Pancreatic ductal adenocarcinomas (PDAC) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC- derived exosomes induce liver pre-metastatic niche formation in naïve mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared to patients whose pancreatic tumors did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

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The exact molecular mechanism of how endostatin inhibits angiogenesis and tumor growth remains uncharacterized. Here, we report that endostatin specifically binds to the cell surface nucleolin with high affinity. Blockage of nucleolin by a neutralizing antibody or knockdown of nucleolin by the RNA interference results in loss of antiendothelial activities of endostatin. Importantly, a neutralizing antinucleolin antibody abrogates the antiangiogenic and antitumor activities of endostatin in vivo. Nucleolin and endostatin are colocalized on the cell surface of endothelial cells of angiogenic blood vessels in the tumor environment. Finally, we found that endostatin is internalized and transported into cell nuclei of endothelial cell via nucleolin. In the nucleus, the phosphorylation of nucleolin, which is critical for cell proliferation, can be inhibited by endostatin. Our studies demonstrate that nucleolin is a novel functional receptor for endostatin, and mediates the antiangiogenic and antitumor activities of endostatin. These findings also provide mechanistic insights of how endostatin specifically inhibits proliferating endothelial cell growth and angiogenesis. IntroductionAngiogenesis, sprouting new blood vessels from existing capillaries, is critical for tumor growth. 1,2 Therefore, antiangiogenic molecules offer new promises as novel therapeutic modalities for the treatment of tumors. Endostatin (ES), a 20-kDa C-terminal globular domain of the collagen XVIII, was originally isolated from the supernatant of a cultured murine hemangioendothelioma cell line for its ability to inhibit tumor angiogenesis. 3 In animal models, tumor dormancy could be induced by repeated administration of ES for several cycles without causing drug resistance. 4 Moreover, low toxicity of ES has been reported in both animal studies and human trials. [4][5][6] ES exhibits potent anti-endothelial cell activities including inhibition of cell proliferation, migration, adhesion, and survival, which are all required for angiogenesis in vivo. 3,[7][8][9][10][11] The exact molecular mechanism of ES still remains an enigma, although a number of ES-binding proteins such as integrins, tropomyosin, glypicans, laminin, and MMP2 have been reported as ES receptors. 9,[12][13][14][15] However, whether these ES receptors are involved in the antitumor function of ES remains elusive. Recently, using DNA gene microarray and proteomic analysis, Huber and colleagues have identified a number of potential intracellular targets of ES (Abdollahi et al 16 ). Nevertheless, several antiangiogenic-related properties of ES remain uncharacterized, and they have raised several critical unexplored issues at the molecular level. These include (1) Why does ES specifically target angiogenic blood vessels but not quiescent blood vessels 17 ? (2) Why does ES specifically inhibit tumor growth and produce little if any toxicity in animal studies and clinical trials 4-6 ? (3) Why are heparin-binding sites required for the angiostatic activities of ES 18 ? To unravel these p...

Purpose: Elevated numbers of tumor-associated macrophages (TAM) in the tumor microenvironment are often correlated with poor prognosis in melanoma. However, the mechanisms by which TAMs modulate melanoma growth are still poorly understood. This study was aimed at examining the function and mechanism of TAM-derived adrenomedullin (ADM) in angiogenesis and melanoma growth.Experimental Design: We established in vitro and in vivo models to investigate the relationship between TAMs and ADM in melanoma, the role and mechanism of ADM in TAM-induced angiogenesis and melanoma growth. The clinical significance of ADM and its receptors was evaluated using melanoma tissue microarrays.Results: ADM was expressed by infiltrating TAMs in human melanoma, and its secretion from macrophages was upregulated upon coculture with melanoma cells, or with melanoma cells conditioned media. Meanwhile, TAMs enhanced endothelial cell migration and tubule formation and also increased B16/F10 tumor growth. Neutralizing ADM antibody and ADM receptor antagonist, AMA, attenuated TAM-induced angiogenesis in vitro and melanoma growth in vivo, respectively. Furthermore, ADM promoted angiogenesis and melanoma growth via both the paracrine effect, mediated by the endothelial nitric oxide synthase signaling pathway, and the autocrine effect, which stimulated the polarization of macrophages toward an alternatively activated (M2) phenotype. Finally, immunofluorescence analysis on human melanomas showed that the expression of ADM in TAMs and its receptors was greatly increased compared with adjacent normal skins.Conclusion: Our study reveals a novel mechanism that TAMs enhance angiogenesis and melanoma growth via ADM and provides potential targets for melanoma therapies.

IntroductionAngiogenesis, the generation of new capillaries from preexisting microvasculature, occurs in a variety of pathologic processes, such as tumor growth and metastasis and various inflammatory disorders. 1,2 Angiogenic vessels differ from normal vessels in their morphologic and molecular characteristics. Some unique molecules have been identified specifically expressing on angiogenic vessels, including some types of endothelial growth factor receptors, integrins, proteolytic enzymes, and extracellular matrix, as well as membrane proteins of unknown functions. 3,4 Recently, cell-surface nucleolin was reported as a specific marker of angiogenic endothelial cells. 5 Nucleolin has been described as a nucleolar protein of eukaryotic cells, involved in regulation of cell proliferation, cytokinesis, replication, embryogenesis, and nucleogenesis. 6,7 Recently, a number of studies have shown that it can also be expressed on the cell surface and serve as a receptor for several ligands such as matrix laminin-1, midkine, and lactoferrin. [8][9][10][11][12][13] However, little information about the physiologic relevance of cell-surface nucleolin in angiogenic is known except that cell-surface nucleolin specifically appears in tumor-induced angiogenic vessels rather than mature vessels or capillaries. 5 Here we report that cell-surface nucleolin is essential for the migration and tubule formation of endothelial cells. During the process of angiogenic, the up-regulation of cell-surface nucleolin is attributed to the shuttle of nucleolin from nucleus to cell membrane. Vascular endothelial growth factor (VEGF), a core regulator in tumor angiogenic and stroma generation, 14,15 can significantly stimulate the translocation of nucleolin when endothelial cells adhere to extracellular matrix. Cell-surface nucleolin was reported to associate with actin cytoskeleton. 9 In this study, we found that nonmuscle myosin heavy chain 9 (MyH9), an actin-based motor protein, provides a linker between cell-surface nucleolin and actin cytoskeleton and mediates its function in angiogenic. MyH9 is one isoform of the class II myosin family, which mediates a variety of cellular processes including protrusion, migration, and modulation of cell locomotion. 16,17 Recent studies showed that MyH9 plays an important role in modulating T-cell motility and tissue organization during embryo development. 18,19 The shuttle of nucleolin between nucleus and cell surface depends on anchoring to MyH9, which appears essential for the angiogenic function of cell-surface nucleolin. Materials and methods Antibodies, proteins, and chemicalsPolyclonal antibodies to nucleolin were from our lab storage. Plasma fibronectin was purified from human blood by gelatin affinity column. Other antibodies, proteins, and chemicals were from Sigma-Aldrich ( For personal use only. on May 11, 2018. by guest www.bloodjournal.org From Identification of isolated proteins with MALDI-TOF mass spectrometryThe major band from coimmunoprecipitation was digested using sequencing grade por...

Before metastasis, certain organs have already been influenced by primary tumors. However, the exact alterations and regulatory mechanisms of the premetastatic organs remain poorly understood. Here, we report that, in the premetastatic stage, angiopoietin 2 (Angpt2), matrix metalloproteinase (MMP) 3, and MMP10 are up-regulated in the lung by primary B16/F10 tumor, which leads to the increased permeability of pulmonary vasculatures and extravasation of circulating tumor cells. Subsequent studies show that Angpt2, MMP3, and MMP10 have a synergistic effect on disrupting vascular integrity in both in vitro and in vivo models. Lentivirus-based in vivo RNA interference of Angpt2, MMP3, and MMP10 attenuates the pulmonary vascular permeability and suppresses the infiltration of myeloid cells in the premetastatic lung. Moreover, knocking down these factors significantly inhibits the spontaneous lung metastasis in the model by orthotopic implantation of MDA-MB-231-Luc-D3H1 cells in nude mice. Further investigations reveal that the malignancy of tumor cells is positively correlated with their capabilities to induce the expression of Angpt2, MMP3, and MMP10. Luciferase reporter assay and chromatin immunoprecipitation assay also suggest that transforming growth factor-B1 and tumor necrosis factor-A signaling are involved in the regulation of these premetastatic factors. Our study shows that pulmonary vascular destabilization in the premetastatic phase promotes the extravasation of tumor cells and facilitates lung metastasis, which may provide potential targets for clinical prevention of metastasis.

Endostatin (ES) was reported to stimulate apoptosis in endothelial cells, but the exact mechanism remains controversial. In the present study, we elucidate the mechanism of ES-induced endothelial cell apoptosis. Our results indicate that ES induces cytochrome c release and caspase-9 activation in human microvascular endothelial cells (HMECs) at the concentration of 1 microM for 24 h, which initiates the apoptosis process. Further, ATP production, mitochondrial membrane potential, and tubule formation assays showed that ES promotes the mitochondrial permeability transition pore (mPTP) opening via voltage-dependent anion channel 1 (VDAC1), a major component of mitochondrial outer membrane. Knocking down VDAC1 by small interfering RNA attenuates ES-induced apoptosis, while overexpression of VDAC1 enhances the sensitivity of endothelial cells to ES. Moreover, we reveal that ES induces the reduction of hexokinase 2 (HK2), which, in turn, promotes VDAC1 phosphorylation and accumulation. Data from two-dimensional electrophoresis, immunoprecipitation, mPTP opening, and caspase-3 activation assays indicate that two serine residues of VDAC1, Ser-12 and Ser-103, can modulate VDAC1 protein level and thus the sensitivity to apoptosis stimuli. On the basis of these findings, we conclude that VDAC1 plays a vital role in modulating ES-induced endothelial cell apoptosis.

In recent years, the utilization of nanomaterials such as carbon nanotubes (CNTs) in the field of neuroscience has forever changed the approach to nerve-related research. The array of novel properties CNTs possess allows them to interact with neurons at the nanodimensional scale. In this study, a CNT rope substrate is developed to allow the electrical stimulation of neural stem cells (NSCs) in culture medium and the in situ observation of the response of these stem cells after stimulation. CNTs are synthesized by chemical vapor deposition and prepared into a ropelike structure with a diameter of 1 mm and length of 1.5 cm. NSCs are differentiated on the CNT rope substrate while the direction of neurite outgrowth, phenotype, and maturity of the NSCs are analyzed. Fluorescence and scanning electron microscopy demonstrate that neurite extension favors the direction of the spiral topography on the CNT rope. NSCs plated on CNT ropes are boosted towards differentiated neurons in the early culture stage when compared to conventional tissue culture plates via the analysis of neuronal gene and protein expressions by quantitative polymerase chain reaction and immunostaining, respectively. Furthermore, a set of electrical stimulation parameters (5 mV, 0.5 mA, 25 ms intermittent stimulation) promotes neuronal maturity while also increasing the speed of neurite outgrowth. These results indicate that an electroconductive CNT rope substrate along with electrical stimulation may have a synergistic effect on promoting neurite elongation and boosting effects on the differentiation of NSCs into mature neuronal cells for therapeutic application in neural regeneration.