MiR-486-5p Targets CD133+ Lung Cancer Stem Cells through the p85/AKT Pathway.

Despite improvements in therapies and screening strategies, lung cancer prognosis still remains dismal, especially for metastatic tumors. Cancer stem cells (CSCs) are endowed with properties such as chemoresistance, dissemination, and stem-like features, that make them one of the main causes of the poor survival rate of lung cancer patients. MicroRNAs (miRNAs), small molecules regulating gene expression, have a role in lung cancer development and progression. In particular, miR-486-5p is an onco-suppressor miRNA found to be down-modulated in the tumor tissue of lung cancer patients. In this study, we investigate the role of this miRNA in CD133+ lung CSCs and evaluate the therapeutic efficacy of coated cationic lipid-nanoparticles entrapping the miR-486-5p miRNA mimic (CCL-486) using lung cancer patient-derived xenograft (PDX) models. In vitro, miR-486-5p overexpression impaired the PI3K/Akt pathway and decreased lung cancer cell viability. Moreover, miR-486-5p overexpression induced apoptosis also in CD133+ CSCs, thus affecting the in vivo tumor-initiating properties of these cells. Finally, we demonstrated that in vivo CCL-486 treatment decreased CD133+ percentage and inhibited tumor growth in PDX models. In conclusion, we provided insights on the efficacy of a novel miRNA-based compound to hit CD133+ lung CSCs, setting the basis for new combined therapeutic strategies.

The Depolarization-Evoked, Ca2+-Dependent Release of Exosomes From Mouse Cortical Nerve Endings: New Insights Into Synaptic Transmission.

Whether exosomes can be actively released from presynaptic nerve terminals is a matter of debate. To address the point, mouse cortical synaptosomes were incubated under basal and depolarizing (25 mM KCl-enriched medium) conditions, and extracellular vesicles were isolated from the synaptosomal supernatants to be characterized by dynamic light scattering, transmission electron microscopy, Western blot, and flow cytometry analyses. The structural and biochemical analysis unveiled that supernatants contain vesicles that have the size and the shape of exosomes, which were immunopositive for the exosomal markers TSG101, flotillin-1, CD63, and CD9. The marker content increased upon the exposure of nerve terminals to the high-KCl stimulus, consistent with an active release of the exosomes from the depolarized synaptosomes. High KCl-induced depolarization elicits the Ca2+-dependent exocytosis of glutamate. Interestingly, the depolarization-evoked release of exosomes from cortical synaptosomes also occurred in a Ca2+-dependent fashion, since the TSG101, CD63, and CD9 contents in the exosomal fraction isolated from supernatants of depolarized synaptosomes were significantly reduced when omitting external Ca2+ ions. Differently, (±)-baclofen (10 µM), which significantly reduced the glutamate exocytosis, did not affect the amount of exosomal markers, suggesting that the GABAB-mediated mechanism does not control the exosome release. Our findings suggest that the exposure of synaptosomes to a depolarizing stimulus elicits a presynaptic release of exosomes that occurs in a Ca2+-dependent fashion. The insensitivity to the presynaptic GABAB receptors, however, leaves open the question on whether the release of exosomes could be a druggable target for new therapeutic intervention for the cure of synaptopathies.

Cotargeting of miR-126-3p and miR-221-3p inhibits PIK3R2 and PTEN, reducing lung cancer growth and metastasis by blocking AKT and CXCR4 signalling.

Lung cancer is the leading cause of cancer-related death worldwide. Late diagnosis and metastatic dissemination contribute to its low survival rate. Since microRNA (miRNA) deregulation triggers lung carcinogenesis, miRNAs might represent an interesting therapeutic tool for lung cancer management. We identified seven miRNAs, including miR-126-3p and miR-221-3p, that are deregulated in tumours compared with normal tissues in a series of 38 non-small-cell lung cancer patients. A negative correlation between these two miRNAs was associated with poor patient survival. Concomitant miR-126-3p replacement and miR-221-3p inhibition, but not modulation of either miRNA alone, reduced lung cancer cell viability by inhibiting AKT signalling. PIK3R2 and PTEN were validated as direct targets of miR-126-3p and miR-221-3p, respectively. Simultaneous miRNA modulation reduced metastatic dissemination of lung cancer cells both in vitro and in vivo through CXCR4 inhibition. Systemic delivery of a combination of miR-126-3p mimic and miR-221-3p inhibitor encapsulated in lipid nanoparticles reduced lung cancer patient-derived xenograft growth through blockade of the PIK3R2-AKT pathway. Our findings reveal that cotargeting miR-126-3p and miR-221-3p to hamper both tumour growth and metastasis could be a new therapeutic approach for lung cancer.

Combined Replenishment of miR-34a and let-7b by Targeted Nanoparticles Inhibits Tumor Growth in Neuroblastoma Preclinical Models.

Neuroblastoma (NB) tumor substantially contributes to childhood cancer mortality. The design of novel drugs targeted to specific molecular alterations becomes mandatory, especially for high-risk patients burdened by chemoresistant relapse. The dysregulated expression of MYCN, ALK, and LIN28B and the diminished levels of miR-34a and let-7b are oncogenic in NB. Due to the ability of miRNA-mimics to recover the tumor suppression functions of miRNAs underexpressed into cancer cells, safe and efficient nanocarriers selectively targeted to NB cells and tested in clinically relevant mouse models are developed. The technology exploits the nucleic acids negative charges to build coated-cationic liposomes, then functionalized with antibodies against GD2 receptor. The replenishment of miR-34a and let-7b by NB-targeted nanoparticles, individually and more powerfully in combination, significantly reduces cell division, proliferation, neoangiogenesis, tumor growth and burden, and induces apoptosis in orthotopic xenografts and improves mice survival in pseudometastatic models. These functional effects highlight a cooperative down-modulation of MYCN and its down-stream targets, ALK and LIN28B, exerted by miR-34a and let-7b that reactivate regulatory networks leading to a favorable therapeutic response. These findings demonstrate a promising therapeutic efficacy of miR-34a and let-7b combined replacement and support its clinical application as adjuvant therapy for high-risk NB patients.

Coated cationic lipid-nanoparticles entrapping miR-660 inhibit tumor growth in patient-derived xenografts lung cancer models.

Lung cancer is the leading cause of cancer-related deaths. Late diagnosis and inadequate therapies contribute to poor outcomes. MicroRNAs (miRNAs) are small non-coding RNAs and are involved in lung cancer development. Because miRNAs simultaneously regulate several cancer-related genes, they represent an interesting therapeutic approach for cancer treatment. We have developed Coated Cationic Lipid-nanoparticles entrapping miR-660 (CCL660) and intraperitoneally administered (1.5 mg/Kg) twice a week for four weeks into SCID mice carrying subcutaneously lung cancer Patients Derived Xenografts (PDXs). Obtained data demonstrated that miR-660 is down-regulated in lung cancer patients and that its replacement inhibited lung cancer growth by inhibiting the MDM2-P53 axis. Furthermore, systemic delivery of CCL660 increased miRNA levels in tumors and significantly reduced tumor growth in two different P53 wild-type PDXs without off-target effects. MiR-660 administration reduced cancer cells proliferation by inhibiting MDM2 and restoring P53 function and its downstream effectors such as p21. Interestingly, anti-tumoral effects of CCL660 also in P53 mutant PDXs but with a functional p21 pathway were observed. Stable miR-660 expression inhibited the capacity of H460 metastatic lung cancer cells to form lung nodules when injected intravenously into SCID mice suggesting a potential role of miR-660 in metastatic dissemination. To investigate the potential toxic effects of both miRNAs and delivery agents, an in vitro approach revealed that miR-660 replacement did not induce any changes in both mouse and human normal cells. Interestingly, lipid-nanoparticle delivery of synthetic miR-660 had no immunological off-target or acute/chronic toxic effects on immunocompetent mice. Altogether, our results highlight the potential role of coated cationic lipid-nanoparticles entrapping miR-660 in lung cancer treatment without inducing immune-related toxic effects.

Overcoming Biological Barriers in Neuroblastoma Therapy: The Vascular Targeting Approach with Liposomal Drug Nanocarriers.

Neuroblastoma is a rare pediatric cancer characterized by a wide clinical behavior and adverse outcome despite aggressive therapies. New approaches based on targeted drug delivery may improve efficacy and decrease toxicity of cancer therapy. Furthermore, nanotechnology offers additional potential developments for cancer imaging, diagnosis, and treatment. Following these lines, in the past years, innovative therapies based on the use of liposomes loaded with anticancer agents and functionalized with peptides capable of recognizing neuroblastoma cells and/or tumor-associated endothelial cells have been developed. Studies performed in experimental orthotopic models of human neuroblastoma have shown that targeted nanocarriers can be exploited for not only decreasing the systemic toxicity of the encapsulated anticancer drugs, but also increasing their tumor homing properties, enhancing tumor vascular permeability and perfusion (and, consequently, drug penetration), inducing tumor apoptosis, inhibiting angiogenesis, and reducing tumor glucose consumption. Furthermore, peptide-tagged liposomal formulations are proved to be more efficacious in inhibiting tumor growth and metastatic spreading of neuroblastoma than nontargeted liposomes. These findings, herein reviewed, pave the way for the design of novel targeted liposomal nanocarriers useful for multitargeting treatment of neuroblastoma.

Enhancement of Tumor Homing by Chemotherapy-Loaded Nanoparticles.

Targeted delivery of anticancer drugs with nanocarriers can reduce side effects and ameliorate therapeutic efficacy. However, poorly perfused and dysfunctional tumor vessels limit the transport of the payload into solid tumors. The use of tumor-penetrating nanocarriers might enhance tumor uptake and antitumor effects. A peptide containing a tissue-penetrating (TP) consensus motif, capable of recognizing neuropilin-1, is here fused to a neuroblastoma-targeting peptide (pep) previously developed. Neuroblastoma cell lines and cells derived from both xenografts and high-risk neuroblastoma patients show overexpression of neuropilin-1. In vitro studies reveal that TP-pep binds cell lines and cells derived from neuroblastoma patients more efficiently than pep. TP-pep, after coupling to doxorubicin-containing stealth liposomes (TP-pep-SL[doxorubicin]), enhances their uptake by cells and cytotoxic effects in vitro, while increasing tumor-binding capability and homing in vivo. TP-pep-SL[doxorubicin] treatment enhances the Evans Blue dye accumulation in tumors but not in nontumor tissues, pointing to selective increase of vascular permeability in tumor tissues. Compared to pep-SL[doxorubicin], TP-pep-SL[doxorubicin] shows an increased antineuroblastoma activity in three neuroblastoma animal models mimicking the growth of neuroblastoma in humans. The enhancement of drug penetration in tumors by TP-pep-targeted nanoparticles may represent an innovative strategy for neuroblastoma.

Targeting Macrophages as a Potential Therapeutic Intervention: Impact on Inflammatory Diseases and Cancer.

Macrophages, cells belonging to the innate immune system, present a high plasticity grade, being able to change their phenotype in response to environmental stimuli. They play central roles during development, homeostatic tissue processes, tissue repair, and immunity. Furthermore, it is recognized that macrophages are involved in chronic inflammation and that they play central roles in inflammatory diseases and cancer. Due to their large involvement in the pathogenesis of several types of human diseases, macrophages are considered to be relevant therapeutic targets. Nanotechnology-based systems have attracted a lot of attention in this field, gaining a pivotal role as useful moieties to target macrophages in diseased tissues. Among the different approaches that can target macrophages, the most radical is represented by their depletion, commonly obtained by means of clodronate-containing liposomal formulations and/or depleting antibodies. These strategies have produced encouraging results in experimental mouse models. In this review, we focus on macrophage targeting, based on the results so far obtained in preclinical models of inflammatory diseases and cancer. Pros and cons of these therapeutic interventions will be highlighted.

A Proof-of-Concept for Epigenetic Therapy of Tissue Fibrosis: Inhibition of Liver Fibrosis Progression by 3-Deazaneplanocin A.

The progression of fibrosis in chronic liver disease is dependent upon hepatic stellate cells (HSCs) transdifferentiating to a myofibroblast-like phenotype. This pivotal process is controlled by enzymes that regulate histone methylation and chromatin structure, which may be targets for developing anti-fibrotics. There is limited pre-clinical experimental support for the potential to therapeutically manipulate epigenetic regulators in fibrosis. In order to learn if epigenetic treatment can halt the progression of pre-established liver fibrosis, we treated mice with the histone methyltransferase inhibitor 3-deazaneplanocin A (DZNep) in a naked form or by selectively targeting HSC-derived myofibroblasts via an antibody-liposome-DZNep targeting vehicle. We discovered that DZNep treatment inhibited multiple histone methylation modifications, indicative of a broader specificity than previously reported. This broad epigenetic repression was associated with the suppression of fibrosis progression as assessed both histologically and biochemically. The anti-fibrotic effect of DZNep was reproduced when the drug was selectively targeted to HSC-derived myofibroblasts. Therefore, the in vivo modulation of HSC histone methylation is sufficient to halt progression of fibrosis in the context of continuous liver damage. This discovery and our novel HSC-targeting vehicle, which avoids the unwanted effects of epigenetic drugs on parenchymal liver cells, represents an important proof-of-concept for epigenetic treatment of liver fibrosis.

A new fluorescence-based optical imaging method to non-invasively monitor hepatic myofibroblasts in vivo.

BACKGROUND & AIMS: Currently, staging of fibrosis in preclinical rodent liver fibrosis models is achieved histologically. Many animals are used at multiple time-points to assess disease progression or therapeutic responses. Hepatic myofibroblasts promote liver fibrosis therefore quantifying these cells in vivo could assess disease or predict therapeutic responses in mice. We fluorescently labelled a single chain antibody (C1-3) that binds hepatic myofibroblasts to monitor fibrogenesis in vivo. METHODS: CCl4 was used to induce acute liver injury in WT and cRel(-/-) mice. Bile duct ligation was used to model chronic fibrosis. Hepatic myofibroblasts were depleted using a liposome-drug delivery system or chemically with sulfasalazine. An IVIS® spectrum visualised fluorophore-conjugated C1-3 in vivo. RESULTS: IVIS detection of fluorescently labelled-C1-3 but not a control antibody discriminates between fibrotic and non-fibrotic liver in acute and chronic liver fibrosis models. cRel(-/-) mice have a fibro-protective phenotype and IVIS signal is reduced in CCl4 injured cRel(-/-) mice compared to wild-type. In vivo imaging of fluorescently labelled-C1-3 successfully predicts reductions in hepatic myofibroblast numbers in fibrotic liver disease in response to therapy. CONCLUSIONS: We report a novel fluorescence imaging method to assess murine hepatic myofibroblast numbers in vivo during liver fibrosis and after therapy. We also describe a novel liposomal antibody targeting system to selectively deliver drugs to hepatic myofibroblasts in vivo. C1-3 binds human hepatic myofibroblast therefore imaging labelled-C1-3 could be used for clinical studies in man to help stage fibrosis, demonstrate efficacy of drugs that promote hepatic myofibroblast clearance or predict early therapeutic responses. LAY SUMMARY: In response to damage and injury scars develop in the liver and the main cell that makes the scar tissue is the hepatic myofibroblast (HM). C1-3 is an antibody fragment that binds to the scar forming HM. We have fluorescently labelled C1-3 and given it to mice that have either normal or scarred livers (which contain HM) and then used a machine called an in vivo imaging system (IVIS) that takes pictures of different wavelengths of light, to visualise the antibody binding to HM inside the living mouse. Using fluorescently labelled C1-3 we can assess HM numbers in the injured liver and monitor response to therapy. We have also used C1-3 to target drugs encapsulated in lipid carriers (liposomes) to the HM to kill the HM and reduce the liver disease.

Neuroblastoma-targeted nanocarriers improve drug delivery and penetration, delay tumor growth and abrogate metastatic diffusion.

Selective tumor targeting is expected to enhance drug delivery and to decrease toxicity, resulting in an improved therapeutic index. We have recently identified the HSYWLRS peptide sequence as a specific ligand for aggressive neuroblastoma, a childhood tumor mostly refractory to current therapies. Here we validated the specific binding of HSYWLRS to neuroblastoma cell suspensions obtained either from cell lines, animal models, or Schwannian-stroma poor, stage IV neuroblastoma patients. Binding of the biotinylated peptide and of HSYWLRS-functionalized fluorescent quantum dots or liposomal nanoparticles was dose-dependent and inhibited by an excess of free peptide. In animal models obtained by the orthotopic implant of either MYCN-amplified or MYCN single copy human neuroblastoma cell lines, treatment with HSYWLRS-targeted, doxorubicin-loaded Stealth Liposomes increased tumor vascular permeability and perfusion, enhancing tumor penetration of the drug. This formulation proved to exert a potent antitumor efficacy, as evaluated by bioluminescence imaging and micro-PET, leading to (i) delay of tumor growth paralleled by decreased tumor glucose consumption, and (ii) abrogation of metastatic spreading, accompanied by absence of systemic toxicity and significant increase in the animal life span. Our findings are functional to the design of targeted nanocarriers with potentiated therapeutic efficacy towards the clinical translation.

The Neuronal Pentraxin-2 Pathway Is an Unrecognized Target in Human Neuroblastoma, Which Also Offers Prognostic Value in Patients.

Neuronal pentraxins (NPTX) and their corresponding receptors (NPTXR) have been studied as synapse-associated proteins in the nervous system, but their role in cancer is largely unknown. By applying a multidisciplinary, high-throughput proteomic approach, we have recently identified a peptide ligand motif for targeted drug delivery to neuroblastoma. Here, we report the sequence similarity between this peptide and a conserved portion of the pentraxin domain that is involved in the homo- and hetero-oligomerization of NPTX2 and NPTXR. We show that, in comparison with normal tissues, NPTX2 and NPTXR are overexpressed in vivo in mouse models, as well as in human Schwannian stroma-poor, stage IV neuroblastoma. Both proteins are concentrated in the vicinity of tumor blood vessels, with NPTXR also present on neuroblastic tumor cells. In vivo targeting of NPTX2 and NPTXR with the selected peptide or with specific antibodies reduces tumor burden in orthotopic mouse models of human neuroblastoma. In vitro interference with this ligand/receptor system inhibits the organization of neuroblastoma cells in tumor-like masses in close contact with vascular cells, as well as their adhesion to normal microenvironment-derived cells, suggesting a role in the cross-talk between tumor and normal cells in the early steps of neuroblastoma development. Finally, we show that NPTX2 is a marker of poor prognosis for neuroblastoma patients.

New therapeutic strategies in neuroblastoma: combined targeting of a novel tyrosine kinase inhibitor and liposomal siRNAs against ALK.

Many different aberrations in the Anaplastic Lymphoma Kinase (ALK) were found to be oncogenic drivers in several cancers including neuroblastoma (NB), therefore ALK is now considered a critical player in NB oncogenesis and a promising therapeutic target. The ALK-inhibitor crizotinib has a limited activity against the various ALK mutations identified in NB patients. We tested: the activity of the novel ALK-inhibitor X-396 administered alone or in combination with Targeted Liposomes carrying ALK-siRNAs (TL[ALK-siRNA]) that are active irrespective of ALK gene mutational status; the pharmacokinetic profiles and the biodistribution of X-396; the efficacy of X-396 versus crizotinib treatment in NB xenografts; whether the combination of X-396 with the TL[ALK-siRNA] could promote long-term survival in NB mouse models. X-396 revealed good bioavailability, moderate half-life, high mean plasma and tumor concentrations. X-396 was more effective than crizotinib in inhibiting in vitro cell proliferation of NB cells and in reducing tumor volume in subcutaneous NB models in a dose-dependent manner. In orthotopic NB xenografts, X-396 significantly increased life span independently of the ALK mutation status. In combination studies, all effects were significantly improved in the mice treated with TL[ALK-siRNA] and X-396 compared to mice receiving the single agents. Our findings provide a rational basis to design innovative molecular-based treatment combinations for clinical application in ALK-driven NB tumors.

Tumor vascular targeted liposomal-bortezomib minimizes side effects and increases therapeutic activity in human neuroblastoma.

Neuroblastoma is a childhood cancer with poor long-term prognosis in advanced stages. A major aim in neuroblastoma therapy is to develop targeted drug delivery systems to ameliorate drug therapeutic index and efficacy. In this study, a novel bortezomib (BTZ) liposomal formulation was set-up and characterized. Since BTZ is freely permeable across the lipidic bilayer, an amino-lactose (LM) was synthesized as complexing agent to entrap BTZ inside the internal aqueous compartment of stealth liposomes. High encapsulation efficiency was achieved by a loading method based on the formation of boronic esters between the boronic acid moiety of BTZ and the hydroxyl groups of LM. Next, NGR peptides were linked to the liposome surface as a targeting-ligand for the tumor endothelial cell marker, aminopeptidase N. Liposomes were characterized for size, Z-potential, polydispersity index, drug content, and release. Lyophilization in the presence of cryoprotectants (trehalose, sucrose) was also examined in terms of particle size changes and drug leakage. BTZ was successfully loaded into non-targeted (SL[LM-BTZ]) and targeted (NGR-SL[LM-BTZ]) liposomes with an entrapment efficiency of about 68% and 57%, respectively. These nanoparticles were suitable for intravenous administration, presenting an average diameter of 170nm and narrow polydispersity. Therefore, orthotopic NB-bearing mice were treated with 1.0 or 1.5mg/kg of BTZ, either in free form or encapsulated into liposomes. BTZ loaded liposomes showed a significant reduction of drug systemic adverse effects with respect to free drug, even at the highest dose tested. Moreover, mice treated with 1.5mg/kg of NGR-SL[LM-BTZ] lived statistically longer than untreated mice (P=0.0018) and SL[LM-BTZ]-treated mice (P=0.0256). Our results demonstrate that the novel vascular targeted BTZ formulation is endowed with high therapeutic index and low toxicity, providing a new tool for future applications in neuroblastoma clinical studies.

Quiescent hepatic stellate cells functionally contribute to the hepatic innate immune response via TLR3.

Toll-like Receptor 3 (TLR3) is a pathogen pattern recognition receptor that plays a key role in innate immunity. TLR3 signalling has numerous functions in liver, both in health and disease. Here we report that TLR3 is expressed by quiescent hepatic stellate cells (HSC) where it functions to induce transcription and secretion of functional interferons as well as a number of other cytokines and chemokines. Upon transdifferentiation into myofibroblasts, HSCs rapidly loose the ability to produce interferon gamma (IFNγ). Mechanistically, this gene silencing may be due to Polycomb complex mediated repression via methylation of histone H3 lysine 27. In contrast to wild type, quiescent HSC isolated from tlr3 knockout mice do not produce IFNγ in response to Poly(I∶C) treatment. Therefore, quiescent HSC may contribute to induction of the hepatic innate immune system in response to injury or infection.

Nanocarrier-mediated targeting of tumor and tumor vascular cells improves uptake and penetration of drugs into neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children, accounting for about 8% of childhood cancers. Despite aggressive treatment, patients suffering from high-risk NB have very poor 5-year overall survival rate, due to relapsed and/or treatment-resistant tumors. A further increase in therapeutic dose intensity is not feasible, because it will lead to prohibitive short-term and long-term toxicities. New approaches with targeted therapies may improve efficacy and decrease toxicity. The use of drug delivery systems allows site specific delivery of higher payload of active agents associated with lower systemic toxicity compared to the use of conventional ("free") drugs. The possibility of imparting selectivity to the carriers to the cancer foci through the use of a targeting moiety (e.g., a peptide or an antibody) further enhances drug efficacy and safety. We have recently developed two strategies for increasing local concentration of anti-cancer agents, such as CpG-containing oligonucleotides, small interfering RNAs, and chemotherapeutics in NB. For doing that, we have used the monoclonal antibody anti-disialoganglioside (GD2), able to specifically recognize the NB tumor and the peptides containing NGR and CPRECES motifs, that selectively bind to the aminopeptidase N-expressing endothelial and the aminopeptidase A-expressing perivascular tumor cells, respectively. The review will focus on the use of tumor- and tumor vasculature-targeted nanocarriers to improve tumor targeting, uptake, and penetration of drugs in preclinical models of human NB.

Enhanced anti-tumor and anti-angiogenic efficacy of a novel liposomal fenretinide on human neuroblastoma.

Neuroblastoma is an embryonal tumor originating from the simpatico-adrenal lineage of the neural crest. It approximately accounts for about 15% of all pediatric oncology deaths. Despite advances in multimodal therapy, metastatic neuroblastoma tumors at diagnosis remain a clinical challenge. Retinoids are a class of compounds known to induce both terminal differentiation and apoptosis/necrosis of neuroblastoma cells. Among them, fenretinide (HPR) has been considered one of the most promising anti-tumor agent but it is partially efficacious due to both poor aqueous solubility and rapid metabolism. Here, we have developed a novel HPR formulation, by which the drug was encapsulated into sterically stabilized nanoliposomes (NL[HPR]) according to the Reverse Phase Evaporation method. This procedure led to a higher structural integrity of liposomes in organic fluids for a longer period of time, in comparison with our previous liposomal formulation developed by the film method. Moreover, NL[HPR] were further coupled with NGR peptides for targeting the tumor endothelial cell marker, aminopeptidase N (NGR-NL[HPR]). Orthotopically xenografted neuroblastoma-bearing mice treated with NGR-NL[HPR] lived statistically longer than mice untreated or treated with free HPR (NGR-NL[HPR] vs both control and HPR: P<0.0001). Also, NL[HPR] resulted in a statistically improved survival (NL[HPR] vs both control and HPR: P<0.001) but to a less extent if compared with that obtained with NGR-NL[HPR] (NGR-NL[HPR] vs NL[HPR]: P<0.01). Staining of tumor sections with antibodies specific for neuroblastoma and for either pericytes or endothelial cells evidenced that HPR reduced neuroblastoma growth through both anti-tumor and anti-angiogenic effects, mainly when delivered by NGR-NL[HPR]. Indeed, in this group of mice a marked reduction of tumor progression, of intra-tumoral vessel counts and VEGF expression, together with a marked down-modulation of matrix metalloproteinases MMP2 and MMP9, was observed. In conclusion, the use of this novel targeted delivery system for the apoptotic and antiangiogenic drug, fenretinide, could be considered as an adjuvant tool in the future treatment of neuroblastoma patients.

Novel phage display-derived neuroblastoma-targeting peptides potentiate the effect of drug nanocarriers in preclinical settings.

Molecular targeting of drug delivery nanocarriers is expected to improve their therapeutic index while decreasing their toxicity. Here we report the identification and characterization of novel peptide ligands specific for cells present in high-risk neuroblastoma (NB), a childhood tumor mostly refractory to current therapies. To isolate such targeting moieties, we performed combined in vitro/ex-vivo phage display screenings on NB cell lines and on tumors derived from orthotopic mouse models of human NB. By designing proper subtractive protocols, we identified phage clones specific either for the primary tumor, its metastases, or for their respective stromal components. Globally, we isolated 121 phage-displayed NB-binding peptides: 26 bound the primary tumor, 15 the metastatic mass, 57 and 23 their respective microenvironments. Of these, five phage clones were further validated for their specific binding ex-vivo to biopsies from stage IV NB patients and to NB tumors derived from mice. All five clones also targeted tumor cells and vasculature in vivo when injected into NB-bearing mice. Coupling of the corresponding targeting peptides with doxorubicin-loaded liposomes led to a significant inhibition in tumor volume and enhanced survival in preclinical NB models, thereby paving the way to their clinical development.

The use of the orthotopic model to validate antivascular therapies for cancer.

The orthotopic model reproduces aspects of the tumour microenvironment and emulates a number of important biological features of cancer progression, angiogenesis, metastasis and resistance. Due to its parallels with human cancer, the model can be used to evaluate therapeutic responses to various therapies. This review outlines the importance of using the orthotopic implantation of tumour cells in mice models for evaluating the effectiveness of antivascular therapies.

Selective therapeutic targeting of the anaplastic lymphoma kinase with liposomal siRNA induces apoptosis and inhibits angiogenesis in neuroblastoma.

The anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is involved in the pathogenesis of different types of human cancers, including neuroblastoma (NB). In NB, ALK overexpression, or point mutations, are associated with poor prognosis and advanced stage disease. Inhibition of ALK kinase activity by small-molecule inhibitors in lung cancers carrying ALK translocations has shown therapeutic potential. However, secondary mutations may occur that, generate tumor resistance to ALK inhibitors. To overcome resistance to ALK inhibitors in NB, we adopted an alternative RNA interference (RNAi)-based therapeutic strategy that is able to knockdown ALK, regardless of its genetic status [mutated, amplified, wild-type (WT)]. NB cell lines, transduced by lentiviral short hairpin RNA (shRNA), showed reduced proliferation and increased apoptosis when ALK was knocked down. In mice, a nanodelivery system for ALK-specific small interfering RNA (siRNA), based on the conjugation of antibodies directed against the NB-selective marker GD(2) to liposomes, showed strong ALK knockdown in vivo in NB cells, which resulted in cell growth arrest, apoptosis, and prolonged survival. ALK knockdown was associated with marked reductions in vascular endothelial growth factor (VEGF) secretion, blood vessel density, and matrix metalloproteinases (MMPs) expression in vivo, suggesting a role for ALK in NB-induced neoangiogenesis and tumor invasion, confirming this gene as a fundamental oncogene in NB.