The mechanisms that sustain endometrial tissues at ectopic sites in patients with endometriosis are poorly understood. Various leukocytes , including macrophages, infiltrate endometriotic lesions. In this study, we depleted mouse macrophages by means of either clodronate liposomes or monoclonal antibodies before the injection of syngeneic endometrial tissue. In the absence of macrophages, tissue fragments adhered and implanted into the peritoneal wall, but endometriotic lesions failed to organize and develop. When we depleted macrophages after the establishment of endometriotic lesions, blood vessels failed to reach the inner layers of the lesions, which stopped growing. Macrophages from patients with endometriosis and experimental mice, but not nonendometriotic patients who underwent surgery for uterine leiomyomas or control mice , expressed markers of alternative activation. These markers included high levels of scavenger receptors, CD163 and CD206, which are involved in both the scavenging of hemoglobin with iron transfer into macrophages and the silent clearance of inflammatory molecules. Macrophages in both inflammatory liquid and ectopic lesions were equally polarized, suggesting a critical role of environmental cues in the peritoneal cavity.Adoptively transferred, alternatively activated macrophages dramatically enhanced endometriotic lesion growth in mice. Inflammatory macrophages effectively protected mice from endometriosis. Therefore, endogenous macrophages involved in tissue remodeling appear as players in the natural history of endometriosis, required for effective vascularization and ectopic lesion growth.

Neuroblastoma, the most common solid tumor of infancy derived from the sympathetic nervous system, continues to present a formidable clinical challenge. Sterically stabilized immunoliposomes (SIL) have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. We showed that SIL loaded with doxorubicin (DXR) and targeted to the disialoganglioside receptor GD 2 [aGD 2 -SIL(DXR)] led to a selective inhibition of the metastatic growth of experimental models of human neuroblastoma. By coupling NGR peptides that target the angiogenic endothelial cell marker aminopeptidase N to the surface of DXR-loaded liposomes [NGR-SL(DXR)], we obtained tumor regression, pronounced destruction of the tumor vasculature, and prolonged survival of orthotopic neuroblastoma xenografts. Here, we showed good liposome stability, long circulation times, and enhanced timedependent tumor accumulation of both the carrier and the drug. Antivascular effects against animal models of lung and ovarian cancer were shown for formulations of NGR-SL(DXR). In the chick embryo chorioallantoic assay, NGR-SL(DXR) substantially reduced the angiogenic potential of various neuroblastoma xenografts, with synergistic inhibition observed for the combination of NGR-SL(DXR) with aGD 2 -SIL(DXR). A significant improvement in antitumor effects was seen in neuroblastoma-bearing animal models when treated with the combined formulations compared with control mice or mice treated with either tumor-or vascular-targeted liposomal formulations, administered separately. The combined treatment resulted in a dramatic inhibition of tumor endothelial cell density. Long-term survivors were obtained only in animals treated with the combined tumor-and vascular-targeted formulations, confirming the pivotal role of combination therapies in treating aggressive metastatic neuroblastoma.

The p73 gene is a p53 homologue which induces apoptosis and inhibits cell proliferation. Although p73 maps at 1p36.3 and is frequently deleted in neuroblastoma (NB), it does not act as a classic oncosuppressor gene. In developing sympathetic neurons of mice, p73 is predominantly expressed as a truncated anti-apoptotic isoform (DNp73), which antagonizes both p53 and the fulllength p73 protein (TAp73). This suggests that p73 may be part of a complex tumor-control mechanism. To determine the role of DNp73 in NB we analyzed the pattern of expression of this gene in vivo and evaluated the prognostic significance of its expression. Our results indicate that DNp73 expression is associated with reduced apoptosis in a NB tumor tissue. Expression of this variant in NB patients significantly correlates with age at diagnosis and VMA urinary excretion. Moreover it is strongly associated with reduced survival (HR=7.93; P50.001) and progression-free survival (HR=5.3; P50.001) and its role in predicting a poorer outcome is independent from age, primary tumor site, stage and MYCN amplification (OS: HR=5.24, P=0.012; PFS: HR=4.36, P=0.005). In conclusion our data seem to indicate that DNp73 is a crucial gene in neuroblastoma pathogenesis.

The immunosurveillance mechanisms governing high-risk neuroblastoma (HR-NB), a major pediatric malignancy, have been elusive. We identify a potential role for natural killer (NK) cells, in particular the interaction between the NK receptor NKp30 and its ligand, B7-H6, in the metastatic progression and survival of HR-NB after myeloablative multimodal chemotherapy and stem cell transplantation. NB cells expressing the NKp30 ligand B7-H6 stimulated NK cells in an NKp30-dependent manner. Serum concentration of soluble B7-H6 correlated with the down-regulation of NKp30, bone marrow metastases, and chemoresistance, and soluble B7-H6 contained in the serum of HR-NB patients inhibited NK cell functions in vitro. The expression of distinct NKp30 isoforms affecting the polarization of NK cell functions correlated with 10-year event-free survival in three independent cohorts of HR-NB in remission from metastases after induction chemotherapy (n = 196, P < 0.001), adding prognostic value to known risk factors such as N-Myc amplification and age >18 months. We conclude that the interaction between NKp30 and B7-H6 may contribute to the fate of NB patients and that both the expression of NKp30 isoforms on circulating NK cells and the concentration of soluble B7-H6 in the serum may be clinically useful as biomarkers for risk stratification.

Bortezomib is an effective inhibitor of neuroblastoma cell growth and angiogenesis. These findings provide the rationale for further clinical investigation of bortezomib in pediatric neuroblastoma.

Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycine-arginine (isoDGR), a ligand of ␣v␤3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the ␣-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind ␣v␤3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind ␣v␤3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (␣v␤5, ␣v␤6, ␣v␤8, and ␣5␤1), although with 10 -100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas ␣-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.Various peptides containing the Asn-Gly-Arg (NGR) motif have been discovered by peptide-phage library panning in tumor-bearing mice (1). The tumor-homing properties of these peptides rely on the interaction with aminopeptidase N (CD13), a membrane protease expressed by the tumor neovasculature (2, 3). Because of this property, these peptides have been exploited for ligand-directed delivery of various drugs and particles to tumor vessels, in the attempt to increase their antitumor activity (4). For instance, we have shown that peptides containing cyclic CNGRC and linear GNGRG motives can be used for delivering tumor necrosis factor ␣ (TNF) 3 (5-7), interferon ␥ (8 -10), and liposomal doxorubicin (11, 12) to tumor neovasculature, improving their therapeutic properties. The CNGRC-TNF conjugate, called NGR-TNF, is currently tested in phase II clinical studies (13-15). Other investigators have used the NGR motif embedded in similar or different molecular scaffolds for delivering chemotherapeutic drugs, antiangiogenic drugs, tissue factor, viruses, and other compounds to tumor vessels (1, 16 -32). Recently, a CNGRC peptide with an acetylated N-terminal ␣-amino group has been successfully exploited also for quantitative molecular magnetic resonance imaging of ...

In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium-sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the DeltapI and DeltaMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors. Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed.

Cancer metabolism is characterized by an accelerated glycolytic rate facing reduced activity of oxidative phosphorylation. This “Warburg effect” represents a standard to diagnose and monitor tumor aggressiveness with 18F-fluorodeoxyglucose whose uptake is currently regarded as an accurate index of total glucose consumption. Studying cancer metabolic response to respiratory chain inhibition by metformin, we repeatedly observed a reduction of tracer uptake facing a marked increase in glucose consumption. This puzzling discordance brought us to discover that 18F-fluorodeoxyglucose preferentially accumulates within endoplasmic reticulum by exploiting the catalytic function of hexose-6-phosphate-dehydrogenase. Silencing enzyme expression and activity decreased both tracer uptake and glucose consumption, caused severe energy depletion and decreased NADPH content without altering mitochondrial function. These data document the existence of an unknown glucose metabolism triggered by hexose-6-phosphate-dehydrogenase within endoplasmic reticulum of cancer cells. Besides its basic relevance, this finding can improve clinical cancer diagnosis and might represent potential target for therapy.