Neuroblastoma Metastasis Research Articles

Abstract 4344: Membrane-type 1 matrix metalloproteinase-mediated pro-invasive properties of neuroblastoma initiating cells

Abstract Introduction. Neuroblastoma is the most common and deadly extracranial solid tumour of childhood. Unlike localized neuroblastomas which have generally good prognosis, metastatic stages are associated with poor outcome. In this context, increasing evidences suggest that neuroblastoma may contain tumor-initiating cells (TICs) that cause relapse and metastasis. Our recent work indicates that CD133, a putative marker of TICs in various cancers, is associated with chemoresistance and poor outcome in neuroblastoma. In addition, we and others have demonstrated that membrane-type 1 matrix metalloproteinase (MT1-MMP) is associated with neuroblastoma progression and metastasis. Purpose. The purpose of this study was to characterize the tumor-initiating properties of CD133high neuroblastoma cells and determinate the role of MT1-MMP in pro-invasive properties of these cells. Methods/Results. Following CD133high cells selection from neuroblastoma cell lines (SK-N-DZ, SK-N-FI, SK-N-SH and SJ-NB10) with FACSAria cell sorter, we have examined their tumor-initiating features with neurosphere-forming assay in serum-free medium, colony-forming assay in soft agar and orthotopic transplantation into the adrenal gland (major site of primary neuroblastoma) of severe immunocompromised NOD/SCID/IL2Rγ- mice (NSG). Contrary to their counterpart CD133low, CD133high cells formed more colonies in soft agar and develop more neurospheres. Interestingly, orthotopic transplantation of as few as 500 CD133high cells into NSG mice resulted in tumor formation into the adrenal gland and dissemination to liver, lungs, brain and bone marrow, while matched control CD133low presented no tumor, suggesting that CD133high cells are TICs in neuroblastoma. Further western blot analyses of neuroblastoma cell lines revealed that CD133 expression correlated with that of MT1-MMP. In addition, CD133high cells growth within three-dimensional type I collagen matrices was markedly higher than CD133low cells and was significantly reduced by anti-MT1-MMP neutralizing antibodies. In the same vein, we performed cell migration assay in Boyden chamber and observed that CD133high cells exhibit more migratory abilities that CD133low cells, this migration being inhibited by anti-MT1-MMP neutralizing antibodies. Conclusions. Altogether, these findings strongly suggest that CD133-expressing TICs of neuroblastoma exhibit high pro-invasive capacities, requiring the involvement of MT1-MMP. Targeting pro-invasive capacities of neuroblastoma TICs with MMPs-activaed pro-drugs in combination with conventional therapy, represents an attractive therapeutic strategy aimed at eliminating both neuroblastoma TICs and the bulk of the tumour. Acknowledgment. Fondation Centre de cancérologie Charles-Bruneau. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4344. doi:10.1158/1538-7445.AM2011-4344

Abstract 5268: CXCR4 and P-glycoprotein N-glycan patterning in developing cancer cells

Abstract Human CXCR4 and P-glycoprotein (Pgp) are N-glycosylated membrane proteins overexpressed in cancer cells. CXCR4 is thought to be an initiator of neuroblastoma metastasis to bone tissue and is overexpressed in neuroblastomas. Pgp is a heavily glycosylated, ATP-dependent drug efflux pump overexpressed in uterine sarcomas. Pgp is responsible for multidrug resistance in some tumors. Our lab is studying the N-glycan profiles of CXCR4 and Pgp in neuroblastoma (IMR-32) and uterine sarcoma cells (MES-SA and MES-SA/Dx5). We believe CXCR4 and Pgp are closely involved in metastasis. To better understand the molecular mechanisms of the metastatic roles of CXCR4 and Pgp, we are characterizing their respective complements of N-glycans. CXCR4 contains a N-linked oligosaccharide on two separate extracellular loops; moreover, Pgp has 3 N-glycans present on a 9 amino acid stretch near its N-terminus. We believe both protein N-glycan moieties are differentially glycosylated enough to act as markers for identifying and staging cancer cells. Our previous studies have shown that Pgp overexpressed in MES-SA/Dx5 cells contains at least two different complex N-glycans; a high mannose tree and a hybrid, sialic acid-capped oligosaccharide. To build on our previous studies, we have developed a novel approach to further characterize CXCR4 and Pgp N-glycans using polyclonal antibodies (pAbs) raised to the specific amino acid sequences containing the sugars in both cancer proteins. We are using these inimitable pAbs to aid in the isolation, sequencing, and profiling of specific N-glycan changes that are associated with the stage of each of the cancer cell lines mentioned above. Our pAb panels bind the native, expressed glycoforms of CXCR4 and Pgp in cells and on Western blots. We are using pAb affinity columns to capture protease-released glycopeptides. Once captured, we intend to sequence each of the released glycopeptides’ oligosaccharides to verify their saccharide sequences in the different cancer cell stages. Furthermore, we predict that the resultant glycopeptide gel and blot patterns will ‘bar code’ the specific cancer stage of the cells lines studied. We envision that these glycopeptide patterns can be used to identify and catalog the various stages of developing cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5268. doi:10.1158/1538-7445.AM2011-5268

Effect of MDM2 and vascular endothelial growth factor inhibition on tumor angiogenesis and metastasis in neuroblastoma

Neuroblastoma is the most common pediatric abdominal tumor and principally a p53 wild-type, highly vascular, aggressive tumor, with limited response to anti-VEGF therapies alone. MDM2 is a key inhibitor of p53 and a positive activator of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) activity with an important role in neuroblastoma pathogenesis. We hypothesized that concurrent inhibition of both MDM2 and VEGF signaling would have cooperative anti-tumor effects, potentiating anti-angiogenic strategies for neuroblastoma and other p53 wild-type tumors. We orthotopically implanted SH-SY5Y neuroblastoma cells into nude mice (n = 40) and treated as follows: control, bevacizumab, Nutlin-3a, combination of bevacizumab plus Nutlin-3a. Expression of HIF-1α and VEGF were measured by qPCR, Western blot, and ELISA. Tumor apoptosis was measured by immunohistochemistry and caspase assay. Angiogenesis was evaluated by immunohistochemistry for vascular markers (CD-31, type-IV collagen, αSMA). Both angiogenesis and metastatic burden were digitally quantified. In vitro, Nutlin-3a suppresses HIF-1α expression with subsequent downregulation of VEGF. Bevacizumab plus Nutlin-3a leads to significant suppression of tumor growth compared to control (P < 0.01) or either agent alone. Combination treated xenograft tumors display a marked decrease in endothelial cells (P < 0.0001), perivascular basement membrane (P < 0.04), and vascular mural cells (P < 0.004). Nutlin-3a alone and in combination with bevacizumab leads to significant tumor apoptosis (P < 0.0001 for both) and significant decrease in incidence of metastasis (P < 0.05) and metastatic burden (P < 0.03). Bevacizumab plus Nutlin-3a cooperatively inhibits tumor growth and angiogenesis in neuroblastoma in vivo with dramatic effects on tumor vascularity. Concomitantly targeting VEGF and p53 pathways potently suppresses tumor growth, and these results support further clinical development of this approach.

Targeting neurotrophin-3 and its dependence receptor tyrosine kinase receptor C: a new antitumoral strategy

Introduction: Tropomyosin-related kinase receptor C (TrkC) is a neurotrophin receptor that belongs to the tyrosine kinase receptor family. This family primarily consists of proto-oncogenes, and TrkC has been involved in oncogenic translocations. However, its expression in tumors is often associated with good prognosis, suggesting it actually acts as a tumor suppressor. TrkC has recently been demonstrated to be a dependence receptor, which regulates neuronal survival. Dependence receptors share the ability to trigger apoptosis in the absence of their ligand, a feature that has been suggested to confer a tumor suppressor function to these receptors. A selective advantage for a tumor cell to survive in an environment with limited ligand availability would hence be either to lose the expression of the dependence receptor, or to gain expression of its ligand.Areas covered: The role of neurotrophin-3 (NT-3) and its dependence receptor TrkC in neuroblastoma, and its suitability as a therapeutic target.Expert opinion: Autocrine production of NT-3 represents a selective advantage for tumor growth and dissemination, in a large fraction of aggressive neuroblastoma. Disruption of the NT-3 autocrine loop in malignant neuroblasts, triggers neuroblastoma cell death, and inhibits neuroblastoma metastasis in animal models. Thus, a novel way of targeting the tyrosine kinase receptor, is via the reactivation of its intrinsic ability to trigger cell death.

Neuroblastoma com metástase orbitária

Neuroblastoma is the most frequent extracranial solid tumor among pediatric patients, and orbital metastatic disease is not uncommon in these children. Physical signs as a consequence of orbital metastases, such as proptosis and periorbital ecchymosis, frequently are encountered. However, subsequent blindness is rare. We report on a 2 year old child with orbital metastasis of a primary neuroblastoma in the left suprarenal region that evaluated to a bilateral blindness. We discuss the importance of the early diagnosis, and more studies [...]

Inhibitory effect of drug-free hybrid liposomes on metastasis of human neuroblastoma

Hybrid liposomes composed of vesicular and micellar molecules have been used as drug-delivery systems. It has become clear that hybrid liposomes alone have an inhibitory effect against the growth of various tumor cells. The present study was designed to determine whether a drug-free hybrid liposome composed of dimyristoylphosphatidylcholine (DMPC) and polyoxyethylenealkyl ether (EO) [90 mol% DMPC/10% C(12)(EO)(21) (HL21), 90 mol% DMPC/10% C(12)(EO)(23) (HL23), or 90 mol% DMPC/10% C(12)(EO)(25) (HL25)], inhibit the liver metastasis of human neuroblastoma cells and thus increases survival. A human neuroblastoma cell, TNB9, and BALB/C-nu/nu athymic mice were used in this study. First, we determined the inhibitory effect of the hybrid liposomes on TNB9 cells in vitro. Next, to determine the inhibitory effect of the hybrid liposomes on metastasis of neuroblastoma cells to the liver, we made a murine hepatic metastasis model by implanting TNB9 cells (2 × 106) in the spleen of the mice and compared anatomic appearance, weights, and histological findings of the livers of treated mice and control mice 60 days after the beginning of a 7-day intraperitoneal injection of a hybrid liposome. We also compared survival rates using the Kaplan-Meier method. In mice implanted with TNB9 neuroblastoma cells and treated with HL21 or HL25, no histological evidence of metastasis was found, the weight of the liver was normal, and survival was a mean of 88 and 87.9 days, respectively. In contrast, mice treated with HL23 and control mice had countless tumor cell masses histologically, their liver weight was increased, and their survival was 73.0 and 68.6 days, respectively. Two kinds of hybrid liposomes, HL21 and HL25, inhibit metastasis of human neuroblastoma cells to the liver, and thus increase survival.

Cooperative Inhibitory Effect Of Bevacizumab And Nutlin-3a on Tumor Growth, Angiogenesis, And Metastasis In An Experimental Model Of Neuroblastoma

Introduction: Vascular endothelial growth factor (VEGF) is critical to tumor angiogenesis, tumor growth and metastasis. However, in neuroblastoma, bevacizumab, a humanized anti-VEGF antibody, has demonstrated limited efficacy alone or in combination with chemotherapy. The oncogene MDM2, a major negative regulator of p53, is also a regulator of hypoxia inducible factor-1α (HIF-1α), and inhibition of MDM2 has been shown in vitro to suppress HIF-1α as well as downstream VEGF expression. We have previously demonstrated that MDM2 inhibition with the small molecule inhibitor, Nutlin-3a, suppresses tumor angiogenesis and tumor growth in an in vivo, orthotopic model of neuroblastoma. Therefore we hypothesize that bevacizumab in combination with Nutlin-3a leads to cooperative inhibition of tumor angiogenesis, tumor growth, and metastasis in an in vivo model of neuroblastoma. Methods: Forty nude mice were intrarenally implanted with the human neuroblastoma cell line, SH-SY5Y. Two weeks after tumor implantation, mice were administered respective treatments for two weeks; control (Nutlin vehicle and bevacizumab vehicle), Nutlin-3a (200 mg/kg/dose BID x 14 days), bevacizumab (5mg/kg/dose BIW x 2 weeks), combination (Nutlin-3a and bevacizumab as above). After two weeks of treatment, all mice were sacrificed. At necropsy, tumors were resected, weighed and preserved. Livers from all mice were resected and preserved. Angiogenesis of preserved xenograft tumors was evaluated by immunohistochemistry for vascular markers, such as endothelial cells (CD-31), perivascular basement membrane (type-IV collagen), and vascular mural cells (αSMA). Tumors were also evaluated for apoptosis. All immunostained tumor tissue underwent digital quantification. Preserved liver tissue underwent H&E staining and histologic analysis for metastasis. Results: The combination of Nutlin-3a and bevacizumab more effectively inhibited xenograft tumor growth than either agent alone when compared to control treatment (79% suppression, p=0.01). Digital quantification of immunostained tumor tissue treated with combination therapy demonstrated significant inhibition of endothelial cells, perivascular basement membrane, and vascular mural cells compared to control treatment tumors (Figure 1). Nutlin-3a alone and in combination with bevacizumab led to significant tumor apoptosis and a significant decrease in both the incidence of metastasis and metastatic burden. Conclusions: We conclude that bevacizumab combined with Nutlin-3a cooperatively inhibits tumor growth, tumor angiogenesis, and metastasis in neuroblastoma. By targeting VEGF and p53 pathways, this novel anti-angiogenic regimen may provide more effective tumor suppression in patients with neuroblastoma and other p53 wild-type tumors.

Unexpected Intraparenchymal Hematoma Caused by Brain Metastasis in a Patient With Neuroblastoma -Case Report-

A 2-year-old girl who had previously been treated for stage IV intra-abdominal neuroblastoma presented with headache and vomiting. Computed tomography (CT) showed hematoma in the right temporal lobe. Two months previously, she had suffered a generalized seizure but brain CT was unremarkable. The hematoma was removed completely without evidence of vascular anomaly or tumor. Four weeks after surgery, magnetic resonance imaging with gadolinium showed a developing mass lesion in the hematoma bed. A second operation for tumor removal confirmed the rare diagnosis of intraparenchymal metastasis. Metastatic neuroblastoma to the central nervous system (CNS) occasionally can cause massive hemorrhage. Therefore, early detection of CNS metastasis can be important. The related factors in this patient with abdominal neuroblastoma included elevated serum lactate dehydrogenase, N-myc gene amplification, and coexisting orbital metastasis, which all occurred within 22 months from initial diagnosis. The median interval from diagnosis of the primary tumor to diagnosis of CNS metastasis in neuroblastoma is 12-22 months, which indicates relatively late metastasis presentation. The initial presentation of this late metastasis is hemorrhage that can have sudden detrimental consequences, so a patient with neuroblastoma must be followed up carefully, particularly if elevated serum lactate dehydrogenase, orbital metastasis, N-myc gene amplification, or clinical symptoms are present, even if brain CT is unremarkable. The understanding that intraparenchymal hematoma can occur prior to detectable CNS metastasis may be important for early detection of this life-threatening condition.

Detection of Bone Marrow Metastases of Neuroblastoma With Immunohistochemical Staining of CD56, Chromogranin A, and Synaptophysin

Neuroblastoma is one of the most common solid tumors occurring in childhood. Bone marrow evaluation is an important part of clinical staging in neuroblastoma patients. In view of the difficulty of detecting neuroblastoma cells with conventional bone marrow aspirate smears and biopsy (BMB) and specimens in cases in which metastasis is not prominent, we propose the use of immunohistochemistry (IHC) as a potential diagnostic tool. We examined 116 BMB and 115 bone marrow clot (BMC) specimens from 60 newly diagnosed neuroblastoma patients for tumor cells. Neuroendocrine IHC markers, such as CD56, chromogranin A, and synaptophysin were applied to diagnose neuroblastoma. Eighteen out of the 60 patients (25.4%) displayed BM metastasis, as observed with conventional hematoxylin and eosin staining. IHC staining of BMC sections was generally more sensitive than that of BMB sections for tumor cell detection. We detected tumors in 5 and 7 additional hematoxylin and eosin-negative BMB and BMC sections, respectively, using CD56. Overall, CD56 or a combination of CD56 and chromogranin A was effective in detecting neuroblastoma cells. IHC analysis of BMB and BMC sections is warranted as a routine component of the diagnostic work-up of neuroblastoma to overcome discrepancies between routine smears and IHC stains.

30 Neuroblastoma and melanoma metastasis: regulation by the tumour microenvironment

Glioblastoma is carcinogenesis of glial cells in central nervous system and has the highest incidence among primary brain tumors. Brain metastasis, such as breast cancer and lung cancer, also leads to high mortality. The available medicines are limited due to blood–brain barrier. Abnormal activation of phosphatidylinositol 3-kinases (PI3K) signaling pathway is prevalent in glioblastoma and metastatic tumors. Here, we characterized a 2-amino-4-methylquinazoline derivative XH30 as a potent PI3K inhibitor with excellent anti-tumor activity against human glioblastoma. XH30 significantly repressed the proliferation of various brain cancer cells and decreased the phosphorylation of key proteins of PI3K signaling pathway, induced cell cycle arrest in G1 phase as well. Additionally, XH30 inhibited the migration of glioma cells and blocked the activation of PI3K pathway by interleukin-17A (IL-17A), which increased the migration of U87MG. Oral administration of XH30 significantly suppressed the tumor growth in both subcutaneous and orthotopic tumor models. XH30 also repressed tumor growth in brain metastasis models of lung cancers. Moreover, XH30 reduced IL-17A and its receptor IL-17RA in vivo. These results indicate that XH30 might be a potential therapeutic drug candidate for glioblastoma migration and brain metastasis.

Abstract 5141: Stathmin mediates neuroblastoma migration and invasion via regulation of the actin network

Abstract Aggressive metastatic neuroblastoma confers a poor clinical prognosis. Stathmin is a microtubule destabilizing protein highly expressed in advanced stage (metastatic) neuroblastoma (1) that promotes metastasis of non-neuronal cancers (2). However, stathmin's role in neuroblastoma metastasis is unclear. The aims of this study were to determine the functional and mechanistic role of stathmin in neuroblastoma metastasis. During metastasis, restructuring of the cell cytoskeleton, in particular the actin network is necessary to drive cell migration/invasion. The Rho-GTPase/LIM kinase/cofilin pathway regulates actin remodeling. Activation of LIM kinases (LIMK1/2) by Rho-GTPases induces actin polymerization via phosphorylation (inactivation) of cofilin. Interestingly, stathmin is also regulated downstream of the Rho-GTPases. We therefore investigated this pathway as a potential mechanism underlying stathmin's role in neuroblastoma cell migration/invasion. Methods: Studies were performed using siRNA to knockdown stathmin in 2 independent neuroblastoma cell lines, BE(2)-C and SY5Y. Stathmin knockdown was confirmed by qPCR and Western blot. SiRNA-treated cell lines were subjected to in vitro migration and invasion assays using fetal calf serum as a chemo-attractant. The expression of actin-regulatory proteins LIMK1/2 and the actin-severing protein cofilin were analyzed by Western blot. Actin filaments within neuroblastoma cells were visualized by phalloidin staining. Results: Stathmin knockdown significantly reduced the migration of BE(2)C (47% reduction p&amp;lt;0.001) and SY5Y (44% reduction p&amp;lt;0.05) cells. Moreover, suppressing stathmin significantly reduced the ability of BE(2)C (62% reduction p&amp;lt;0.05) and SY5Y (55% reduction p&amp;lt;0.05) cells to invade an extra-cellular matrix. Stathmin knockdown BE(2)C cells displayed altered phosphorylation of both LIMK1/2 and cofilin upon serum-activation, compared to control cells. Cofilin phosphorylation was induced 5 minutes post serum activation in both control and stathmin knockdown cells. After 5 minutes, cofilin phosphorylation declined in control cells but was maintained in stathmin knockdown BE(2)C cells up to 60 minutes post serum-activation. The most notable difference was observed 15 minutes post serum-activation, a time point at which dense actin structures were visible at the cell periphery in phalloidin-stained stathmin knockdown cells but not controls cells. These data show that stathmin is a necessary intermediate for actin restructuring required for the migration and invasion of neuroblastoma cells. Conclusion: Stathmin plays a pro-migratory and invasive role via regulation of the actin network in neuroblastoma. These identify stathmin as a promising new therapeutic target to treat metastatic neuroblastoma. 1. N. Hailat et al. J Clin Invest 1991; 88: 341 2. B. Belletti et al. Mol Biol Cell. 2008: 19: 2003 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5141.

Abstract 557: Galectin-3 binding protein produced by neuroblastoma cells stimulates the expression of interleukin-6 in bone marrow mesenchymal cells

Abstract There is recent evidence that bone marrow-derived mesenchymal cells (BMMC) play an important role in bone metastasis in several cancers, including myeloma and neuroblastoma. We previously reported that in neuroblastoma these cells are a source of interleukin-6 (IL-6) that promotes the growth and survival of neuroblastoma cells and bone degradation. We have recently isolated and identified Galectin-3 binding protein (Gal-3BP) as a soluble factor produced by a neuroblastoma cell line as a factor upregulating the production of IL-6 by BMMC. Here we document the production of Gal-3BP in several neuroblastoma cell lines, in particular those without MYCN amplification that correlates with their ability to induce the production of IL-6 in BMMC. The stimulation of IL-6 by Gal-3BP is mediated by Gal-3, a multifunctional glycoprotein that binds Gal-3BP and is present in BMMC. It is also dependent on the activation of the Ras/Raf/MEK/ERK1/2 pathway by Gal-3BP/Gal-3 and is blocked by the ERK1/2 inhibitor PD980559. Transcriptional activation of IL-6 by Gal-3BP/Gal-3 requires a 115 bp region (−97 to −212) in the IL-6 promoter that contains recognition motifs for a cAMP responsive element binding protein (CREB) and a CCAAT/enhancer binding protein (C/EBP). The expression of Gal-3BP was also examined by immunohistochemistry in 38 primary neuroblastoma tumors with (n=9) and without (n=29) MYCN amplification. This analysis revealed that Gal-3BP is paradoxically more abundant in MYCN non-amplified tumors. This unanticipated observation suggests a significant difference in the Gal-3BP/Gal-3/IL-6 pathway between subgroups of neuroblastoma tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 557.

Establishment of subcutaneously transplanted and metastatic neuroblastoma models in nude mice

To establish a tumor-bearing nude mouse model of human neuroblastoma in order to study the mechanisms of neuroblastoma invasion and metastasis, and to investigate potential therapeutic modalities in the experimental animal models. A human neuroblastoma cell line was cultured in vitro. 1 x 10(7) cells undergoing exponential growth were collected in 0.1 ml of suspension and subcutaneously inoculated into the right flank next to the forelimb in nude mice. The biological characteristics of the developed tumors were observed, and histopathological and DNA microarray analyses were performed. The expressions of NSE in the subcutaneous tumor, metastatic tumor and the primary neuroblastoma tumor tissues from a pediatric patient were analyzed by immunohistochemistry. Tumors successfully grew in 36 out of 48 injected mice, with a total tumor-formation rate of 75.0%. Metastasis occurred in 10 cases, and the metastatic rate was 20.8%. Tumors in five injected mice grew locally without metastasis. These tumors had large volume and the tumor weight reached up to half of the body weight of the host animal. Four mice exhibited systemic metastasis without tumor growth at the primary inoculation site. There were six mice with locally growing tumor accompanied by metastasis. We have successfully established a human neuroblastoma xenograft model in nude mice with high tumor growth and metastatic rates. This model depicting the natural cell growth, local infiltration and distant metastasis characteristics of human neuroblastoma, providing an ideal animal model for in vivo studies of neuroblastoma. In addition, the results of this study indicate the heterogeneous nature of neuroblastoma, it may play an important role in metastasis of this tumor.

Identification of miRNAs that are associated with tumor metastasis in Neuroblastoma

The discovery of microRNAs (miRNAs) has opened a new avenue for both diagnosis and treatment of cancers. Accumulating experimental evidences indicate that miRNAs are aberrantly expressed in different tumor types and have a critical role in tumorigenesis. However, the miRNA expression profile in neuroblastoma (NB), one of the most common cancer forms in children, has not been well characterized. In the present study, we established a heterotopic transplant mice model of NB and employed miRNA microarray analysis to identify miRNAs that are differentially expressed in metastatic NB compared to primary NB. A list of 54 miRNAs was found to be significantly altered in metastatic tumors compared to primary tumors. These differentially-expressed miRNAs were selectively validated by a stem-loop qRT-PCR assay. Furthermore, we predicted a list of potential miRNA–target pairs that may be involved in the metastasis of neuroblastoma by three different computer–aided algorithms. Taken together, our results indicate that a unique panel of miRNAs are associated with metastatic NB and these differentially-expressed miRNAs may play an important role in the NB metastatic process. This finding may also shed light in our understanding of the nature of metastatic NB and provide a potential novel target for therapeutic treatment of this tumor.

Gene‐expression‐based analysis of local and metastatic neuroblastoma variants reveals a set of genes associated with tumor progression in neuroblastoma patients

Metastasis is the primary cause of mortality in Neuroblastoma (NB) patients, but the metastatic process in NB is poorly understood. Metastsis is a multistep process that requires the coordinated action of many genes. The identification of genes that promote or suppress tumor metastasis can advance our understanding of this process. In the present study, we utilized a human NB xenograft model comprising local and metastatic NB variants, which was recently developed in our laboratory. We set out to identify molecular correlates of NB metastasis and to determine the clinical relevance of these molecules. We first performed genome-wide expression profiles of metastatic and nonmetastatic NB variants that have an identical genetic background. We found that some of the proteins highly expressed in the metastatic NB variants are localized in the cytoplasm and endoplasmic reticulum. Other proteins are linked to metabolic processes and signaling pathways, thereby supporting the invasive and metastatic state of the cells. Subsequently, we intersected the differentially expressed genes in the human xenografted variants with genes differentially expressed in Stage 1 and Stage 4 primary tumors of NB patients. By using the same gene-expression platform, molecular correlates associated with metastatic progression in primary NB tumors were identified. The resulting smaller gene set was clinically relevant as it discriminated between high- and low-risk NB patients.

Environmental endocrine disruptors promote invasion and metastasis of SK-N-SH human neuroblastoma cells

Neuroblastoma (NB) is the most common pediatric extracranial cancer. Metastasis is the main cause of mortality in NB patients. Currently, little is known about the risk factors and their mechanisms that cause metastasis. Environmental endocrine disruptors (EED) are recently identified risk factors associated with various human diseases including malignant tumors. Our previous studies have implicated the role of di(2-ethylhexyl) phthalate (DEHP) and bisphenol A (BPA), two of the most common EED, in neuroblastoma cell proliferation. Here, we further investigated the effects of DEHP, BPA as well as 17beta-estradiol (E2) on the invasion and metastasis of human neuroblastoma SK-N-SH cells in vitro. SK-N-SH cells expressed estrogen receptor (ER)-beta, matrix metalloproteinases-2 (MMP-2), MMP-9 and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) at readily detectable levels. 50 microM DEHP, 0.1 microM BPA and 10 microM E2 exposure all resulted in enhanced motility and invasiveness of SK-N-SH cells (P<0.001), elevated expression of MMP-2 and MMP-9, and decreased expression of TIMP-2 (P<0.01). Furthermore, phosphorylation of Akt (Ser473) was also induced following the exposure (P<0.01). Importantly, both ER antagonist ICI182,780 and phosphoinositide 3-kinase (PI3K) specific inhibitor LY294002 significantly inhibited the DEHP, BPA, or E2-induced cell migration and invasion, as well as the disregulation of MMP-2, MMP-9 and TIMP-2 expression. ICI182,780 may have worked through abolishing Akt (Ser473) phosphorylation. In conclusion, DEHP, BPA, and E2 potently promote invasion and metastasis of neuroblastoma cells through overexpression of MMP-2 and MMP-9 as well as downregulation of TIMP-2. ER-dependent pathway and PI3K/Akt pathway are involved, which may become potential therapeutic targets for neuroblastoma treatment.

Sustained and NK/CD4+ T Cell-Dependent Efficient Prevention of Lung Metastasis Induced by Dendritic Cells Harboring Recombinant Sendai Virus

We recently demonstrated efficient antitumor immunity against murine tumors using dendritic cells (DCs) activated by recombinant Sendai viruses (rSeVs), and proposed a new concept, "immunostimulatory virotherapy," for cancer immunotherapy. However, there has been little information on the efficacy of this method in preventing metastatic diseases. In this study, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV (rSeV/dF) using a murine model of lung metastasis. Bolus and i.v. administration of DCs harboring rSeV/dF-expressing GFP without pulsation of tumor Ag (DC-rSeV/dF-GFP) 2 days before tumor inoculation showed efficient prevention against lung metastasis of c1300 neuroblastoma, but not of RM-9 prostatic cancer. We found that the timing of DC therapy was critical for the inhibition of pulmonary metastasis of RM-9, and that the optimal effect of DCs was seen 28 days before tumor inoculation. Interestingly, the antimetastatic effect was sustained for over 3 mo, even when administered DCs were already cleared from the lung and organs related to the immune system. Although NK cell activity had already declined to baseline at the time of tumor inoculation, Ab-mediated depletion studies revealed that CD4+ cells as well as the presence of, but not the activation of, NK cells were crucial to the prevention of lung metastasis. These results are the first demonstration of efficient inhibition of lung metastasis via bolus administration of virally activated DCs that was sustained and NK/CD4+ cell-dependent, and may suggest a potentially new mechanism of DC-based immunotherapy for advanced malignancies.

Neuroimaging Patterns of Central Nervous System Metastases in Neuroblastoma: Report of 2 Recent Cases and Literature Review

The authors describe imaging patterns of intracranial metastases in 2 children with grade 4 neuroblastoma. Central nervous system metastases from neuroblastoma are extremely rare and may involve the cerebral parenchyma, leptomeninges, or dura. Cerebral parenchymal metastases can be cystic with mural nodules or solid with hemorrhagic elements. The first patient in our study had multiple cystic parenchymal metastases with calcific mural nodules, while the second patient developed solid hemorrhagic parenchymal metastatic lesions along with extensive leptomeningeal and dural deposits. Central nervous system involvement in both patients occurred within a time span ranging from 12 to 14 months from the time of initial diagnosis.

In vivo evaluation of microspheres containing the angiogenesis inhibitor, TNP-470, and the metastasis suppression with liver metastatic model implanted neuroblastoma

TNP-470 (AGM-1470, O-(chloroacetylcarbamoyl) fumagillol), which strongly inhibits the angiogenesis, is promising as a new drug for tumor dormancy therapy; however, TNP-470 is very unstable in vitro and in vivo. We previously prepared TNP-470 containing microspheres composed of poly (lactic acid) with medium-chain triglyceride, and demonstrated that the microspheres released TNP-470 over the long-term in vitro. The present study was undertaken to evaluate the release profile of TNP-470 in vivo and the inhibitory effect on hepatic metastasis of neuroblastoma. It was found that the microspheres could maintain high levels of TNP-470 in the blood plasma for over 4 weeks in vivo. In addition, hepatic metastasis of neuroblastoma was strongly inhibited at 2 weeks after intraperitoneal injection of the microspheres. Following 2 weeks of treatment, the liver weights of mice injected with TNP-DDS (TNP-DDS (H), and TNP-DDS (L) groups) and those injected with only physiological saline (C-1300 group) after implantation of neuroblastoma cells were 1.18 ± 0.13 g, 1.28 ± 0.10 g, and 2.54 ± 0.97 g, respectively ( p < 0.05; C-1300 group compared with the TNP-DDS (H) and the TNP-DDS (L) groups, respectively). It was evident that microspheres containing TNP-470 have an excellent potential for clinical application in tumor dormancy therapy.

Nódulos subcutáneos como forma de presentación de síndrome linfoproliferativo maligno

Skin involvement in children with malignant processes usually appears at the same time or after the diagnosis of the primary tumour. We present the case of a girl with cutaneous involvement prior to the diagnosis of a malignant lymphoproliferative process. A previously healthy 5-month old girl who presented with an inflammatory subcutaneous lesion on the right foot. During hospital admission due to bronchiolitis at 7 months with associated pancytopenia while the myelogram showed myeloid and megakaryocytic hypoplasia, the abdominal and foot ultrasound were normal. After completing corticoid therapy for her respiratory process and transfusional support, the foot lesion had disappeared at discharge. Two months later she had a local recurrence with associated scattered subcutaneous nodules. The skin biopsy confirmed malignant infiltration; the myelogram showed 6% blast infiltration, and both abdominal ultrasound and CT scan demonstrated lymph node involvement. Immunophenotype confirmed the diagnosis of Precursor B Cell Lymphoblastic Leukemia-Lymphoma. Although complete remission was achieved at the end of the induction chemotherapy according EuroLB-02 protocol for stage IV, the patient presented a refractory leukaemia relapse thirteen months after diagnosis. Commentary: Malignancy should be suspected in the presence of a skin lesion with torpid evolution and biopsy should be considered. Differential diagnosis of malignant skin lesions in children, especially in infants, must include mainly secondary involvement of leukaemia, lymphoma, metastases of neuroblastoma or rhabdomyosarcoma and less frequently other primary processes. In our patient with an isolated cutaneous presentation, the progression of her malignant lymphoproliferative process could be modified by the corticotherapy given before the definitive diagnosis.