Serena Marchiò

Ligand-targeted theranostic nanomedicines against cancer.

Nanomedicines have significant potential for cancer treatment. Although the majority of nanomedicines currently tested in clinical trials utilize simple, biocompatible liposome-based nanocarriers, their widespread use is limited by non-specificity and low target site concentration and thus, do not provide a substantial clinical advantage over conventional, systemic chemotherapy. In the past 20years, we have identified specific receptors expressed on the surfaces of tumor endothelial and perivascular cells, tumor cells, the extracellular matrix and stromal cells using combinatorial peptide libraries displayed on bacteriophage. These studies corroborate the notion that unique receptor proteins such as IL-11Rα, GRP78, EphA5, among others, are differentially overexpressed in tumors and present opportunities to deliver tumor-specific therapeutic drugs. By using peptides that bind to tumor-specific cell-surface receptors, therapeutic agents such as apoptotic peptides, suicide genes, imaging dyes or chemotherapeutics can be precisely and systemically delivered to reduce tumor growth in vivo, without harming healthy cells. Given the clinical applicability of peptide-based therapeutics, targeted delivery of nanocarriers loaded with therapeutic cargos seems plausible. We propose a modular design of a functionalized protocell in which a tumor-targeting moiety, such as a peptide or recombinant human antibody single chain variable fragment (scFv), is conjugated to a lipid bilayer surrounding a silica-based nanocarrier core containing a protected therapeutic cargo. The functionalized protocell can be tailored to a specific cancer subtype and treatment regimen by exchanging the tumor-targeting moiety and/or therapeutic cargo or used in combination to create unique, theranostic agents. In this review, we summarize the identification of tumor-specific receptors through combinatorial phage display technology and the use of antibody display selection to identify recombinant human scFvs against these tumor-specific receptors. We compare the characteristics of different types of simple and complex nanocarriers, and discuss potential types of therapeutic cargos and conjugation strategies. The modular design of functionalized protocells may improve the efficacy and safety of nanomedicines for future cancer therapy.

PRUNE2 is a human prostate cancer suppressor regulated by the intronic long noncoding RNA PCA3.

Significance Prostate cancer has an unpredictable natural history: While most tumors are clinically indolent, some patients display lethal phenotypes. Serum prostate-specific antigen is the most often used test in prostate cancer but screening is controversial. Treatment options are limited for metastatic disease, hence the need for early diagnosis. Prostate cancer antigen 3 (PCA3), a long noncoding RNA, is the most specific biomarker identified and approved as a diagnostic test. However, its inherent biological function (if any) has remained elusive. We uncovered a negative transdominant oncogenic role for PCA3 that down-regulates an unrecognized tumor suppressor gene, PRUNE2 (a human homolog of the Drosophila prune gene) thereby promoting malignant cell growth. This work defines a unique biological function for PCA3 in prostate cancer. Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention.

Identification of CD36 molecular features required for its in vitro angiostatic activity.

Thrombospondin‐1 (TSP‐1), a natural inhibitor of angiogenesis, acts directly on endothelial cells (EC) via CD36 to inhibit their migration and morphogenesis induced by basic fibroblast growth factor. Here we show that CD36 triggered by TSP‐1 inhibits in vitro angiogenesis stimulated by vascular endothelial growth factor‐A (VEGF‐A). To demonstrate that the TSP‐1 inhibitory signal was mediated by CD36, we transduced CD36 in CD36‐deficient endothelial cells. Both TSP‐1 and the agonist anti‐CD36 mAb SMO, which mimics TSP‐1 activity, reduced the VEGF‐A165‐induced migration and sprouting of CD36‐ECs. To address the mechanisms by which CD36 may exert its angiostatic function, we investigated the functional components of the C‐terminal cytoplasmic tail by site‐directed mutagenesis. Our results indicate that C464, R467, and K469 of CD36 are required for the inhibitory activity of TSP‐1. In contrast, point mutation of C466 did not alter TSP‐1 ability to inhibit EC migration and sprouting. Moreover, we show that activation of CD36 by TSP‐1 down‐modulates the VEGF receptor‐2 (VEGFR‐2) and p38 mitogen‐associated protein kinase phosphorylation induced by VEGF‐A165, and this effect was specifically abolished by point mutation at C464. These results identify specific amino acids of the C‐terminal cytoplasmic tail of CD36 crucial for the in vitro angiostatic activity of TSP‐1 and extend our knowledge of regulation of VEGFR‐2‐mediated biological activities on ECs.

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.

Luminescent Silica Nanoparticles for Cancer Diagnosis

Fluorescence imaging techniques are becoming essential for preclinical investigations, necessitating the development of suitable tools for in vivo measurements. Nanotechnology entered this field to help overcome many of the current technical limitations, and luminescent nanoparticles (NPs) are one of the most promising materials proposed for future diagnostic implementation. NPs also constitute a versatile platform that can allow facile multi-functionalization to perform multimodal imaging or theranostics (simultaneous diagnosis and therapy). In this contribution we have mainly focused on dye doped silica or silica-based NPs conjugated with targeting moieties to enable imaging of specific cancer cells. We also cite and briefly discuss a few non-targeted systems for completeness. We summarize common synthetic approaches to these materials, and then survey the most recent imaging applications of silica-based nanoparticles in cancer. The field of theranostics is particularly important and stimulating, so, even though it is not the central topic of this paper, we have included some significant examples. We conclude with a short section on NP-based systems already in clinical trials and examples of specific applications in childhood tumors. This review aims to describe and discuss, through focused examples, the great potential of these materials in the medical field, with the aim to encourage further research to implement applications, which today are still rare.

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.

Angiopoietin-like 7, a novel pro-angiogenetic factor over-expressed in cancer

Angiopoietin-like (ANGPTL) proteins are secreted proteins showing structural similarity to members of the angiopoietin family. Some ANGPTL proteins possess pleiotropic activities, being involved in cancer lipid, glucose energy metabolisms, and angiogenesis. ANGPTL7 is the less characterized member of the family whose functional role is only marginally known. In this study, we provide experimental evidences that ANGPTL7 is over-expressed in different human cancers. To understand the role played by ANGPTL7 in tumor biology, we asked whether ANGPTL7 is endogenously expressed by malignant cells or in response to environmental stimuli. We found that ANGPTL7 is marginally expressed under standard growth condition while it is specifically up-regulated by hypoxia. Interestingly, the protein is secreted and partially associated with the exosomal fraction, suggesting that it could be found in the systemic circulation of oncologic patients and act in an endocrine way. Moreover, we found that ANGPTL7 exerts a pro-angiogenetic effect on human differentiated endothelial cells by stimulating their proliferation, motility, invasiveness, and capability to form capillary-like networks while it does not stimulate progenitor endothelial cells. Finally, we showed that ANGPTL7 promotes vascularization in vivo in the mouse Matrigel sponge assay, thereby accrediting this molecule as a pro-angiogenic factor.