Long pentraxin-3 inhibits epithelial-mesenchymal transition in melanoma cells.

During melanoma progression, malignant melanocytes are reprogrammed into mesenchymal-like cells through to an epithelial-mesenchymal transition (EMT) process associated with the acquisition of an invasive, prometastatic phenotype. The fibroblast growth factor-2 (FGF2)/FGF receptor (FGFR) system plays a pivotal role in melanoma, leading to autocrine/paracrine induction of tumor cell proliferation and angiogenesis. Long pentraxin-3 (PTX3) interacts with FGF2, and other FGF family members, inhibiting FGF-dependent neovascularization and tumor growth. Here, PTX3 protein and the PTX3-derived acetylated pentapeptide Ac-ARPCA-NH2 inhibit FGF2-driven proliferation and downstream FGFR signaling in murine melanoma B16-F10 cells. Moreover, human PTX3-overexpressing hPTX_B16-F10 cells are characterized by the reversed transition from a mesenchymal to an epithelial-like appearance, inhibition of cell proliferation, loss of clonogenic potential, reduced motility and invasive capacity, downregulation of various mesenchymal markers, and upregulation of the epithelial marker E-cadherin. Accordingly, PTX3 affects cell proliferation and EMT transition in human A375 and A2058 melanoma cells. Also, hPTX_B16-F10 cells showed a reduced tumorigenic and metastatic activity in syngeneic C57BL/6 mice. In conclusion, PTX3 inhibits FGF/FGFR-driven EMT in melanoma cells, hampering their tumorigenic and metastatic potential. These data represent the first experimental evidence about a nonredundant role of the FGF/FGFR system in the modulation of the EMT process in melanoma and indicate that PTX3 or its derivatives may represent the basis for the design of novel therapeutic approaches in FGF/FGFR-dependent tumors, including melanoma.

Inhibition of angiogenesis by ß-galactosylceramidase deficiency in globoid cell leukodystrophy.

Globoid cell leukodystrophy (Krabbe disease) is a neurological disorder of infants caused by genetic deficiency of the lysosomal enzyme ß-galactosylceramidase leading to accumulation of the neurotoxic metabolite 1-ß-d-galactosylsphingosine (psychosine) in the central nervous system. Angiogenesis plays a pivotal role in the physiology and pathology of the brain. Here, we demonstrate that psychosine has anti-angiogenic properties by causing the disassembling of endothelial cell actin structures at micromolar concentrations as found in the brain of patients with globoid cell leukodystrophy. Accordingly, significant alterations of microvascular endothelium were observed in the post-natal brain of twitcher mice, an authentic model of globoid cell leukodystrophy. Also, twitcher endothelium showed a progressively reduced capacity to respond to pro-angiogenic factors, defect that was corrected after transduction with a lentiviral vector harbouring the murine ß-galactosylceramidase complementary DNA. Finally, RNA interference-mediated ß-galactosylceramidase gene silencing causes psychosine accumulation in human endothelial cells and hampers their mitogenic and motogenic response to vascular endothelial growth factor. Accordingly, significant alterations were observed in human microvasculature from brain biopsy of a globoid cell leukodystrophy case. Together these data demonstrate that ß-galactosylceramidase deficiency induces significant alterations in endothelial neovascular responses that may contribute to central nervous system and systemic damages that occur in globoid cell leukodystrophy.

Long pentraxin-3 as an epithelial-stromal fibroblast growth factor-targeting inhibitor in prostate cancer.

Fibroblast growth factors (FGFs) exert autocrine/paracrine functions in prostate cancer by stimulating angiogenesis and tumour growth. Here dihydrotestosterone (DHT) up-regulates FGF2 and FGF8b production in murine TRAMP-C2 prostate cancer cells, activating a FGF-dependent autocrine loop of stimulation. The soluble pattern recognition receptor long pentraxin-3 (PTX3) acts as a natural FGF antagonist that binds FGF2 and FGF8b via its N-terminal domain. We demonstrate that recombinant PTX3 protein and the PTX3-derived pentapeptide Ac-ARPCA-NH2 abolish the mitogenic response of murine TRAMP-C2 cells and human LNCaP prostate cancer cells to DHT and FGFs. Also, PTX3 hampers the angiogenic activity of DHT-activated TRAMP-C2 cells on the chick embryo chorioallantoic membrane (CAM). Accordingly, human PTX3 overexpression inhibits the mitogenic activity exerted by DHT or FGFs on hPTX3_TRAMP-C2 cell transfectants and their angiogenic activity. Also, hPTX3_TRAMP-C2 cells show a dramatic decrease of their angiogenic and tumourigenic potential when grafted in syngeneic or immunodeficient athymic male mice. A similar inhibitory effect is observed when TRAMP-C2 cells overexpress only the FGF-binding N-terminal PTX3 domain. In keeping with the anti-tumour activity of PTX3 in experimental prostate cancer, immunohistochemical analysis of prostate needle biopsies from primary prostate adenocarcinoma patients shows that parenchymal PTX3 expression, abundant in basal cells of normal glands, is lost in high-grade prostatic intraepithelial neoplasia and in invasive tumour areas. These results identify PTX3 as a potent FGF antagonist endowed with anti-angiogenic and anti-neoplastic activity in prostate cancer.

Long pentraxin-3 inhibits FGF8b-dependent angiogenesis and growth of steroid hormone-regulated tumors.

Fibroblast growth factor-8b (FGF8b) exerts nonredundant autocrine/paracrine functions in steroid hormone-regulated tumors. Previous observations had shown that the soluble pattern recognition receptor long pentraxin-3 (PTX3) is a natural selective antagonist for a restricted number of FGF family members, inhibiting FGF2 but not FGF1 and FGF4 activity. Here, we assessed the capacity of PTX3 to antagonize FGF8b and to inhibit the vascularization and growth of steroid hormone-regulated tumors. Surface plasmon resonance analysis shows that PTX3 binds FGF8b with high affinity (K(d) = 30-90 nmol/L). As a consequence, PTX3 prevents the binding of FGF8b to its receptors, inhibits FGF8b-driven ERK1/2 activation, cell proliferation, and chemotaxis in endothelial cells, and suppresses FGF8b-induced neovascularization in vivo. Also, PTX3 inhibits dihydrotestosterone (DHT)- and FGF8b-driven proliferation of androgen-regulated Shionogi 115 (S115) mouse breast tumor cells. Furthermore, DHT-treated, PTX3 overexpressing hPTX3_S115 cell transfectants show a reduced proliferation rate in vitro and a limited angiogenic activity in the chick embryo chorioallantoic membrane and murine s.c. Matrigel plug assays. Accordingly, hPTX3_S115 cells show a dramatic decrease of their tumorigenic activity when grafted in immunodeficient male mice. These results identify PTX3 as a novel FGF8b antagonist endowed with antiangiogenic and antineoplastic activity with possible implications for the therapy of hormonal tumors.

Antiangiogenic activity of a neutralizing human single-chain antibody fragment against fibroblast growth factor receptor 1.

Fibroblast growth factor receptor-1 (FGFR-1) transduces proangiogenic and proliferative signals in human cancers. Thus, FGFR-1 may represent a target for the development of antiangiogenic/antineoplastic therapies. We screened a human single-chain fragment variable (scFv) antibody phage display library against the extracellular domain of the FGFR-1-IIIc isoform that harbors the FGF binding site. Several phages were isolated and tested for specificity and sensitivity, and the most promising antibody fragment RR-C2 was characterized for its biochemical and biological properties. ScFv RR-C2 specifically recognizes FGFR-1α and FGFR-1ß isoforms in ELISA, Western blotting, and surface plasmon resonance analysis with a K(d) value of 300 and 144 nmol/L for the 2 receptor isoforms, respectively. The antibody fragment also recognizes FGFR-1 when the receptor is exposed on the cell surface, thus preventing the formation of the ternary complex among FGFR-1, its ligand FGF2, and cell surface heparan sulfate proteoglycans. Accordingly, scFv RR-C2 specifically inhibits FGF2-mediated mitogenic activity in endothelial cells of human, bovine, and murine origin in a nanomolar range of concentrations. Also, the antibody fragment prevents FGF2-triggered sprouting of both human umbilical vein endothelial cell spheroids and of murine endothelium from aortic rings. Finally, the antibody fragment hampers the angiogenic activity exerted both by FGF2 in the chick embryo chorioallantoic membrane assay and by S115 mouse mammary tumor cells in the Matrigel plug assay. Taken together, the data show that scFv RR-C2 recognizes and neutralizes FGFR-1 activity in different animal species, including humans, thus representing a novel tool for the development of antiangiogenic/antineoplastic therapies.

IL-7 is superior to IL-2 for ex vivo expansion of tumour-specific CD4(+) T cells.

It is well established that tumours hinder both natural and vaccine-induced tumour-specific CD4(+) T-cell responses. Adoptive T-cell therapy has the potential to circumvent functional tolerance and enhance anti-tumour protective responses. While protocols suitable for the expansion of cytotoxic CD8(+) T cells are currently available, data on tumour-specific CD4(+) T cells remain scarce. We report here that CD4(+) T cells sensitized to tumour-associated Ag in vivo, proliferate in vitro in response to IL-7 without the need for exogenous Ag stimulation and accumulate several folds while preserving a memory-like phenotype. Both cell proliferation and survival accounts for the outgrowth of tumour-sensitized T cells among other memory and naive lymphocytes following exposure to IL-7. Also IL-2, previously used to expand anti-tumour CTL, promotes tumour-specific CD4(+) T-cell accumulation. However, IL-7 is superior to IL-2 at preserving lymphocyte viability, in vitro and in vivo, maintaining those properties, that are required by helper CD4(+) T cells to confer therapeutic efficacy upon transplantation in tumour-bearing hosts. Together our data support a unique role for IL-7 in retrieving memory-like CD4(+) T cells suitable for adoptive T-cell therapy.

Delivering cytokines at tumor site: The immunocytokine-conjugated anti-EDB-fibronectin antibody case.

Although considerable efforts have been made in the discovery of new agents for cancer treatment, several promising therapeutics cannot be applied systemically because of their severe side effects. This is the case for various recombinant pro-inflammatory cytokines that, despite their potent anti-cancer activity, can not find their way to clinical exploitation due to their devastating toxicity shown during dose escalation to therapeutically active concentrations. To circumvent these problems, an elegant and efficient way to accumulate therapeutic agents at the tumor site, thus reducing systemic side effects, is their conjugation to tumor-specific antibodies. Here, we review preclinical data about immunocytokines conjugated to a promising single-chain human antibody that selectively targets tumor-associated stroma and blood vessels by binding with high affinity and specificity to the extra domain-B (EDB) of fibronectin.

Anti-FGF2 approaches as a strategy to compensate resistance to anti-VEGF therapy: long-pentraxin 3 as a novel antiangiogenic FGF2-antagonist.

Angiogenesis, the formation of new blood vessels from the endothelium of the existing vasculature, plays a pivotal role in tumor growth, progression and metastasis. Over the last 30 years, numerous pro- and antiangiogenic molecules, their ligands, and intracellular signaling pathways have been identified, and significant efforts have been undertaken to develop antiangiogenic strategies for cancer therapy. Agents that selectively target vascular endothelial growth factor (VEGF) and its receptors have shown promising activity in clinical trials and have been approved for use in selected cancer indications. However, patients may ultimately develop resistance to these drugs. One proposed mechanism of tumor escape from anti-VEGF therapy is the up-regulation of fibroblast growth factor-2 (FGF2). FGF2 is a pleiotropic, angiogenesis inducer belonging to the family of the heparin-binding FGF growth factors. FGF2 is expressed by numerous tumor types and exerts its proangiogenic activity by interacting with tyrosine kinase receptors, heparan-sulfate proteoglycans, and integrins expressed on the endothelial cell surface. Experimental evidence suggests that targeting FGF2, in addition to VEGF, might provide synergistic effects in the treatment of angiogenesis-related diseases, including cancer. Several FGF2 inhibitors, with different chemical structure and mechanism of action, have been identified. Recent observations have shown the ability of the soluble pattern recognition receptor long-pentraxin-3 (PTX3) to bind FGF2, thus acting as a FGF2 antagonist. PTX3 binds FGF2 with high affinity and specificity. This interaction prevents the binding of FGF2 to its cognate tyrosine kinase receptors, leading to inhibition of the angiogenic activity of the growth factor. Further, preliminary observations support the hypothesis that PTX3 may inhibit FGF2-mediated tumor angiogenesis and growth. The identification of the FGF2-binding domain in the unique N-terminal extension of PTX3 has allowed the design of PTX3-derived synthetic peptides endowed with significant antiangiogenic activity in vitro and in vivo. These findings may provide the basis for the development of novel antiangiogenic FGF2 antagonists, with potential implications for cancer therapy.

alphavbeta3 Integrin-dependent antiangiogenic activity of resveratrol stereoisomers.

Angiogenesis is target for antineoplastic and chemopreventive therapies. The natural phytoalexin resveratrol is found in grapes and red wine as cis and trans stereoisomers. trans-Resveratrol shows antiangiogenic activity, but its mechanism of action is not fully elucidated. Recently, trans-resveratrol has been shown to interact with the beta3 integrin subunit, raising the possibility that inhibition of endothelial alphavbeta3 integrin function may concur to its angiosuppressive activity. To get novel insights about the antiangiogenic activity of resveratrol, we compared cis- and trans-resveratrol stereoisomers for their effect on the angiogenesis process and endothelial alphavbeta3 integrin function. trans-Resveratrol inhibits endothelial cell proliferation and the repair of mechanically wounded endothelial cell monolayers. Also, it prevents endothelial cell sprouting in fibrin gel, collagen gel invasion, and morphogenesis on Matrigel. In vivo, trans-resveratrol inhibits vascularization of the chick embryo area vasculosa and murine melanoma B16 tumor growth and neovascularization. In all the assays, cis-resveratrol exerts a limited, if any, effect. In keeping with these observations, trans-resveratrol, but not cis-resveratrol, inhibits alphavbeta3 integrin-dependent endothelial cell adhesion and the recruitment of enhanced green fluorescent protein-tagged beta3 integrin in focal adhesion contacts. In conclusion, stereoisomery affects the antiangiogenic activity of resveratrol, the trans isomer being significantly more potent than the cis isoform. The different antiangiogenic potential of resveratrol stereoisomers is related, at least in part, to their different capacity to affect alphavbeta3 integrin function. This may have profound implications for the design of synthetic antiangiogenic/angiopreventive phytoalexin derivatives.

Synthetic CD4+ T cell-targeted antigen-presenting cells elicit protective antitumor responses.

CD4(+) helper T cells are critical for protective immune responses and yet suboptimally primed in response to tumors. Cell-based vaccination strategies are under evaluation in clinical trials but limited by the need to derive antigen-presenting cells (APC) from patients or compatible healthy donors. To overcome these limitations, we developed CD4(+) T cell-targeted synthetic microbead-based artificial APC (aAPC) and used them to activate CD4(+) T lymphocytes specific for a tumor-associated model antigen (Ag) directly from the naive repertoire. In vitro, aAPC specifically primed Ag-specific CD4(+) T cells that were activated to express high levels of CD44, produced mainly interleukin 2, and could differentiate into Th1-like or Th2-like cells in combination with polarizing cytokines. I.v. administration of aAPC led to Ag-specific CD4(+) T-cell activation and proliferation in secondary lymphoid organs, conferred partial protection against subcutaneous tumors, and prevented the establishment of lung metastasis. Taken together, our data support the use of cell-free, synthetic aAPC as a specific and versatile alternative to expand peptide-specific CD4(+) T cells in adoptive and active immunotherapy.

Uptake of 18F-Fluorocholine, 18F-FET, and 18F-FDG in C6 gliomas and correlation with 131I-SIP(L19), a marker of angiogenesis.

UNLABELLED: Targeting extracellular structures that are involved in angiogenic processes, such as the extra domain B of fibronectin, is a promising approach for the diagnosis of solid tumors. The aim of this study was to determine uptake of the (18)F-labeled PET tracers (18)F-fluorocholine (N,N-dimethyl-N-(18)F-fluoromethyl-2-hydroxyethylammonium), (18)F-fluoro-ethyl-l-tyrosine (FET), and (18)F-FDG in C6 gliomas of the rat and to correlate it with uptake of the anti-extra domain B antibody (131)I-SIP(L19) as a marker of neoangiogenesis. METHODS: C6 gliomas were orthotopically induced in 17 rats. Uptake of all tracers was measured using quantitative autoradiography, and uptake of (18)F-fluorocholine, (18)F-FET, and (18)F-FDG was correlated with uptake of (131)I-SIP(L19) on a pixelwise basis. RESULTS: The mean (131)I-SIP(L19), (18)F-fluorocholine, (18)F-FET, and (18)F-FDG standardized uptake values in the tumor and the contralateral normal cortex (in parentheses) were 0.31 +/- 0.22 (not detectable), 2.00 +/- 0.53 (0.49 +/- 0.07), 3.67 +/- 0.36 (1.42 +/- 0.22), and 7.23 +/- 1.22 (3.64 +/- 0.51), respectively. The (131)I-SIP(L19) uptake pattern correlated best with (18)F-fluorocholine uptake (z = 0.80, averaged z-transformed Pearson correlation coefficient) and (18)F-FET uptake (z = 0.79) and least with (18)F-FDG (z = 0.37). CONCLUSION: One day after intravenous injection, (131)I-SIP(L19) displayed a very high tumor-to-cortex ratio, which may be used in the diagnostic work-up of brain tumor patients. Of the 3 investigated (18)F tracers, (18)F-fluorocholine and (18)F-FET correlated better with the pattern of (131)I-SIP(L19) uptake than did (18)F-FDG. Whether this means that (18)F-fluorocholine and (18)F-FET are better suited than (18)F-FDG to monitor antiangiogenic therapy should be investigated in future studies.

Selective occlusion of tumor blood vessels by targeted delivery of an antibody-photosensitizer conjugate.

The irregular vasculature and high interstitial pressure of solid tumors hinder the delivery of cytotoxic agents to cancer cells. As a consequence, the doses of chemotherapy necessary to achieve complete tumor eradication are associated with unacceptably high toxicities. The selective thrombosis of tumor blood vessels has been postulated as an alternative avenue for combating cancer, depriving tumors of nutrients and oxygen and causing an avalanche of tumor cell deaths. The human antibody L19, specific to the EDB domain of fibronectin, a marker of angiogenesis, is capable of selective in vivo localization around tumor blood vessels and is thus a suitable agent for delivering toxic payloads to the tumor neovasculature. Here we show that a chemical conjugate of the L19 antibody with the photosensitizer bis(triethanolamine)Sn(IV) chlorin e(6), after intravenous injection and irradiation with red light, caused an arrest of tumor growth in mice with subcutaneous tumors. By contrast, a photosensitizer conjugate obtained with an antibody of identical pharmacokinetic properties but irrelevant specificity did not exhibit a significant therapeutic effect. These results confirm that vascular targeting strategies, aimed at the selective occlusion/disruption of tumor blood vessels, have a significant anticancer therapeutic potential and encourage the use of antibody-photosensitizer conjugates for the therapy of superficial tumors and possibly other angiogenesis-related pathologies.

Molecular imaging of atherosclerotic plaques using a human antibody against the extra-domain B of fibronectin.

Current imaging modalities of human atherosclerosis, such as angiography, ultrasound, and computed tomography, visualize plaque morphology. However, methods that provide insight into plaque biology using molecular tools are still insufficient. The extra-domain B (ED-B) is inserted into the fibronectin molecule by alternative splicing during angiogenesis and tissue remodeling but is virtually undetectable in normal adult tissues. Angiogenesis and tissue repair are also hallmarks of advanced plaques. For imaging atherosclerotic plaques, the human antibody L19 (specific against ED-B) and a negative control antibody were labeled with radioiodine or infrared fluorophores and injected intravenously into atherosclerotic apolipoprotein E-null (ApoE-/-) or normal wild-type mice. Aortas isolated 4 hours, 24 hours, and 3 days after injection exhibited a selective and stable uptake of L19 when using radiographic or fluorescent imaging. L19 binding was confined to the plaques as assessed by fat staining. Comparisons between fat staining and autoradiographies 24 hours after 125I-labeled L19 revealed a significant correlation (r=0.89; P<0.0001). Minimal antibody uptake was observed in normal vessels from wild-type mice receiving the L19 antibody and in atherosclerotic vessels from ApoE-/- mice receiving the negative control antibody. Immunohistochemical studies revealed increased expression of ED-B not only in murine but also in human plaques, in which it was found predominantly around vasa vasorum and plaque matrix. In summary, we demonstrate selective targeting of atheromas in mice using the human antibody to the ED-B domain of fibronectin. Thus, our findings may set the stage for antibody-based molecular imaging of atherosclerotic plaques in the intact organism.

Engineering a thermostable human prolyl endopeptidase for antibody-directed enzyme prodrug therapy.

We present a new antibody-directed enzyme prodrug therapy strategy (ADEPT) based on a post-proline cleaving endopeptidase and prodrugs, in which cytotoxic moieties are linked to a proline-containing peptide. Human prolyl endopeptidase was expressed in Escherichia coli and purified to homogeneity. The enzyme was active in buffer and in human serum but was rapidly thermally inactivated by incubation at 37 degrees C, thus preventing applications in vivo. While prolyl endopeptidase display on filamentous phage abolished viral infectivity and prevented directed evolution strategies based on phage display, we robotically screened 10752 individual colonies of mutant enzymes using a fluorogenic assay to improve enzyme stability. A single amino acid mutation (Glu289 --> Gly) improved protein stability, resulting in a half-life of 16 h at 37 degrees C in phosphate buffer. Two prodrugs were synthesized, in which an N-protected glycine-proline dipeptide was covalently coupled to doxorubicin and melphalan. (Benzyloxycarbonyl)glycylprolylmelphalan, but not the more sterically hindered doxorubicin prodrug, could be efficiently activated by prolyl endopeptidase [specific activity = 813.3 nmol min(-1) (mg of enzyme)(-1) at 25 degrees C]. The melphalan prodrug was essentially nontoxic to CHO, F9 teratocarcinoma, MCF7 breast adenocarcinoma, and p3U1 mouse myeloma cells up to millimolar concentrations, while prodrug incubation with the engineered prolyl endopeptidase mutant led to a cell killing profile superimposable to the one of melphalan. The prolyl endopeptidase mutant was then chemically coupled to the human antibody L19, specific to the EDB domain of fibronectin, a marker of angiogenesis. The resulting immunoconjugate retains antigen binding and enzymatic activity, thus opening the way to anticancer ADEPT applications.

Molecular targeting of angiogenesis.

The majority of pharmacological approaches for the treatment of solid tumors suffer from poor selectivity, thus limiting dose escalation (i.e., the doses of drug which are required to kill tumor cells cause unacceptable toxicities to normal tissues). The situation is made more dramatic by the fact that the majority of anticancer drugs accumulate preferentially in normal tissues rather than in neoplastic sites, due to the irregular vasculature and to the high interstitial pressure of solid tumors. One avenue towards the development of more efficacious and better tolerated anti-cancer drugs relies on the targeted delivery of therapeutic agents to the tumor environment, thus sparing normal tissues. Molecular markers which are selectively expressed in the stroma and in neo-vascular sites of aggressive solid tumors appear to be particularly suited for ligand-based tumor targeting strategies. Tumor blood vessels are accessible to agents coming from the bloodstream, and their occlusion may result in an avalanche of tumor cell death. Furthermore, endothelial cells and stromal cells are genetically more stable than tumor cells and can produce abundant markers, which are ideally suited for tumor targeting strategies. This review focuses on recent advances in the development of ligands for the selective targeting of tumor blood vessels and new blood vessels in other angiogenesis-related diseases.