Marco Soster

Combined targeting of perivascular and endothelial tumor cells enhances anti-tumor efficacy of liposomal chemotherapy in neuroblastoma.

The therapeutic index of anti-cancer drugs is increased when encapsulating them in tumor-targeted liposomes. Liposome-entrapped doxorubicin (DXR), targeting the tumor vasculature marker, aminopeptidase N (APN), displayed enhanced anti-tumor effects and prolonged survival in human neuroblastoma (NB)-bearing mice. Here we exploited a peptide ligand of aminopeptidase A (APA), discovered by phage display technology for delivery of liposomal DXR to perivascular tumor cells. Immunohistochemistry, performed in NB-bearing mice, showed APA expression in the vascular wall of NB primary and metastatic lesions. APA-targeted peptides displayed specific binding to APA-transfected cells in vitro, and also accumulation in the tumor of NB-bearing mice. Consequently, novel, APA-targeted, DXR-liposomes were developed and in vivo proof-of-principle was established, alone and in combination with APN-targeted DXR-loaded liposomes, in NB-bearing mice. Mice receiving APA-targeted liposomal DXR exhibited an increased life span in comparison to control mice, but to a lesser extent relative to that in mice treated with APN-targeted formulation, moreover the greatest increase in TUNEL-positive tumor cells was observed in animals treated with APN-targeted formulations. Mice treated with a combination of APA- and APN-targeted, liposomal DXR had a significant increase in life span compared to each treatment administered separately. There was a significant increase in the level of apoptosis in the tumors of mice on the combination therapy, and a pronounced destruction of the tumor vasculature with nearly total ablation of endothelial cells and pericytes. The availability of novel ligands binding to additional tumor vasculature-associated antigens will allow the design of sophisticated combinations of ligand-targeted liposomal anti-cancer drugs.

The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 Upregulation in Osteosarcoma Preclinical Models

Purpose: The multikinase inhibitor sorafenib displays antitumor activity in preclinical models of osteosarcoma. However, in sorafenib-treated patients with metastatic-relapsed osteosarcoma, disease stabilization and tumor shrinkage were short-lived and drug resistance occurred. We explored the sorafenib treatment escape mechanisms to overcome their drawbacks. Experimental Design: Immunoprecipitation, Western blotting, and immunohistochemistry were used to analyze the mTOR pathway [mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2)]. Cell viability, colony growth, and cell migration were evaluated in different osteosarcoma cell lines (MNNG-HOS, HOS, KHOS/NP, MG63, U-2OS, SJSA-1, and SAOS-2) after scalar dose treatment with sorafenib (10–0.625 μmol/L), rapamycin-analog everolimus (100–6.25 nmol/L), and combinations of the two. Cell cycle, reactive oxygen species (ROS) production, and apoptosis were assessed by flow cytometry. Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice injected with MNNG-HOS cells were used to determine antitumor and antimetastatic effects. Angiogenesis and vascularization were evaluated in vitro by exploiting endothelial branching morphogenesis assays and in vivo in xenografted mice and chorioallantoic membranes. Results: After sorafenib treatment, mTORC1 signaling was reduced (downstream target P-S6), whereas mTORC2 was increased (phospho-mTOR Ser2481) in MNNG-HOS xenografts compared with vehicle-treated mice. Combining sorafenib with everolimus resulted in complete abrogation of both mTORC1 [through ROS-mediated AMP-activated kinase (AMPK) activation] and mTORC2 (through complex disassembly). The sorafenib/everolimus combination yielded: (i) enhanced antiproliferative and proapoptotic effects, (ii) impaired tumor growth, (iii) potentiated antiangiogenesis, and (iv) reduced migratory and metastatic potential. Conclusion: mTORC2 activation is an escape mechanism from sorafenib treatment. When sorafenib is combined with everolimus, its antitumor activity is increased by complete inhibition of the mTOR pathway in the preclinical setting. Clin Cancer Res; 19(8); 2117–31. ©2013 AACR.

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.

Targeted dual-color silica nanoparticles provide univocal identification of micrometastases in preclinical models of colorectal cancer

Background and methods Despite the recent introduction of targeted bio-drugs, the scarcity of successful therapeutic options for advanced colorectal cancer remains a limiting factor in patient management. The efficacy of curative surgical interventions can only be extended through earlier detection of metastatic foci, which is dependent on both the sensitivity and specificity of the diagnostic tools. Results We propose a high-performance imaging platform based on silica-poly(ethylene glycol) nanoparticles doped with rhodamine B and cyanine 5. Simultaneous detection of these dyes is the basis for background subtraction and signal amplification, thus providing high-sensitivity imaging. The functionalization of poly(ethylene glycol) tails on the external face of the nanoparticles with metastasis-specific peptides guarantees their homing to and accumulation at target tissues, resulting in specific visualization, even of submillimetric metastases. Conclusions The results reported here demonstrate that our rationally designed modular nanosystems have the ability to produce a breakthrough in the detection of micrometastases for subsequent translation to clinics in the immediate future.

A complex of α6 integrin and E‐cadherin drives liver metastasis of colorectal cancer cells through hepatic angiopoietin‐like 6

Homing of colorectal cancer (CRC) cells to the liver is a non‐random process driven by a crosstalk between tumour cells and components of the host tissue. Here we report the isolation of a liver metastasis‐specific peptide ligand (CGIYRLRSC) that binds a complex of E‐cadherin and α6 integrin on the surface of CRC cells. We identify angiopoietin‐like 6 protein as a peptide‐mimicked natural ligand enriched in hepatic blood vessels of CRC patients. We demonstrate that an interaction between hepatic angiopoietin‐like 6 and tumoural α6 integrin/E‐cadherin drives liver homing and colonization by CRC cells, and that CGIYRLRSC inhibits liver metastasis through interference with this ligand/receptor system. Our results indicate a mechanism for metastasis whereby a soluble factor accumulated in normal vessels functions as a specific ligand for circulating cancer cells. Consistently, we show that high amounts of coexpressed α6 integrin and E‐cadherin in primary tumours represent a poor prognostic factor for patients with advanced CRC.

Nanobubbles: a promising efficienft tool for therapeutic delivery

In recent decades ultrasound-guided delivery of drugs loaded on nanocarriers has been the focus of increasing attention to improve therapeutic treatments. Ultrasound has often been used in combination with microbubbles, micron-sized spherical gas-filled structures stabilized by a shell, to amplify the biophysical effects of the ultrasonic field. Nanometer size bubbles are defined nanobubbles. They were designed to obtain more efficient drug delivery systems. Indeed, their small sizes allow extravasation from blood vessels into surrounding tissues and ultrasound-targeted site-specific release with minimal invasiveness. Additionally, nanobubbles might be endowed with improved stability and longer residence time in systemic circulation. This review will describe the physico-chemical properties of nanobubbles, the formulation parameters and the drug loading approaches, besides potential applications as a therapeutic tool.

Molecularly imprinted cyclodextrin nanosponges for the controlled delivery of L-DOPA: perspectives for the treatment of Parkinson's disease.

ABSTRACT Background: L-DOPA is an amino acid precursor to the neurotransmitter dopamine that is extensively used as a prodrug for the treatment of Parkinson’s disease. However, L-DOPA is an unstable compound: when exposed to light or added to aqueous solutions, it may degrade, compromising its therapeutic properties. Methods: In this work, a new type of drug-loaded cyclodextrin-based nanosponge, obtained using molecular imprinting, is described for the prolonged and controlled release of L-DOPA. The molecularly imprinted nanosponges (MIP-NSs) were synthesized by cross-linking β-cyclodextrin with 1,1ʹ-carbonyldiimidazole in DMF in the presence of L-DOPA as a template molecule. TGA, DSC and FTIR analyses were performed to characterize the interactions between L-DOPA and the two nanosponge structures. Quantitative NMR spectroscopy was used to determine the amount and the affinity of L-DOPA entrapped in the nanosponges. The in vitro L-DOPA release kinetics from the NSs were quantitatively determined by HPLC analysis. Results: The MIP-NSs show a slower and more prolonged release profile than the non-imprinted nanosponges. No degradation of the L-DOPA hosted in the MIP-NSs was observed after long-term storage at room temperature. Conclusions: The MIP-NSs are a promising alternative for the storage and controlled delivery of L-DOPA.

The combination of sorafenib and everolimus shows antitumor activity in preclinical models of malignant pleural mesothelioma

BackgroundMalignant Pleural Mesothelioma (MPM) is an aggressive tumor arising from mesothelial cells lining the pleural cavities characterized by resistance to standard therapies. Most of the molecular steps responsible for pleural transformation remain unclear; however, several growth factor signaling cascades are known to be altered during MPM onset and progression. Transducers of these pathways, such as PIK3CA-mTOR-AKT, MAPK, and ezrin/radixin/moesin (ERM) could therefore be exploited as possible targets for pharmacological intervention. This study aimed to identify ‘druggable’ pathways in MPM and to formulate a targeted approach based on the use of commercially available molecules, such as the multikinase inhibitor sorafenib and the mTOR inhibitor everolimus.MethodsWe planned a triple approach based on: i) analysis of immunophenotypes and mutational profiles in a cohort of thoracoscopic MPM samples, ii) in vitro pharmacological assays, ii) in vivo therapeutic approaches on MPM xenografts. No mutations were found in ‘hot spot’ regions of the mTOR upstream genes (e.g. EGFR, KRAS and PIK3CA).ResultsPhosphorylated mTOR and ERM were specifically overexpressed in the analyzed MPM samples. Sorafenib and everolimus combination was effective in mTOR and ERM blockade; exerted synergistic effects on the inhibition of MPM cell proliferation; triggered ROS production and consequent AMPK-p38 mediated-apoptosis. The antitumor activity was displayed when orally administered to MPM-bearing NOD/SCID mice.ConclusionsERM and mTOR pathways are activated in MPM and ‘druggable’ by a combination of sorafenib and everolimus. Combination therapy is a promising therapeutic strategy against MPM.

Antitumor Activity of Src Inhibitor Saracatinib (AZD-0530) in Preclinical Models of Biliary Tract Carcinomas

Biliary tract carcinoma (BTC) has a poor prognosis due to limited treatment options. There is, therefore, an urgent need to identify new targets and to design innovative therapeutic approaches. Among potential candidate molecules, we evaluated the nonreceptor tyrosine kinase Src, observing promising antitumor effects of its small-molecule inhibitor saracatinib in BTC preclinical models. The presence of an active Src protein was investigated by immunohistochemistry in 19 surgical samples from patients with BTC. Upon saracatinib treatment, the phosphorylation of Src and of its downstream transducers was evaluated in the BTC cell lines TFK-1, EGI-1, HuH28, and TGBC1-TKB. The effect of saracatinib on proliferation and migration was analyzed in these same cell lines, and its antitumor activity was essayed in EGI-1 mouse xenografts. Saracatinib-modulated transcriptome was profiled in EGI-1 cells and in tumor samples of the xenograft model. Src was activated in about 80% of the human BTC samples. In cultured BTC cell lines, low-dose saracatinib counteracted the activation of Src and of its downstream effectors, increased the fraction of cells in G0–G1 phase, and inhibited cell migration. At high concentrations (median dose from 2.26–6.99 μmol/L), saracatinib was also capable of inhibiting BTC cell proliferation. In vivo, saracatinib treatment resulted in delayed tumor growth, associated with an impaired vascular network. Here, we provide a demonstration that the targeted inhibition of Src kinase by saracatinib is of therapeutic benefit in preclinical models of BTC. We propose our results as a basis for the design of saracatinib-based clinical applications. Mol Cancer Ther; 11(7); 1528–38. ©2012 AACR.

Dextran-shelled oxygen-loaded nanodroplets reestablish a normoxia-like pro-angiogenic phenotype and behavior in hypoxic human dermal microvascular endothelium

In chronic wounds, hypoxia seriously undermines tissue repair processes by altering the balances between pro-angiogenic proteolytic enzymes (matrix metalloproteinases, MMPs) and their inhibitors (tissue inhibitors of metalloproteinases, TIMPs) released from surrounding cells. Recently, we have shown that in human monocytes hypoxia reduces MMP-9 and increases TIMP-1 without affecting TIMP-2 secretion, whereas in human keratinocytes it reduces MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. Provided that the phenotype of the cellular environment is better understood, chronic wounds might be targeted by new oxygenating compounds such as chitosan- or dextran-shelled and 2H,3H-decafluoropentane-cored oxygen-loaded nanodroplets (OLNs). Here, we investigated the effects of hypoxia and dextran-shelled OLNs on the pro-angiogenic phenotype and behavior of human dermal microvascular endothelium (HMEC-1 cell line), another cell population playing key roles during wound healing. Normoxic HMEC-1 constitutively released MMP-2, TIMP-1 and TIMP-2 proteins, but not MMP-9. Hypoxia enhanced MMP-2 and reduced TIMP-1 secretion, without affecting TIMP-2 levels, and compromised cell ability to migrate and invade the extracellular matrix. When taken up by HMEC-1, nontoxic OLNs abrogated the effects of hypoxia, restoring normoxic MMP/TIMP levels and promoting cell migration, matrix invasion, and formation of microvessels. These effects were specifically dependent on time-sustained oxygen diffusion from OLN core, since they were not achieved by oxygen-free nanodroplets or oxygen-saturated solution. Collectively, these data provide new information on the effects of hypoxia on dermal endothelium and support the hypothesis that OLNs might be used as effective adjuvant tools to promote chronic wound healing processes.