Paola Orecchia

Periostin promotes fibrosis and predicts progression in patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease without effective therapeutics. Periostin has been reported to be elevated in IPF patients relative to controls, but its sources and mechanisms of action remain unclear. We confirm excess periostin in lungs of IPF patients and show that IPF fibroblasts produce periostin. Blood was obtained from 54 IPF patients (all but 1 with 48 wk of follow-up). We show that periostin levels predict clinical progression at 48 wk (hazard ratio = 1.47, 95% confidence interval = 1.03-2.10, P < 0.05). Monocytes and fibrocytes are sources of periostin in circulation in IPF patients. Previous studies suggest that periostin may regulate the inflammatory phase of bleomycin-induced lung injury, but periostin effects during the fibroproliferative phase of the disease are unknown. Wild-type and periostin-deficient (periostin(-/-)) mice were anesthetized and challenged with bleomycin. Wild-type mice were injected with bleomycin and then treated with OC-20 Ab (which blocks periostin and integrin interactions) or control Ab during the fibroproliferative phase of disease, and fibrosis and survival were assessed. Periostin expression was upregulated quickly after treatment with bleomycin and remained elevated. Periostin(-/-) mice were protected from bleomycin-induced fibrosis. Instillation of OC-20 during the fibroproliferative phase improved survival and limited collagen deposition. Chimeric mouse studies suggest that hematopoietic and structural sources of periostin contribute to lung fibrogenesis. Periostin was upregulated by transforming growth factor-β in lung mesenchymal cells, and periostin promoted extracellular matrix deposition, mesenchymal cell proliferation, and wound closure. Thus periostin plays a vital role in late stages of pulmonary fibrosis and is a potential biomarker for disease progression and a target for therapeutic intervention.

Identification of a novel cell binding site of periostin involved in tumour growth

INTRODUCTION Periostin (PN), a member of the fasciclin family of proteins, is a TGF-β-induced extracellular matrix protein involved in cell survival, angiogenesis, invasion and metastasis. It is considered a potent angiogenic factor and a marker of tumour progression in many types of human cancer. Many different kinds of cells bind to PN by means of the integrins αvβ3 and αvβ5, but the periostin epitope recognised by these integrins is not formally demonstrated. The aim of our study was to identify which domain of PN could be involved in cell adhesion and its potential role in tumour growth. METHODS We generated the monoclonal antibody OC-20 (mAb OC-20) by hybridoma technology. Different PN recombinant fragments were used to characterise the periostin epitope recognised by the mAb OC-20 and to localise a new cell binding site of the protein. A murine model of human melanoma was used in the preclinical in vivo experiments. RESULTS We formally demonstrate that the periostin epitope recognised by OC-20 is a new binding site for the integrins αvβ3 and αvβ5, localised in the second FAS1 domain (FAS1-2) of the protein. Moreover the in vivo use of this antibody significantly inhibits tumour growth and angiogenesis. CONCLUSION Our results show that the FAS1-2 domain of PN plays a role in tumour progression. Moreover this novel antibody may likewise prove to be very useful in clarifying the role of PN in angiogenesis and may contribute to the design of novel anti-angiogenesis drugs.

Group 3 innate lymphoid cells regulate neutrophil migration and function in human decidua.

Innate lymphoid cells (ILCs) have a central role in innate defenses against pathogens, lymphoid organogenesis, and tissue remodeling. They have been detected in human decidua, however, their role in this tissue remains unclear. Successful pregnancy requires an early inflammatory phase favoring implantation and tissue remodeling as well as a subsequent regulatory phase to prevent fetal rejection and supporting neoangiogenesis. Here, we show that, during the first trimester of pregnancy, neutrophils infiltrate decidua basalis and are more abundant in normal pregnancy than in spontaneous miscarriages. Decidual neutrophils localize in proximity of NCR+ILC3, which may influence neutrophil migration and survival given their production of CXCL8 and granulocyte macrophage colony-stimulating factor (GM-CSF). Moreover, NCR+ILC3-derived GM-CSF was found to induce the expression of heparin-binding EGF-like growth factor and IL1ra in neutrophils, two proteins/cytokines involved in tissue remodeling and maintenance of pregnancy. Our data suggest that the simultaneous presence of NCR+ILC3 and neutrophils in decidual tissues and their possible cross talk, may have a role in the early phases of pregnancy.

Proteomic analysis of the nuclear matrix in the early stages of rat liver carcinogenesis: Identification of differentially expressed and MAR-binding proteins

Tumor progression is characterized by definite changes in the protein composition of the nuclear matrix (NM). The interactions of chromatin with the NM occur via specific DNA sequences called MARs (matrix attachment regions). In the present study, we applied a proteomic approach along with a Southwestern assay to detect both differentially expressed and MAR-binding NM proteins, in persistent hepatocyte nodules (PHN) in respect with normal hepatocytes (NH). In PHN, the NM undergoes changes both in morphology and in protein composition. We detected over 500 protein spots in each two dimensional map and 44 spots were identified. Twenty-three proteins were differentially expressed; among these, 15 spots were under-expressed and 8 spots were over-expressed in PHN compared to NH. These changes were synchronous with several modifications in both NM morphology and the ability of NM proteins to bind nuclear RNA and/or DNA containing MARs sequences. In PHN, we observed a general decrease in the expression of the basic proteins that bound nuclear RNA and the over-expression of two species of Mw 135 kDa and 81 kDa and pI 6.7-7.0 and 6.2-7.4, respectively, which exclusively bind to MARs. These results suggest that the deregulated expression of these species might be related to large-scale chromatin reorganization observed in the process of carcinogenesis by modulating the interaction between MARs and the scaffold structure.

A novel human anti-syndecan-1 antibody inhibits vascular maturation and tumour growth in melanoma.

INTRODUCTION Syndecan-1 is a cell membrane protein that, after its shedding by heparanase enzymes, is accumulated in the extracellular matrix of some tumours, e.g. myeloma and lung carcinoma, where it modulates several key processes of tumourigenesis such as cancer cell proliferation and apoptosis, angiogenesis and metastasis. Few studies have focused on syndecan-1 in malignant melanoma, a tumour for which new therapeutic targets are desperately needed. We aimed to investigate the role of syndecan-1 in melanoma and to evaluate the potential therapeutic efficacy of a novel fully human anti-syndecan-1 recombinant antibody in this deadly disease. METHODS The OC-46F2 recombinant antibody was generated by selecting a human antibody phage display library on human melanoma cells and by its expression in mammalian cells. The specific antigen recognised by the antibody was identified by mass spectrometry. Murine models of human melanoma and ovarian carcinoma were used in the pre-clinical in vivo experiments. RESULTS The fully human antibody OC-46F2, specific for the extracellular domain of syndecan-1, inhibited vascular maturation and tumour growth in an experimental human melanoma model. The therapeutic efficacy of this antibody was also demonstrated in an experimental ovarian carcinoma model. A co-distribution of syndecan-1 with vascular endothelial growth factor receptor 2 (VEGFR2) observed in the intratumour melanoma microenvironment was absent in the tumours from mice treated with OC-46F2 scFv. CONCLUSION These findings highlight the role of syndecan-1 as a potential therapeutic target in melanoma and ovarian carcinoma and provide a new tool able to block vessel maturation, one of the mechanisms that underpin the angiogenic process essential for solid tumour growth.

In vivo generation of decidual Natural Killer cells from resident hematopoietic progenitors

Decidual natural killer cells accumulate at the fetal-maternal interface and play a key role in a successful pregnancy. However, their origin is still unknown. Do they derive from peripheral natural killer cells recruited in decidua or do they represent a distinct population that originates in situ? Here, we identified natural killer precursors in decidua and uterus of pregnant mice. These precursors underwent rapid in situ differentiation and large proportions of proliferating immature natural killer cells were present in decidua and uterus as early as gestation day 4.5. Here, we investigated the origin of decidua- and uterus-natural killer cells by performing transfer experiments of peripheral mature natural killer cells or precursors from EGFP+ mice. Results showed that mature natural killer cells did not migrate into decidua and uterus, while precursors were recruited in these organs and differentiated towards natural killer cells. Moreover, decidua- and uterus-natural killer cells displayed unique phenotypic and functional features. They expressed high levels of the activating Ly49D receptor in spite of their immature phenotype. In addition, decidua- and uterus-natural killer cells were poorly cytolytic and produced low amounts of IFN-γ, while they released factors (GM-CSF, VEGF, IP-10) involved in neo-angiogenesis and tissue remodeling. Our data reveal in situ generation of decidual natural killer cells and provide an important correlation between mouse and human decidual natural killer cells, allowing further studies to be carried out on their role in pregnancy-related diseases.

Schedule-dependent therapeutic efficacy of L19mTNF-α and melphalan combined with gemcitabine

L19‐tumor necrosis factor alpha (L19mTNF‐α; L), a fusion protein consisting of mouse TNFα and the human antibody fragment L19 directed to the extra domain‐B (ED‐B) of fibronectin, is able to selectively target tumor vasculature and to exert a long‐lasting therapeutic activity in combination with melphalan (M) in syngeneic mouse tumor models. We have studied the antitumor activity of single L19mTNF‐α treatment in combination with melphalan and gemcitabine (G) using different administration protocols in two histologically different murine tumor models: WEHI‐164 fibrosarcoma and K7M2 osteosarcoma. All responding mice showed significant reduction in myeloid‐derived suppressor cells (MDSCs) and an increase in CD4+ and CD8+ T cells in the tumor infiltrates, as well as significant reduction in regulatory T cells (Treg) at the level of draining lymph nodes. What is important is that all cured mice rejected tumor challenge up to 1 year after therapy. Targeted delivery of L19mTNF‐α synergistically increases the antitumor activity of melphalan and gemcitabine, but optimal administration schedules are required. This study provides information for designing clinical studies using L19mTNF‐α in combination with chemotherapeutic drugs.

Targeting Syndecan-1, a molecule implicated in the process of vasculogenic mimicry, enhances the therapeutic efficacy of the L19-IL2 immunocytokine in human melanoma xenografts

Anti-angiogenic therapy of solid tumors has until now failed to produce the long lasting clinical benefits desired, possibly due to the complexity of the neoangiogenic process. Indeed, a prominent role is played by “vasculogenic” or “vascular” mimicry (VM), a phenomenon in which aggressive cancer cells form an alternative microvascular circulation, independently of endothelial cell angiogenesis. In this study we observed, in melanoma patient cell lines having vasculogenic/stem-cell like phenotype and in melanoma tumors, the syndecan-1 co-expression with VM markers, such as CD144 and VEGFR-2. We show that melanoma cells lose their ability to form tubule-like structures in vitro after blocking syndecan-1 activity by the specific human recombinant antibody, OC-46F2. Moreover, in a human melanoma xenograft model, the combined therapy using OC-46F2 and L19-IL2, an immunocytokine specific for the tumor angiogenic-associated B-fibronectin isoform(B-FN), led to a complete inhibition of tumor growth until day 90 from tumor implantation in 71% of treated mice, with statistically significant differences compared to groups treated with OC-46F2 or L19-IL2 as monotherapy. Furthermore, in the tumors recovered from mice treated with OC-46F2 either as monotherapy or in combination with L19-IL2, we observed a dramatic decrease of vascular density and loss of VM structures. These findings indicate for the first time a role of syndecan-1 in melanoma VM and that targeting syndecan-1, together with B-FN, could be promising in improving the treatment of metastatic melanoma.

Tumor Vasculature Targeted TNFα Therapy: Reversion of Microenvironment Anergy and Enhancement of the Anti-tumor Efficiency

Tumor cells and tumor-associated stromal cells such as immune, endothelial and mesenchimal cells create a tumor microenvironment (TME) which allows tumor cell promotion, growth and dissemination while dampening the anti-tumor immune response. Efficient anti-tumor interventions have to keep into consideration the complexity of the TME and take advantage of immunotherapy and chemotherapy combined approaches. Thus, the aim of tumor therapy is to directly hit tumor cells and reverse endothelial and immune cell anergy. Selective targeting of tumor vasculature using TNFα-associated peptides or antibody fragments in association with chemotherapeutic agents has been shown able to exert a potent stimulatory effect on endothelial cells as well as on innate and adaptive immune responses. These drug combinations reducing the dose of single agents employed have led to minimize the associated side-effects. In this review we will analyze different TNFα-mediated tumor vessel-targeted therapies in both humans and tumor mouse models, with emphasis on the role played by the cross-talk between natural killer and dendritic cells and on the ability of TNFα to trigger tumor vessel activation and normalization. The improvement of the TNFα-based therapy with anti-angiogenic immunomodulatory drugs that may convert the TME from immunosuppressive to immunostimulant, will be discussed as well.