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Tumour-associated macrophages (TAM) isolated from five murine sarcomas had a relatively high frequency of I-A+ cells, with mean values of 27% (mFS6), 52% (MN/MCA1), 68% (N3), 62% (N4) and 98% (J3) for TAM compared to 12% for resident peritoneal macrophages. Expression of I-E in TAM was also high (29%) in the only sarcoma (N4) examined in this respect. Expression of I-A by TAM declined in culture but exposure to lymphokine supernatants maintained and increased the frequency of I-A+ cells in TAM. Transplantation of tumours into nude mice caused a marked decrease in the percentage of I-A+ TAM in the case of the N4 sarcoma (8% compared to 48%), whereas for the MN/MCA1 sarcoma the diminution was only marginal (from 53 to 41%), TAM from murine sarcomas did not constitutively release appreciable levels of interleukin-1 (IL-1) activity. Upon stimulation with bacterial lipopolysaccharides or silica, TAM showed a limited capacity to produce and release IL-1 activity compared to peritoneal macrophages. Thus the expression of I-A antigens and the IL-1-producing capacity are uncoupled in TAM from murine sarcomas. These properties of TAM could play an important role in the generation of anti-tumour immunity and/or of suppressive T-cell circuits. [ABSTRACT FROM AUTHOR]

Background: The pancreatic microenvironment has a defensive role against cancer but it can acquire tumor-promoting properties triggered by multiple mechanisms including alterations in the equilibrium between proteases and their inhibitors. The identification of proteolytic events, targets and pathways would set the basis for the design of new therapeutic approaches., Methods and Results: Here we demonstrate that spheroids isolated from human and murine healthy pancreas and co-transplanted orthotopically with pancreatic ductal adenocarcinoma (PDAC) in mouse pancreas inhibited tumor growth. The effect was mediated by trypsin-generated fibronectin (FN) fragments released by pancreatic spheroids. Tumor inhibition was observed also in a model of acute pancreatitis associated with trypsin activation. Mass spectrometry proteomic analysis of fragments and mAb against different FN epitopes identified the FN type III domain as responsible for the activity. By inhibiting integrin α5β1, FAK and FGFR1 signaling, the fragments induced tumor cell detachment and reduced cell proliferation. Consistent with the mutual relationship between the two pathways, FGF2 restored both FGFR1 and FAK signaling and promoted PDAC cell adhesion and proliferation. FAK and FGFR inhibitors additively inhibited PDAC growth in vitro and in orthotopic in vivo models., Conclusions: This study identifies a novel role for pancreatic trypsin and fibronectin cleavage as a mechanism of protection against cancer by the pancreatic microenvironment. The finding of a FAK-FGFR cross-talk in PDAC support the combination of FAK and FGFR inhibitors for PDAC treatment to emulate the protective effect of the normal pancreas against cancer., (© 2023. Italian National Cancer Institute ‘Regina Elena’.)

The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signalling pathway drives severe pathologies, including cancer development and angiogenesis-driven pathologies. The perturbation of the FGF2/FGFR axis via extracellular allosteric small inhibitors is a promising strategy for developing FGFR inhibitors with improved safety and efficacy for cancer treatment. We have previously investigated the role of new extracellular inhibitors, such as rosmarinic acid (RA), which bind the FGFR-D2 domain and directly compete with FGF2 for the same binding site, enabling the disruption of the functional FGF2/FGFR interaction. To select ligands for the previously identified FGF2/FGFR RA binding site, NMR data-driven virtual screening has been performed on an in-house library of non-commercial small molecules and metabolites. A novel drug-like compound, a resorcinol derivative named RBA4 has been identified. NMR interaction studies demonstrate that RBA4 binds the FGF2/FGFR complex, in agreement with docking prediction. Residue-level NMR perturbations analysis highlights that the mode of action of RBA4 is similar to RA in terms of its ability to target the FGF2/FGFR-D2 complex, inducing perturbations on both proteins and triggering complex dissociation. Biological assays proved that RBA4 inhibited FGF2 proliferative activity at a level comparable to the previously reported natural product, RA. Identification of RBA4 chemical groups involved in direct interactions represents a starting point for further optimization of drug-like extracellular inhibitors with improved activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper: [Collina Simona reports financial support was provided by University of Pavia.]., (Copyright © 2023 Elsevier Inc. All rights reserved.)

NMR-based approaches play a pivotal role in providing insight into molecular recognition mechanisms, affording the required atomic-level description and enabling the identification of promising inhibitors of protein-protein interactions. The aberrant activation of the fibroblast growth factor 2 (FGF2)/fibroblast growth factor receptor (FGFR) signaling pathway drives several pathologies, including cancer development, metastasis formation, resistance to therapy, angiogenesis-driven pathologies, vascular diseases, and viral infections. Most FGFR inhibitors targeting the intracellular ATP binding pocket of FGFR have adverse effects, such as limited specificity and relevant toxicity. A viable alternative is represented by targeting the FGF/FGFR extracellular interactions. We previously identified a few small-molecule inhibitors acting extracellularly, targeting FGFR or FGF. We have now built a small library of natural and synthetic molecules that potentially act as inhibitors of FGF2/FGFR interactions to improve our understanding of the molecular mechanisms of inhibitory activity. Here, we provide a comparative analysis of the interaction mode of small molecules with the FGF2/FGFR complex and the single protein domains. DOSY and residue-level NMR analysis afforded insights into the capability of the potential inhibitors to destabilize complex formation, highlighting different mechanisms of inhibition of FGF2-induced cell proliferation.

Blood vessels in tumors are formed through a variety of different mechanisms, each generating vessels with peculiar structural, molecular, and functional properties. This heterogeneity has a major impact on tumor response or resistance to antineoplastic therapies and is now emerging as a promising target for strategies to prevent drug resistance and improve the distribution and efficacy of antineoplastic treatments. This review presents evidence of how different mechanisms of tumor vessel formation (vasculogenesis, glomeruloid proliferation, intussusceptive angiogenesis, vasculogenic mimicry, and vessel co-option) affect tumor responses to antiangiogenic and antineoplastic therapies, but also how therapies can promote alternative mechanisms of vessel formation, contributing to tumor recurrence, malignant progression, and acquired drug resistance. We discuss the possibility of tailoring treatment strategies to overcome vasculature-mediated drug resistance or to improve drug distribution and efficacy.

Thrombospondins (TSPs) are a family of five multimeric matricellular proteins. Through a wide range of interactions, TSPs play pleiotropic roles in embryogenesis and in tissue remodeling in adult physiology as well as in pathological conditions, including cancer development and metastasis. TSPs are active in bone remodeling, the process of bone resorption (osteolysis) and deposition (osteogenesis) that maintains bone homeostasis. TSPs are particularly involved in aberrant bone remodeling, including osteolytic and osteoblastic skeletal cancer metastasis, frequent in advanced cancers such as breast and prostate carcinoma. TSPs are major players in the bone metastasis microenvironment, where they finely tune the cross talk between tumor cells and bone resident cells in the metastatic niche. Each TSP family member has different effects on the differentiation and activity of bone cells-including the bone-degrading osteoclasts and the bone-forming osteoblasts-with different outcomes on the development and growth of osteolytic and osteoblastic metastases. Here, we overview the involvement of TSP family members in the bone tissue microenvironment, focusing on their activity on osteoclasts and osteoblasts in bone remodeling, and present the evidence to date of their roles in bone metastasis establishment and growth.

The prominent desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) is a determinant factor in tumor progression and a major barrier to the access of chemotherapy. The PDAC microenvironment therefore appears to be a promising therapeutic target. CCN2/CTGF is a profibrotic matricellular protein, highly present in the PDAC microenvironment and associated with disease progression. Here we have investigated the therapeutic value of the CCN2-targeting BLR100 and BLR200, two modified synthetic peptides derived from active regions of CCN3, an endogenous inhibitor of CCN2. In a murine orthotopic PDAC model, the two peptides, administered as monotherapy at low doses (approximating physiological levels of CCN3), had tumor inhibitory activity that increased with the dose. The peptides affected the tumor microenvironment, inhibiting fibrosis and vessel formation and reducing necrosis. Both peptides were active in preventing ascites formation. An increased activity was obtained in combination regimens, administering BLR100 or BLR200 with the chemotherapeutic drug gemcitabine. Pharmacokinetic analysis indicated that the improved activity of the combination was not mainly determined by the substantial increase in gemcitabine delivery to tumors, suggesting other effects on the tumor microenvironment. The beneficial remodeling of the tumor stroma supports the potential value of these CCN3-derived peptides for targeting pathways regulated by CCN2 in PDAC.

Trabectedin is a marine-derived antineoplastic drug. Besides targeting the cancer cells, trabectedin has a peculiar activity on the tumor microenvironment with marked effects on the vasculature and the immune response. Because a favorable microenvironment is a key factor in the progression of cutaneous melanoma, we hypothesized that trabectedin might affect the growth and metastasis of this highly aggressive cancer. This study shows that trabectedin inhibited the subcutaneous growth of the murine melanoma B16-BL6 and K1735-M2. In line with its known activities on the environment of other tumor types, it caused a significant reduction of tumor blood vessel density and tumor-associated macrophages. Trabectedin had a significant antimetastatic activity, inhibiting the formation of lung colonies following intravenous injection of B16-BL6 or K1735-M2 cells. The drug was also active in a clinically relevant spontaneous metastasis assay, where it inhibited lung metastasis when administered before (neoadjuvant) or after (adjuvant) surgical removal of the primary tumor. Relevant to its antimetastatic activity, trabectedin inhibited melanoma cell invasiveness in vitro, associated with increased tissue inhibitor of metalloproteinase-1 production and alteration in cell shape and cytoskeleton organization. This study shows that trabectedin affects melanoma growth and metastasis, acting with tumor-dependent mechanisms on both the tumor cells and the vascular and the inflammatory tumor microenvironment., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Control of synapse number and function in the developing central nervous system is critical to the formation of neural circuits. Astrocytes play a key role in this process by releasing factors that promote the formation of excitatory synapses. Astrocyte-secreted thrombospondins (TSPs) induce the formation of structural synapses, which however remain post-synaptically silent, suggesting that completion of early synaptogenesis may require a two-step mechanism. Here, we show that the humoral innate immune molecule Pentraxin 3 (PTX3) is expressed in the developing rodent brain. PTX3 plays a key role in promoting functionally-active CNS synapses, by increasing the surface levels and synaptic clustering of AMPA glutamate receptors. This process involves tumor necrosis factor-induced protein 6 (TSG6), remodeling of the perineuronal network, and a β1-integrin/ERK pathway. Furthermore, PTX3 activity is regulated by TSP1, which directly interacts with the N-terminal region of PTX3. These data unveil a fundamental role of PTX3 in promoting the first wave of synaptogenesis, and show that interplay of TSP1 and PTX3 sets the proper balance between synaptic growth and synapse function in the developing brain., (© 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

In patients with diabetes, impaired activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), the plasma metalloprotease that cleaves highly thrombogenic von Willebrand factor multimers, is a major risk factor of cardiovascular events. Here, using Adamts13 -/- mice made diabetic by streptozotocin, we investigated the impact of the lack of ADAMTS13 on the development of diabetes-associated end-organ complications. Adamts13 -/- mice experienced a shorter life span than their diabetic wild-type littermates. It was surprising that animal death was not related to the occurrence of detectable thrombotic events. The lack of ADAMTS13 drastically increased the propensity for ventricular arrhythmias during dobutamine-induced stress in diabetic mice. Cardiomyocytes of diabetic Adamts13 -/- mice exhibited an aberrant distribution of the ventricular gap junction connexin 43 and increased phosphorylation of Ca 2+ /calmodulin-dependent kinase II (CaMKII), and with the consequent CaMKII-induced disturbance in Ca 2+ handling, which underlie propensity for arrhythmia. In vitro, thrombospondin 1 (TSP1) promoted, in a paracrine manner, CaMKII phosphorylation in murine HL-1 cardiomyocytes, and ADAMTS13 acted to inhibit TSP1-induced CaMKII activation. In conclusion, the deficiency of ADAMTS13 may underlie the onset of lethal arrhythmias in diabetes through increased CaMKII phosphorylation in cardiomyocytes. Our findings disclose a novel function for ADAMTS13 beyond its antithrombotic activity., (© 2018 by the American Diabetes Association.)

The clinical management of pancreatic ductal adenocarcinoma (PDAC) is hampered by the lack of reliable biomarkers. This study investigated the value of soluble stroma-related molecules as PDAC biomarkers. In the first exploratory phase, 12 out of 38 molecules were associated with PDAC in a cohort of 25 PDAC patients and 16 healthy subjects. A second confirmatory phase on an independent cohort of 131 PDAC patients, 30 chronic pancreatitis patients, and 131 healthy subjects confirmed the PDAC association for MMP7, CCN2, IGFBP2, TSP2, sICAM1, TIMP1, and PLG Multivariable logistic regression model identified biomarker panels discriminating respectively PDAC versus healthy subjects (MMP7 + CA19.9, AUC = 0.99, 99% CI = 0.98-1.00) (CCN2 + CA19.9, AUC = 0.96, 99% CI = 0.92-0.99) and PDAC versus chronic pancreatitis (CCN2 + PLG+FN+Col4 + CA19.9, AUC = 0.94, 99% CI = 0.88-0.99). Five molecules were associated with PanIN development in two GEM models of PDAC (PdxCre/LSL-Kras G12D and PdxCre/LSL-Kras G12D/+ /LSL-Trp53 R172H/+ ), suggesting their potential for detecting early disease. These markers were also elevated in patient-derived orthotopic PDAC xenografts and associated with response to chemotherapy. The identified stroma-related soluble biomarkers represent potential tools for PDAC diagnosis and for monitoring treatment response of PDAC patients., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Cancer cells use two major types of movement: Mesenchymal, which is typical of cells of mesenchymal origin and depends on matrix metalloproteinase (MMP) activity, and amoeboid, which is characteristic of cells with a rounded shape and relies on the activity of Rho-associated kinase (ROCK). The present authors previously demonstrated that, during neoplastic transformation, telomerase-immortalized human fibroblasts (cen3tel cells) acquired a ROCK-dependent/MMP independent mechanism of invasion, mediated by the downregulation of the ROCK cellular inhibitor Round (Rnd)3/RhoE. In the present study, cen3tel transformation was also demonstrated to be paralleled by downregulation of Snail, a major determinant of the mesenchymal movement. To test whether Snail levels could determine the type of movement adopted by mesenchymal tumor cells, Snail was ectopically expressed in tumorigenic cells. It was observed that ectopic Snail did not increase the levels of typical mesenchymal markers, but induced cells to adopt an MMP-dependent mechanism of invasion. In cells expressing ectopic Snail, invasion became sensitive to the MMP inhibitor Ro 28-2653 and insensitive to the ROCK inhibitor Y27632, suggesting that, once induced by Snail, the mesenchymal movement prevails over the amoeboid one. Snail-expressing cells had a more aggressive behavior in vivo , and exhibited increased tumor growth rate and metastatic ability. These results confirm the high plasticity of cancer cells, which can adopt different types of movement in response to changes in the expression of specific genes. Furthermore, the present findings indicate that Rnd3 and Snail are possible regulators of the type of invasion mechanism adopted by mesenchymal tumor cells.

The FGFs/FGFRs system is a recognized actionable target for therapeutic approaches aimed at inhibiting tumor growth, angiogenesis, metastasis, and resistance to therapy. We previously identified a non-peptidic compound (SM27) that retains the structural and functional properties of the FGF2-binding sequence of thrombospondin-1 (TSP-1), a major endogenous inhibitor of angiogenesis. Here we identified new small molecule inhibitors of FGF2 based on the initial lead. A similarity-based screening of small molecule libraries, followed by docking calculations and experimental studies, allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting its binding to both heparan sulfate proteoglycans and FGFR-1. The compounds inhibit FGF2 activity in in vitro and ex vivo models of angiogenesis, with improved potency over SM27. Comparative analysis of the selected hits, complemented by NMR and biochemical analysis of 4 newly synthesized functionalized phenylamino-substituted naphthalenes, allowed identifying the minimal stereochemical requirements to improve the design of naphthalene sulfonates as FGF2 inhibitors.

Vascular endothelial growth factor C (VEGFC) has been reported to promote tumor progression in several tumor types, mainly through the stimulation of lymphangiogenesis and lymphatic metastasis. However, the expression and biological significance of the VEGFC/VEGF receptor (VEGFR)-3 pathway in ovarian cancer growth and dissemination are unclear, and have been investigated in this study. Soluble VEGFC was detected in the plasma and ascites of patients with ovarian carcinoma, and VEGFR3 expression was found in their tumor tissues. In human ovarian carcinoma xenograft models, high levels of soluble VEGFC in ascites and serum were detected, in association with disease progression, tumor burden, and volume of ascites. Peak VEGFC expression preceded para-aortic lymph node infiltration by HOC8 neoplastic cells. Histological detection of tumor cells in blood and lymphatic vessels indicated both hematogenous and lymphatic dissemination. Overexpression of VEGFC in the VEGFR3-positive and luciferase-expressing IGROV1 cells promoted carcinoma dissemination after orthotopic transplantation in the ovary of immunodeficient mice. In vitro, VEGFC released by the tumor cells stimulated tumor cell migration in an autocrine manner. Cediranib, an inhibitor of VEGFR1-3 and c-kit, inhibited in vivo metastasis of VEGFC-overexpressing IGROV1 and in vitro autocrine effects. These findings suggest that the VEGFC/VEGFR3 pathway acts as an enhancer of ovarian cancer progression through autocrine and paracrine mechanisms, hence offering a potential target for therapy., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

E-3810 is a novel small molecule that inhibits VEGF receptor-1, -2, and -3 and fibroblast growth factor receptor-1 tyrosine kinases at nmol/L concentrations currently in phase clinical II. In preclinical studies, it had a broad spectrum of antitumor activity when used as monotherapy in a variety of human xenografts. We here investigated the activity of E-3810 combined with different cytotoxic agents in a MDA-MB-231 triple-negative breast cancer xenograft model. The molecule could be safely administered with 5-fluorouracil, cisplatin, and paclitaxel. The E-3810-paclitaxel combination showed a striking activity with complete, lasting tumor regressions; the antitumor activity of the combination was also confirmed in another triple-negative breast xenograft, MX-1. The activity was superior to that of the combinations paclitaxel+brivanib and paclitaxel+sunitinib. Pharmacokinetics studies suggest that the extra antitumor activity of the combination is not due to higher paclitaxel tumor levels, which in fact were lower in mice pretreated with all three kinase inhibitors, and the paclitaxel plasma levels excluded reduced drug availability. Pharmacodynamic studies showed that E-3810, brivanib, and sunitinib given as single agents or in combination with paclitaxel reduced the number of vessels, but did not modify vessel maturation. Reduced tumor collagen IV and increased plasma collagen IV, associated with increased matrix metalloproteinases (MMP), particularly host MMP-9, indicate a proteolytic remodeling of the extracellular matrix caused by E-3810 that in conjunction with the cytotoxic effect of paclitaxel on the tumor cells (caspase-3/7 activity) may contribute to the striking activity of their combination. These data support the therapeutic potential of combining E-3810 with conventional chemotherapy., (©2012 AACR.)

Taxanes are potent inhibitors of cell motility, a property implicated in their antiangiogenic and antimetastatic activity and unrelated to their antiproliferative effect. The molecular mechanism of this anti-motility activity is poorly understood. In this study, we found that paclitaxel induced tubulin acetylation in endothelial and tumor cells, at concentrations that affected cell motility but not proliferation (10(-8) to 10(-9) M, for 4 hours). Induction of tubulin acetylation correlated with inhibition of motility but not proliferation based on a comparison of highly and poorly cytotoxic taxanes (paclitaxel and IDN5390) and tumor cell lines sensitive and resistant to paclitaxel (1A9 and 1A9 PTX22). Consistent with the hypothesis that tubulin deacetylase activity might affect cell response to the anti-motility activity of taxanes, we found that overexpression of the tubulin deacetylase SIRT2 increased cell motility and reduced cell response to the anti-motility activity of paclitaxel. Conversely, the SIRT2 inhibitor splitomicin reduced cell motility and potentiated the anti-motility activity of paclitaxel. The inhibitory effect was further potentiated by the addition of the HDAC6 inhibitor trichostatin A. Paclitaxel and splitomicin promoted translocation into the nucleus--and hence activation--of FOXO3a, a negative regulator of cell motility. This study indicates a role for SIRT2 in the regulation of cell motility and suggests that therapies combining sirtuin inhibitors and taxanes could be used to treat cell motility-based pathologic processes such as tumor angiogenesis, invasion, and metastasis.

Fibroblast growth factors (FGFs) are recognized targets for the development of therapies against angiogenesis-driven diseases, including cancer. The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity. Here by using a combination of techniques including Nuclear Magnetic Resonance, Molecular Dynamics, Surface Plasmon Resonance and cell-based binding assays we clarify the molecular mechanism of inhibition of an angiostatic small molecule, sm27, mimicking the endogenous inhibitor of angiogenesis, thrombospondin-1. NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions. The functional consequence of the inhibitor binding is an impaired FGF2 interaction with both its receptors, as demonstrated by SPR and cell-based binding assays. We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

Background: Mesenchymal and amoeboid movements are two important mechanisms adopted by cancer cells to invade the surrounding environment. Mesenchymal movement depends on extracellular matrix protease activity, amoeboid movement on the RhoA-dependent kinase ROCK. Cancer cells can switch from one mechanism to the other in response to different stimuli, limiting the efficacy of antimetastatic therapies., Methodology and Principal Findings: We investigated the acquisition and molecular regulation of the invasion capacity of neoplastically transformed human fibroblasts, which were able to induce sarcomas and metastases when injected into immunocompromised mice. We found that neoplastic transformation was associated with a change in cell morphology (from fibroblastic to polygonal), a reorganization of the actin cytoskeleton, a decrease in the expression of several matrix metalloproteases and increases in cell motility and invasiveness. In a three-dimensional environment, sarcomagenic cells showed a spherical morphology with cortical actin rings, suggesting a switch from mesenchymal to amoeboid movement. Accordingly, cell invasion decreased after treatment with the ROCK inhibitor Y27632, but not with the matrix protease inhibitor Ro 28-2653. The increased invasiveness of tumorigenic cells was associated with reduced expression of Rnd3 (also known as RhoE), a cellular inhibitor of ROCK. Indeed, ectopic Rnd3 expression reduced their invasive ability in vitro and their metastatic potential in vivo., Conclusions: These results indicate that, during neoplastic transformation, cells of mesenchymal origin can switch from a mesenchymal mode of movement to an amoeboid one. In addition, they point to Rnd3 as a possible regulator of mesenchymal tumor cell invasion and to ROCK as a potential therapeutic target for sarcomas.

Inhibitors of angiogenesis are an important addition to conventional chemotherapy. Among different "druggable" angiogenic factors, fibroblast growth factor-2 (FGF-2) is an attractive target for novel therapies because of its intricated involvement in tumor neovascularization, tumor cell proliferation and migration, and the acquisition of resistance to antiangiogenic therapies. FGF-2 bioavailability and activity is affected by several natural ligands, including the endogenous inhibitor of angiogenesis thrombospondin-1 (TSP-1). We hypothesized that the FGF-2-binding sequence of TSP-1 might serve as a template for the development of non-peptide inhibitors of angiogenesis. Computational biology and nuclear magnetic resonance spectroscopy approaches, major investigative tools in the characterizations of protein-protein interaction (PPI), were used to map the residues at the TSP-1/FGF-2 interface. The translation of this three-dimensional information into a pharmacophore model allowed screening a small molecule databases, identifying three FGF-2-binding, antiangiogenic small molecules, mimetic of TSP-1. Pharmacophore-based approaches are thus feasible tools to exploit naturally occurring PPI, by generating a set of lead compounds mimetic of endogenous proteins, as a starting point for the development of novel therapeutic agents.

Uncontrolled neovascularization occurs in several angiogenesis-dependent diseases, including cancer. Neovascularization is tightly controlled by the balance between angiogenic growth factors and antiangiogenic agents. The various natural angiogenesis inhibitors identified so far affect neovascularization by different mechanisms of action. Thrombospondin-1 (TSP-1) is a matricellular modular glycoprotein that acts as a powerful endogenous inhibitor of angiogenesis. It acts both indirectly, by sequestering angiogenic growth factors and effectors in the extracellular environment, and directly, by inducing an antiangiogenic program in endothelial cells following engagement of specific receptors including CD36, CD47, integrins and proteoglycans (all involved in angiogenesis ). In view of its central, multifaceted role in angiogenesis, TSP-1 has served as a source of antiangiogenic tools, including TSP-1 fragments, synthetic peptides and peptidomimetics, gene therapy strategies, and agents that up-regulate TSP-1 expression. This review discusses TSP-1-based inhibitors of angiogenesis, their mechanisms of action and therapeutic potential, drawing our experience with angiogenic growth factor-interacting TSP-1 peptides, and the possibility of exploiting them to design novel antiangiogenic agents.

Vesicles shed by cancer cells are known to mediate several tumor-host interactions. Tumor microenvironment may, in turn, influence the release and the activity of tumor-shed microvesicles. In this study, we investigated the molecular mediators of the pH-dependent proinvasive activity of tumor-shed vesicles. Gelatinase zymography showed increased microvesicle activity of matrix metalloproteinases 9 and 2 as a result of acid exposure (pH 5.6) compared to pH 7.4. Thus, we reasoned that the cysteine protease cathepsin B might play a role in mediating the pH-dependent activation of gelatinases. Cathepsin B expression in tumor-shed microvesicles was confirmed by Western blot analysis and zymography. The activity of vesicle-associated cathepsin B measured using Z-Arg-Arg-pNA as substrate was significantly increased at acidic pH values. Inhibition of protease activity by the cysteine protease inhibitor, E-64, and treatment of ovarian cancer cells with small interfering RNA against cathepsin B suppressed the ability of tumor-shed microvesicles to stimulate both gelatinase activation and the invasiveness of endothelial cells observed at low pH values. We conclude that microvesicle shedding is a major secretory pathway for cathepsin B release from tumor cells. Hence, the acidic microenvironment found in most solid tumors may contribute to cathepsin B-mediated proinvasive capabilities of tumor-shed vesicles.

Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP) regulate each other, contributing to tumor progression. We have previously reported that MMP9 induces the release of tumor VEGF, promoting ascites formation in human ovarian carcinoma xenografts. The aim of this study was to investigate whether tumor-derived VEGF regulated the expression of gelatinase by the stroma, influencing the invasive properties of ovarian tumors. Tumor variants derived from 1A9 human ovarian carcinoma, stably expressing VEGF(121) in the sense (1A9-VS-1) and antisense orientations (1A9-VAS-3), were used. In vivo, zymographic analysis of tumors from 1A9-VS-1 implanted in the peritoneal cavity of nude mice showed higher levels of gelatinases, particularly murine MMP9, indicating that VEGF stimulates host expression of the matrix-degrading enzyme. Murine MMP9 expression was also high in the ovaries of mice bearing 1A9-VS-1 tumors. The effect on host MMP9 activity was organ-specific. The levels of host pro-MMP9 in ovaries correlated with the plasma levels of tumor VEGF and with the selective invasion of the ovaries. Induction of host MMP9 expression in tumors and ovaries was independent of the site of tumor growth as it was seen in mice carrying both intraperitoneal and subcutaneous tumors. The anti-VEGF antibody bevacizumab (Avastin) inhibited MMP9 expression and tumor invasion in the ovaries of mice bearing 1A9-VS-1 tumors. These findings point to a complex cross-talk between VEGF and MMPs in the progression of ovarian tumor and suggest the possibility of using VEGF inhibitors to affect MMP-dependent tumor invasion.

Tumor angiogenesis is regulated by a dynamic cross-talk between tumor cells and the host microenvironment. Because membrane vesicles shed by tumor cells are known to mediate several tumor-host interactions, we determined whether vesicles might also stimulate angiogenesis. Vesicles shed by human ovarian carcinoma cell lines CABA I and A2780 stimulated the motility and invasiveness of endothelial cells in vitro. Enzyme-linked immunosorbent assay and Western blot analysis revealed relevant amounts of vascular endothelial growth factor (VEGF) and the two matrix metalloproteinases MMP-2 and MMP-9, but not fibroblast growth factor-2, contained in shed vesicles. An A2780 cell-derived clone transfected to overexpress VEGF shed the same amount of vesicles as did a control clone, but contained significantly more VEGF within the vesicles. Despite a greater amount of VEGF in vesicles of the overexpressing clone, vesicles of both clones stimulated endothelial cell motility to comparable levels, suggesting that VEGF was stored within the vesicle and was unavailable. Only following vesicle burst induced by acidic pH (a characteristic of the tumor microenvironment) was VEGF released, leading to significantly higher stimulation of cell motility. Thus, tumor-shed membrane vesicles carry VEGF and release it in a bioactive form in conditions typical of the tumor microenvironment.

Unlabelled: We studied the biological features and the immunophenotype of a cell culture established from the lesion of soft tissues of a woman affected by Gorham-Stout syndrome. We found that these cells belonged to a monocytic lineage with some characteristics of immature osteoclasts and were able to release large amounts of osteoclastogenic and angiogenic molecules that may contribute to disease progression., Introduction: Gorham-Stout syndrome is a rare disease characterized by osteolysis and proliferation of vascular or lymphatic vessels, with a severe outcome. Its etiology and the identification of the cell types involved are completely unknown., Materials and Methods: A cell culture from a lesion of soft tissues was established, and its behavior in vitro and in immunodeficient mice was studied. We analyzed (1) the cell phenotype by flow cytometry; (2) the adhesive and migratory properties on different substrates; (3) the ability to differentiate into mature osteoclasts; (4) the production of osteclastogenic and angiogenic molecules; (5) the in vivo angiogenic activity of the cells subcutaneously implanted in mouse in a Matrigel plug; and (6) the ability to recapitulate the disease when transplanted in nude mice., Results and Conclusions: The established culture consisted of a morphologically homogeneous cell population belonging to a monocytic lineage having some features of an osteoclast-like cell type. Cells had an invasive phenotype, were angiogenic, and produced osteoclastogenic (IL-6, TGF-beta1, IL-1beta) and angiogenic (vascular endothelial growth factor-A [VEGF-A], CXCL-8) molecules when challenged with inflammatory cytokines. Immunodeficient mice injected with these cells did not show any bone lesions or vascular alteration, but had high amounts of circulating human IL-6 and VEGF-A. Cells isolated from a cutaneous lymphangiomatosis did not show any of these findings. These data suggest that cells of monocyte-macrophage lineage play an essential role in the pathogenesis of Gorham-Stout disease, whose progression is propelled by cytokine circuits that accelerate angiogenesis and osteoclastogenesis.

ZD6126 is a vascular targeting agent, developed for the treatment of solid tumors. In vivo, ZD6126 is rapidly converted into the tubulin-binding agent N-acetylcolchinol. We have previously reported that in vitro N-acetylcolchinol disrupts microtubules and induces rapid changes in endothelial cell morphology, which in a tumor would lead to a rapid loss of tumor vessel integrity and subsequent extensive tumor necrosis. The aim of this study was to investigate the effect of cytotoxic antineoplastic drugs-cisplatin, doxorubicin, vincristine, paclitaxel, and docetaxel-on endothelial cell response to N-acetylcolchinol. We found that cisplatin and doxorubicin did not interfere with the ability of N-acetylcolchinol to cause morphologic changes in human umbilical vein endothelial cells, whereas vincristine showed additive effects. In contrast, the microtubule-stabilizing agents paclitaxel (1-10 micromol/L) and docetaxel (0.1-1 micromol/L) prevented the morphologic changes induced by N-acetylcolchinol in human umbilical vein endothelial cells. The effect was observed when cells were exposed to paclitaxel and N-acetylcolchinol together or when paclitaxel was given shortly before N-acetylcolchinol. Paclitaxel and N-acetylcolchinol interacted at the level of microtubule organization, as shown in immunofluorescence analysis of the cytoskeleton. The protective effect was reversible because 4 hours after paclitaxel wash out, cells recovered the sensitivity to N-acetylcolchinol. In vivo, pretreatment of mice with paclitaxel inhibited the vascular targeting activity of ZD6126 on newly formed vessels in the Matrigel plug assay and ZD6126-induced necrosis in tumors. These findings indicate that paclitaxel, depending on the timing and schedule of administration, can affect the vascular targeting activity of ZD6126, which may have an effect on the optimal scheduling of therapies based on the combined use of microtubule-stabilizing and microtubule-destabilizing agents.