotic resistance, thereby permitting study of its tumor biology and of novel approaches to therapy. Keywords: collagen; death receptor DR5; mTOR; multicellular resistance; PI3K/Akt survival pathway; TNF-related apoptosis-inducing ligand (TRAIL); tumor-associated macrophage; tumor fragment spheroid Resistance to apoptosis, or programmed cell death, is now considered to be a critical step in the generation and maintenance of cancer (1). Resistance to apoptosis may underlie the resistance of tumors to chemotherapy and radiotherapy (2). Mechanisms of resistance have been identified on a cellular level, for example via P-glycoprotein efflux pumps, DNA repair mechanisms, or from expression of anti-apoptotic proteins such as Bcl-2 (3). Additional mechanisms of resistance are now recognized to involve stimuli from the cell's external environment, termed multicellular resistance (3). These multicellular resistance mechanisms have been attributed to cell-cell contacts, cell-matrix contacts, and the three-dimensional shape found in tissues but (Received in original form November 14, 2004 and in final form July 30, 2005) *These authors contributed equally to this work. Two types of in vitro models used to study the complex resistance found in tumors are multicellular spheroids and tumor fragment spheroids (5, 6). In the first, cells are allowed to grow into three-dimensional structures called multicellular spheroids (5). In thes...

GBL withdrawal can be managed in ambulatory settings, but needs to be backed up with seamless access to in-patient treatment if required.