New targeting agent for the selective drug delivery of nanocarriers for treating neuroblastoma

Novel targeting agents against neuroblastoma based on the meta-iodobenzylguanidine (MIBG) moiety were synthesized and biologically evaluated for nanocarrier vectorization. These compounds have been anchored on the surface of drug loaded mesoporous silica nanocarriers, resulting in the improved cellular uptake in tumoral cells. Neuroblastoma (NB) is the most frequent extracranial pediatric tumor. Advanced forms of the disease (metastatic and/or refractory) have a dismal prognosis despite the combination of chemotherapy, radiotherapy, surgery and bone narrow transplants. These treatments carry severe side effects and, in some cases, compromise the life of the patient. MIBG has been widely applied in the medical diagnosis of NB due to its affinity for tumor cells through the norepinephrine transporter (NET), which is expressed in 90% of NB tumors. The exclusive accumulation of MIBG in neuroblastoma has been widely studied; however, its properties have been never exploited as a targeting agent in nanocarrier drug delivery systems. Several structural analogues of MIBG have been prepared and attached on the surface of nanocarriers. Their selective internalization has been tested against human neuroblastoma cells, which show, in the best case, cellular uptake four times higher than that of the naked nanosystem. Furthermore, in vivo experiments showed preferential and selective accumulation and retention of the targeted nanosystem comparing with the naked and only PEGylated counterpart systems. This novel nanosystem could be easily applicable to all kinds of drug delivery nanocarriers, providing a universal tool for neuroblastoma chemotherapies that is superior to classical approaches through a novel nanosystem exclusively designed to target this terrible malignancy

Threshold responses to morphogen gradients by zero-order ultrasensitivity

Translating a graded morphogen distribution into tight response borders is central to all developmental processes. Yet, the molecular mechanisms generating such behavior are poorly understood. During patterning of the Drosophila embryonic ventral ectoderm, a graded mitogen-activated protein kinase (MAPK) activation is converted into an all-or-none degradation switch of the Yan transcriptional repressor. Replacing the cardinal phosphorylated amino acid of Yan by a phosphomimetic residue allowed its degradation in a MAPK-independent manner, consistent with Yan phosphorylation being the critical event in generating the switch. Several alternative threshold mechanisms that could, in principle, be realized by this phosphorylation, including first order, cooperativity, positive feedback and zero-order ultrasensitivity, were analyzed. We found that they can be distinguished by their kinetics and steady-state responses to Yan overexpression. In agreement with the predictions for zero-order kinetics, an increase in Yan levels did not shift the degradation border, but significantly elevated the time required to reach steady state. We propose that a reversible loop of Yan phosphorylation implements a zero-order ultrasensitivity-like threshold mechanism, with the capacity to form sharp thresholds that are independent of the level of Yan

Hybrid Enzyme-Polymeric Capsules/Mesoporous Silica Nanodevice for In Situ Cytotoxic Agent Generation

A novel nanocarrier based on functionalized mesoporous silica nanoparticles able to transport a non-toxic pro-drug and the enzyme responsible for its activation is presented. This nanodevice is able to generate in situ cytotoxic species once accumulated in the tumoral cell. Enzymes are sensitive macromolecules which can suffer denaturalization in biological media due to the presence of proteases or other aggressive agents. Moreover, the direct attachment of enzymes to the silica surface can reduce their activity by conformational changes or active site blockage. For these reasons, in order to create a robust system able to work in living organisms, the enzymes are previously coated with a protective polymeric shell which allows the attachment on the silica surface preserving their activity. The efficacy of this hybrid nanodevice for antitumoral purposes is tested against several human tumoral cells as neuroblastoma and leukemia showing significant efficacy. It converts this device in a promising candidate for further in vivo studies for oncology therapy

Enrichment of neural-related genes in human mesenchymal stem cells from neuroblastoma patients.

Neuroblastoma (NB) is one of the most common pediatric solid tumors and, like most human cancers, is char-acterized by a broad variety of genomic alterations. Although mesenchymal stem cells (MSCs) are known to interact with cancer cells, the relationship between MSCs and metastatic NB cancer cells in bone marrow (BM) is unknown. To obtain genetic evidence about this interaction, we isolated ΒΜ-derived MSCs from children with NB and compared their global expression patterns with MSCs obtained from normal pediatric donors, using the Agilent 44K microarrays. Significance analysis of microarray results with a false discovery rate (FDR) <5% identified 496 differentially expressed genes showing either a 2-fold upregulation or downregulation between both groups of samples. Comparison of gene ontology categories of differ-entially expressed genes revealed the upregulation of genes categorized as ‘neurological system process’, ‘cell adhesion’, ‘apoptosis’, ‘cell surface receptor linked signal transduction’, ‘intrinsic to membrane’ and ‘extracellular region’. Among the downregulated genes, several immunology-related terms were the most abundant. These findings provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in ΒΜ as well as identify relevant biological processes potentially altered in MSCs in response to NBThis study was supported by grants from the Fondo de Investigaciones Sanitarias (FIS; PI05/2217 and PI08/0029 to J.G.C.), MICINN (PLE2009-0115) and the Madrid Regional Government (S-BIO-0204-2006 and P2010/BMD-2420) in Spain. The experiments were approved by the appropriate committees.S

Molecular scaffolds as double targeting agents for the diagnosis and treatment of Neuroblastoma.

The selective delivery of therapeutic and imaging agents to tumoral cells has been postulated as some of the most important challenges in nanomedicine field. The cellular selectivity of nanocarriers is usually achieved by their surface decoration with targeting moieties able to recognize these cells in a specific manner. Neuroblastoma (NB) is the most frequent extra-cranial pedriatic tumor, almost incurable at advanced stages. Meta-iodo-benzilguanidine (MIBG) is widely used for the diagnosis of this disease due to its strong affinity by the norepinephrine transporter (NET) usually overexpressed on the membrane of the malignant cells. Herein, a complete family of novel Y-shaped scaffolds which contains structural MIBG analogues covalently attached in each end of the Y-structure has been synthesized. The enhanced cellular uptake capacities of these double-targeting ligands have been evaluated in vitro and in vivo yielding one specific Y-shape structure which presents capacity to be engulfed by the malignant cells and be accumulated in the tumoral tissue, significantly higher than the structure which contains only one single targeting agent. This Y-shaped ligand can provide a powerful tool for the current and treatment diagnosis of this disease

Bone marrow mesenchymal stem cells from infants with MLL-AF4+ acute leukemia harbor and express the MLL-AF4 fusion gene

MLL-AF4 fusion is a hallmark genetic abnormality in infant B-acute lymphoblastic leukemia (B-ALL) known to arise in utero. The cellular origin of leukemic fusion genes during human development is difficult to ascertain. The bone marrow (BM) microenvironment plays an important role in the pathogenesis of several hematological malignances. BM mesenchymal stem cells (BM-MSC) from 38 children diagnosed with cytogenetically different acute leukemias were screened for leukemic fusion genes. Fusion genes were absent in BM-MSCs of childhood leukemias carrying TEL-AML1, BCR-ABL, AML1-ETO, MLL-AF9, MLL-AF10, MLL-ENL or hyperdiploidy. However, MLL-AF4 was detected and expressed in BM-MSCs from all cases of MLL-AF4+ B-ALL. Unlike leukemic blasts, MLL-AF4+ BM-MSCs did not display monoclonal Ig gene rearrangements. Endogenous or ectopic expression of MLL-AF4 exerted no effect on MSC culture homeostasis. These findings suggest that MSCs may be in part tumor-related, highlighting an unrecognized role of the BM milieu on the pathogenesis of MLL-AF4+ B-ALL. MLL-AF4 itself is not sufficient for MSC transformation and the expression of MLL-AF4 in MSCs is compatible with a mesenchymal phenotype, suggesting a differential impact in the hematopoietic system and mesenchyme. The absence of monoclonal rearrangements in MLL-AF4+ BM-MSCs precludes the possibility of cellular plasticity or de-differentiation of B-ALL blasts and suggests that MLL-AF4 might arise in a population of prehematopoietic precursors

Patient-derived mesenchymal stem cells as delivery vehicles for oncolytic virotherapy: novel state-of-the-art technology.

Oncolytic virotherapy is gaining interest in the clinic as a new weapon against cancer. In vivo administration of oncolytic viruses showed important limitations that decrease their effectiveness very significantly: the antiviral immune response causes the elimination of the therapeutic effect, and the poor natural ability of oncolytic viruses to infect micrometastatic lesions significantly minimizes the effective dose of virus. This review will focus on updating the technical and scientific foundations of one of the strategies developed to overcome these limitations, ie, using cells as vehicles for oncolytic viruses. Among many candidates, a special type of adult stem cell, mesenchymal stem cells (MSCs), have already been used in the clinic as cell vehicles for oncolytic viruses, partly due to the fact that these cells are actively being evaluated for other indications. MSC carrier cells are used as Trojan horses loaded with oncoviruses, are administered systemically, and release their cargos at the right places. MSCs are equipped with an array of molecules involved in cell arrest in the capillaries (integrins and selectins), migration toward specific parenchymal locations within tissues (chemokine receptors), and invasion and degradation of the extracellular matrix (proteases). In addition to anatomical targeting capacity, MSCs have a well-recognized role in modulating immune responses by affecting cells of the innate (antigen-presenting cells, natural killer cells) and adaptive immune system (effector and regulatory lymphocytes). Therefore, carrier MSCs may also modulate the immune responses taking place after therapy, ie, the antiviral and the antitumor immune responses.S

Patient-derived mesenchymal stem cells as delivery vehicles for oncolytic virotherapy: novel state-of-the-art technology

Oncolytic virotherapy is gaining interest in the clinic as a new weapon against cancer. In vivo administration of oncolytic viruses showed important limitations that decrease their effectiveness very significantly: the antiviral immune response causes the elimination of the therapeutic effect, and the poor natural ability of oncolytic viruses to infect micrometastatic lesions significantly minimizes the effective dose of virus. This review will focus on updating the technical and scientific foundations of one of the strategies developed to overcome these limitations, ie, using cells as vehicles for oncolytic viruses. Among many candidates, a special type of adult stem cell, mesenchymal stem cells (MSCs), have already been used in the clinic as cell vehicles for oncolytic viruses, partly due to the fact that these cells are actively being evaluated for other indications. MSC carrier cells are used as Trojan horses loaded with oncoviruses, are administered systemically, and release their cargos at the right places. MSCs are equipped with an array of molecules involved in cell arrest in the capillaries (integrins and selectins), migration toward specific parenchymal locations within tissues (chemokine receptors), and invasion and degradation of the extracellular matrix (proteases). In addition to anatomical targeting capacity, MSCs have a well-recognized role in modulating immune responses by affecting cells of the innate (antigen-presenting cells, natural killer cells) and adaptive immune system (effector and regulatory lymphocytes). Therefore, carrier MSCs may also modulate the immune responses taking place after therapy, ie, the antiviral and the antitumor immune responses

Mesenchymal stromal cells derived from the bone marrow of acute lymphoblastic leukemia patients show altered BMP4 production: correlations with the course of disease.

The relevance of tumor microenvironment for the development and progression of tumor cells in hematological malignancies has been extensively reported. Identification of factors involved in the information exchange between the malignant cells and the bone marrow mesenchymal stem cells (BM-MSCs) and the knowledge on their functioning may provide important information to eliminate leukemic cells from protective BM niches. We evaluated changes in BM-MSCs obtained from children with acute lymphoblastic leukemia (ALL) at different times in the course of disease. Whereas ALL-MSCs did not exhibit phenotypic changes compared to BM-derived MSCs isolated from healthy donors, they exhibited increased adipogenic capacity. In addition, the viability of healthy CD34+ hematopoietic progenitors was significantly reduced when co-cultured with ALL-MSCs. ALL-MSCs grow less efficiently, although gradually recover normal growth with treatment. Accordingly, proliferation is particularly low in MSCs obtained at diagnosis and in the first days of treatment (+15 days), recovering to control levels after 35 days of treatment. Correlating these results with bone morphogenetic protein 4 (BMP4) production, a molecule demonstrated to affect MSC biology, we found higher production of BMP4 in ALL-MSCs derived from patients over the course of disease but not in those free of leukemia. However, no significant differences in the expression of different members of the BMP4 signaling pathway were observed. Furthermore, an inverse correlation between high levels of BMP4 production in the cultures and MSC proliferation was found, as observed in MSCs derived from patients at diagnosis that produce high BMP4 levels. In addition, co-culturing ALL-MSC with the REH leukemia cell line, but not CD34+ hematopoietic progenitors, powerfully enhanced BMP4 production, suggesting an intimate crosstalk among ALL-MSCs isolated from BM colonized by ALL cells that presumably also occurs in situ conditions. Our data may support the participation of BMP4 in BM niche, but the mechanism remains to be elucidated.This work was supported by grands BFU2009-10315, SAF2012-33180, and BFU2010-18250 from the Spanish Ministry of Science and Innovation, Spanish Association Against Cancer 2010 AECC; P2010/BMD-2420-CellCAM from Regional Government of Madrid and RD12/0019/0007-TerCell Network from the Health Institute Carlos III. MNVG contract UCM number 50053735 by Comunidad de Madrid. AE grant number AP2010-0795 of Ministry of Education, Culture and Sports. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S