Manuela Nebuloni

Regulation of the Chemokine Receptor CXCR4 by Hypoxia

Cell adaptation to hypoxia (Hyp) requires activation of transcriptional programs that coordinate expression of genes involved in oxygen delivery (via angiogenesis) and metabolic adaptation (via glycolysis). Here, we describe that oxygen availability is a determinant parameter in the setting of chemotactic responsiveness to stromal-derived factor 1 (CXCL12). Low oxygen concentration induces high expression of the CXCL12 receptor, CXC receptor 4 (CXCR4), in different cell types (monocytes, monocyte-derived macrophages, tumor-associated macrophages, endothelial cells, and cancer cells), which is paralleled by increased chemotactic responsiveness to its specific ligand. CXCR4 induction by Hyp is dependent on both activation of the Hyp-inducible factor 1 α and transcript stabilization. In a relay multistep navigation process, the Hyp–Hyp-inducible factor 1 α–CXCR4 pathway may regulate trafficking in and out of hypoxic tissue microenvironments.

Role of Macrophage Targeting in the Antitumor Activity of Trabectedin

There is widespread interest in macrophages as a therapeutic target in cancer. Here, we demonstrate that trabectedin, a recently approved chemotherapeutic agent, induces rapid apoptosis exclusively in mononuclear phagocytes. In four mouse tumor models, trabectedin caused selective depletion of monocytes/macrophages in blood, spleens, and tumors, with an associated reduction of angiogenesis. By using trabectedin-resistant tumor cells and myeloid cell transfer or depletion experiments, we demonstrate that cytotoxicity on mononuclear phagocytes is a key component of its antitumor activity. Monocyte depletion, including tumor-associated macrophages, was observed in treated tumor patients. Trabectedin activates caspase-8-dependent apoptosis; selectivity for monocytes versus neutrophils and lymphocytes is due to differential expression of signaling and decoy TRAIL receptors. This unexpected property may be exploited in different therapeutic strategies.

p50 nuclear factor-κB overexpression in tumor-associated macrophages inhibits M1 inflammatory responses and antitumor resistance

Tumor-associated macrophages (TAM) are a major inflammatory infiltrate in tumors and a major component of the protumor function of inflammation. TAM in established tumors generally have an M2 phenotype with defective production of interleukin-12 (IL-12) and high IL-10. Here, we report that defective responsiveness of TAM from a murine fibrosarcoma and human ovarian carcinoma to M1 activation signals was associated with a massive nuclear localization of the p50 nuclear factor-kappaB (NF-kappaB) inhibitory homodimer. p50 overexpression inhibited IL-12 expression in normal macrophages. TAM isolated from p50(-/-) mice showed normal production of M1 cytokines, associated with reduced growth of transplanted tumors. Bone marrow chimeras showed that p50 inactivation in hematopoietic cells was sufficient to result in reduced tumor growth. Thus, p50 NF-kappaB overexpression accounts for the inability of TAM to mount an effective M1 antitumor response capable of inhibiting tumor growth.

Cardioprotective Function of the Long Pentraxin PTX3 in Acute Myocardial Infarction

Background— Despite widespread clinical use as a prognostic marker in ischemic heart disorders, the actual pathogenetic role of the short pentraxin, C-reactive protein, has not undergone stringent genetic testing because of evolutionary divergence between mouse and humans. The long pentraxin PTX3 is conserved in evolution, is expressed in the heart under inflammatory conditions, and is a candidate prognostic marker in acute myocardial infarction. It was therefore important to assess whether PTX3 plays a pathogenetic role in acute myocardial infarction. Methods and Results— In a model of acute myocardial infarction caused by coronary artery ligation and reperfusion, tissue mRNA expression and circulating levels of PTX3 increased. The interleukin-1R–MyD88 pathway plays a pivotal role in the induction of PTX3 transcript after ischemia. ptx3-deficient mice showed exacerbated heart damage (33% larger infarcts in null mice; P=0.0047). Increased myocardial damage in ptx3-deficient mice was associated with a greater no-reflow area, increased neutrophil infiltration, decreased number of capillaries, and increased number of apoptotic cardiomyocytes. In addition, ptx3-deficient mice with acute myocardial infarction showed higher circulating levels of interleukin-6 and increased C3 deposition in lesional tissue. The phenotype was reversed by exogenous PTX3. Conclusions— Thus, PTX3 plays a nonredundant, regulatory, cardioprotective role in acute myocardial infarction in mice. Our results suggest that modulation of the complement cascade contributes to the cardioprotective function of PTX3.

Tumor-Conditioned Macrophages Secrete Migration-Stimulating Factor: A New Marker for M2-Polarization, Influencing Tumor Cell Motility

Tumor-associated macrophages (TAMs) are key orchestrators of the tumor microenvironment directly affecting neoplastic cell growth, neoangiogenesis, and extracellular matrix remodeling. In turn, the tumor milieu strongly influences maturation of TAMs and shapes several of their features. To address the early macrophage (Mϕ) differentiation phase in a malignant context, we mimicked a tumor microenvironment by in vitro coculturing human blood monocytes with conditioned media from different cancer cell lines. Only 2 out of 16 tumor cell lines induced Mϕ differentiation due to secreted M-CSF isoforms, including high molecular mass species. A global gene profiling of tumor-conditioned Mϕ was performed. Comparison with other datasets (polarized M1-Mϕ, M2-Mϕ, and TAMs isolated from human tumors) highlighted the upregulation of several genes also shared by TAM and M2-polarized Mϕ. The most expressed genes were selenoprotein 1, osteoactivin, osteopontin, and, interestingly, migration-stimulating factor (MSF), a poorly studied oncofoetal isoform of fibronectin. MSF (present in fetal/cancer epithelial and stromal cells but not in healthy tissues) was never identified in Mϕ. MSF production was confirmed by immunohistochemistry in human TAMs. MSF was induced by M-CSF, IL-4, and TGFβ but not by proinflammatory stimuli. RNA and protein analysis clearly demonstrated that it is specifically associated with the M2 polarization of Mϕ. Tumor-conditioned Mϕ-derived MSFs strongly stimulated tumor cell migration, thus contributing to the motile phenotype of neoplastic cells. In conclusion, MSF is a new molecule associated with the M2 polarization of Mϕ and expressed by TAMs. Its biological function may contribute to Mϕ-mediated promotion of cancer cell invasion and metastasis.

Direct contribution of epithelium to organ fibrosis: epithelial-mesenchymal transition.

Fibrosis of epithelial parenchymal organs and end-stage organ failure represent the final common pathway of many chronic diseases and are a major determinant of morbidity and mortality worldwide. Fibrosis is a complex response initiated to protect the host from an injurious event; nevertheless, it leads to serious organ damage when it becomes independent from the initiating stimulus. It involves massive deposition of matrix by an expanded pool of fibrogenic cells, disruption of the normal tissue architecture, and parenchymal destruction. Fibroblasts, the effector cells of matrix production, when engaged in fibrogenesis, display the highly activated phenotype characteristic of myofibroblasts. These cells are present in a large number in sites with ongoing inflammation, reparative reaction, and fibrosis, but their origin has not yet been definitely elucidated. Although proliferation of preexisting stromal fibroblasts and, probably, recruitment of bone marrow-derived fibrogenic cells may account for a portion of them, emerging evidence seems to indicate that an important number of matrix-producing fibroblasts/myofibroblasts arises through a mechanism of epithelial-mesenchymal transition. Through this process, epithelial cells would lose intercellular cohesion and would translocate from the epithelial compartment into the interstitium where, gaining a full mesenchymal phenotype, they could participate in the synthesis of the fibrotic matrix. Epithelial-mesenchymal transition is induced by the integrated actions of many stimuli including transforming growth factor-beta and matrix-generated signals that are also known to be implicated in inflammation, repair responses, and fibrosis. The consequences of epithelial-mesenchymal transition in chronic fibrosing diseases could be two-fold as follows: on one hand, by supplementing new mesenchymal cells, it might feed the expanding pool of interstitial fibroblasts/myofibroblasts responsible for the matrix accumulation; on the other hand, it could cause loss of epithelial cells, thus, contributing to the parenchyma destruction seen in advanced fibrosis. Markers of epithelium undergoing epithelial-mesenchymal transition include loss of E-cadherin and cytokeratin; de novo expression of fibroblast-specific protein 1/S100A4, vimentin, and alpha-smooth muscle actin; basement membrane component loss; and production of interstitial-type matrix molecules such as fibronectin and type I/III collagen. Evidence of epithelial-mesenchymal transition has been reported in the kidney, lung, liver, eye, and serosal membranes suggesting that epithelial-mesenchymal transition could be involved in the pathogenesis of fibrotic disorders in these organs. Thus, because of its fibrogenic potential, the detection of epithelial-mesenchymal transition in biopsy specimens could be useful diagnostically and represent a new biomarker of progression in chronic fibrosing diseases.

Antitumor and anti-inflammatory effects of trabectedin on human myxoid liposarcoma cells.

Inflammatory mediators present in the tumor milieu may promote cancer progression and are considered promising targets of novel biological therapies. We previously reported that the marine antitumor agent trabectedin, approved in Europe in 2007 for soft tissue sarcomas and in 2009 for ovarian cancer, was able to downmodulate the production of selected cytokines/chemokines in immune cells. Patients with myxoid liposarcoma (MLS), a subtype characterized by the expression of the oncogenic transcript FUS-CHOP, are highly responsive to trabectedin. The drug had marked antiproliferative effects on MLS cell lines at low nanomolar concentrations. We tested the hypothesis that trabectedin could also affect the inflammatory mediators produced by cancer cells. Here, we show that MLS express several cytokines, chemokines, and growth factors (CCL2, CCL3, CCL5, CXCL8, CXCL12, MIF, VEGF, SPARC) and the inflammatory and matrix-binder protein pentraxin 3 (PTX3), which build up a prominent inflammatory environment. In vitro treatment with noncytotoxic concentrations of trabectedin selectively inhibited the production of CCL2, CXCL8, IL-6, VEGF, and PTX3 by MLS primary tumor cultures and/or cell lines. A xenograft mouse model of human MLS showed marked reduction of CCL2, CXCL8, CD68+ infiltrating macrophages, CD31+ tumor vessels, and partial decrease of PTX3 after trabectedin treatment. Similar findings were observed in a patient tumor sample excised after several cycles of therapy, indicating that the results observed in vitro might have in vivo relevance. In conclusion, trabectedin has dual effects in liposarcoma: in addition to direct growth inhibition, it affects the tumor microenvironment by reducing the production of key inflammatory mediators.

HER2 Expression in Breast Cancer Cells Is Downregulated Upon Active Targeting by Antibody-Engineered Multifunctional Nanoparticles in Mice

Subcellular destiny of targeted nanoparticles in cancer cells within living organisms is still an open matter of debate. By in vivo and ex vivo experiments on tumor-bearing mice treated with antibody-engineered magnetofluorescent nanocrystals, in which we combined fluorescence imaging, magnetic relaxation, and trasmission electron microscopy approaches, we provide evidence that nanoparticles are effectively delivered to the tumor by active targeting. These nanocrystals were demonstrated to enable contrast enhancement of the tumor in magnetic resonance imaging. In addition, we were able to discriminate between the fate of the organic corona and the metallic core upon cell internalization. Accurate immunohistochemical analysis confirmed that hybrid nanoparticle endocytosis is mediated by the complex formation with HER2 receptor, leading to a substantial downregulation of HER2 protein expression on the cell surface. These results provide a direct insight into the pathway of internalization and degradation of targeted hybrid nanoparticles in cancer cells in vivo and suggest a potential application of this immunotheranostic nanoagent in neoadjuvant therapy of cancer.

Protection against inflammation- and autoantibody-caused fetal loss by the chemokine decoy receptor D6

Fetal loss in animals and humans is frequently associated with inflammatory conditions. D6 is a promiscuous chemokine receptor with decoy function, expressed in lymphatic endothelium, that recognizes and targets to degradation most inflammatory CC chemokines. Here, we report that D6 is expressed in placenta on invading extravillous trophoblasts and on the apical side of syncytiotrophoblast cells, at the very interface between maternal blood and fetus. Exposure of D6−/− pregnant mice to LPS or antiphospholipid autoantibodies results in higher levels of inflammatory CC chemokines and increased leukocyte infiltrate in placenta, causing an increased rate of fetal loss, which is prevented by blocking inflammatory chemokines. Thus, the promiscuous decoy receptor for inflammatory CC chemokines D6 plays a nonredundant role in the protection against fetal loss caused by systemic inflammation and antiphospholipid antibodies.

Tumour homing and therapeutic effect of colloidal nanoparticles depend on the number of attached antibodies

Active targeting of nanoparticles to tumours can be achieved by conjugation with specific antibodies. Specific active targeting of the HER2 receptor is demonstrated in vitro and in vivo with a subcutaneous MCF-7 breast cancer mouse model with trastuzumab-functionalized gold nanoparticles. The number of attached antibodies per nanoparticle was precisely controlled in a way that each nanoparticle was conjugated with either exactly one or exactly two antibodies. As expected, in vitro we found a moderate increase in targeting efficiency of nanoparticles with two instead of just one antibody attached per nanoparticle. However, the in vivo data demonstrate that best effect is obtained for nanoparticles with only exactly one antibody. There is indication that this is based on a size-related effect. These results highlight the importance of precisely controlling the ligand density on the nanoparticle surface for optimizing active targeting, and that less antibodies can exhibit more effect.