Yanping Ding

Pulmonary vascular destabilization in the premetastatic phase facilitates lung metastasis.

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-beta1 and tumor necrosis factor-alpha 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.

Tumor-Associated Macrophages Promote Angiogenesis and Melanoma Growth via Adrenomedullin in a Paracrine and Autocrine Manner

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. Clin Cancer Res; 17(23); 7230–9. ©2011 AACR.

Overexpression of Platelet-Derived Growth Factor-BB Increases Tumor Pericyte Content via Stromal-Derived Factor-1α/CXCR4 Axis

Platelet-derived growth factor-BB (PDGF-BB) is a well-characterized growth factor displaying potent biological effects on angiogenesis. Recent studies reveal that overexpression of PDGF-BB within tumors results in increased pericyte coverage, suggesting that PDGF-BB signaling is also essential for the cancerous pericyte recruitment process. However, the molecular mechanism underlying this regulation remains obscure. In the current study, we show that tumor-derived PDGF-BB induces SDF-1alpha expression in endothelial cells (EC), resulting in the formation of SDF-1alpha chemotaxis gradient, which coincides with the PDGF-BB-induced pericyte recruitment during angiogenesis. PDGF-BB dramatically up-regulates SDF-1alpha secretion through the activation of PDGFRbeta in tumor-associated ECs, whereas this up-regulation can be substantially inhibited by either blockade of the phosphatidylinositol 3-kinase/Akt/mTOR pathway with chemical inhibitors or the inactivation of HIF-1alpha through small interfering RNA interference. On the other hand, we reveal that SDF-1alpha can increase pericytes motility in vitro. Blockade of the SDF-1alpha/CXCR4 axis prevents the PDGF-BB-induced pericyte recruitment not only in three in vitro recruitment models but also in the PDGF-BB-overexpressing tumor xenograft models. These results highlight that the involvement of SDF-1alpha/CXCR4 axis is essential for the pericyte recruitment within the PDGF-BB-overexpressing tumors and raise the possibility that blockade of the SDF-1alpha/CXCR4 axis may provide a therapeutic synergy with antiangiogenic molecules that selectively target ECs.

Using Functional Nanomaterials to Target and Regulate the Tumor Microenvironment: Diagnostic and Therapeutic Applications

Malignant tumors remain a major health burden throughout the world and effective therapeutic strategies are urgently needed. Cancer nanotechnology, as an integrated platform, has the potential to dramatically improve cancer diagnosis, imaging, and therapy, while reducing the toxicity associated with the current approaches. Tumor microenvironment is an ensemble performance of various stromal cells and extracellular matrix. The recent progress in understanding the critical roles and the underlying mechanisms of the tumor microenvironment on tumor progression has resulted in emerging diagnostic and therapeutic nanomaterials designed and engineered specifically targeting the microenvironment components. Meanwhile, the bio-physicochemical differences between tumor and normal tissues have recently been exploited to achieve specific tumor-targeting for cancer diagnosis and treatment. Here, the major players in the tumor microenvironment and their biochemical properties, which can be utilized for the design of multifunctional nanomaterials with the potential to target and regulate this niche, are summarized. The recent progress in engineering intelligent and versatile nanomaterials for targeting and regulating the tumor microenvironment is emphasized. Although further investigations are required to develop robust methods for more specific tumor-targeting and well-controlled nanomaterials, the applications of tumor microenvironment regulation-based nanotechnology for safer and more effective anticancer nanomedicines have been proven successful and will eventually revolutionize the current landscape of cancer therapy.

Triple-punch strategy for triple negative breast cancer therapy with minimized drug dosage and improved antitumor efficacy.

Effective therapeutics against triple negative breast cancer (TNBC), which has no standard-of-care therapy, needs to be developed urgently. Here we demonstrated a strategy of integrating indocyanine green (ICG), paclitaxel (PTX), and survivin siRNA into one thermosensitive poly(2-(2-methoxyethoxy)ethyl methacrylate-co-oligo(ethylene glycol) methacrylate)-co-2-(dimethylamino)ethyl methacrylate-b-poly(D,L-lactide-co-glycolide) (P (MEO2MA-co-OEGMA-co-DMAEMA)-b-PLGA) nanoparticle (NP-IPS) for triple-punch strategy against TNBC. The NP-IPS significantly enhanced the stability of ICG. Controlled release of the PTX in tumor regions was triggered by the hyperthermia produced by laser irradiated ICG. The NP-IPS exhibited remarkable antitumor efficacy (almost complete ablation of the tumor xenografts) due to the combinational effects of chemotherapy, photothermal therapy, and gene therapy with low drug dose (ICG, 0.32 μmol/kg; PTX, 0.54 μmol/kg; siRNA, 1.5 mg/kg) and minimal side effects. Taken together, our current study demonstrates a nanoplatform for triple-therapy, which reveals a promising strategy for TNBC treatment.

The nuclear translocation of endostatin is mediated by its receptor nucleolin in endothelial cells

Endostatin, the C-terminal fragment of collagen XVIII, is a potent anti-angiogenic factor that significantly modulates the gene expression pattern in endothelial cells. Upon cell surface binding, endostatin can not only function extracellularly, but also translocate to the nucleus within minutes. However, the mechanism by which this occurs is partially understood. Here we systematically investigated the nuclear translocation mechanism of endostatin. By chemical inhibition and RNA interference, we firstly observed that clathrin-mediated endocytosis, but not caveolae-dependent endocytosis or macropinocytosis, is essential for the nuclear translocation of endostatin. We then indentified that nucleolin and integrin α5β1, two widely accepted endostatin receptors, mediate this clathrin-dependent uptake process, which also involves urokinase plasminogen activator receptor (uPAR). Either mutagenesis study, fluorescence resonance energy transfer assay, or fluorescence cell imaging demonstrates that nucleolin and integrin α5β1 interact with uPAR simultaneously upon endostatin stimulation. Blockade of uPAR decreases not only the interaction between nucleolin and integrin α5β1, but also the uptake process, suggesting that the nucleolin/uPAR/integrin α5β1 complex facilitates the internalization of endostatin. After endocytosis, nucleolin further regulates the nuclear transport of endostatin. RNA interference and mutational analysis revealed that the nuclear translocation of endostatin involves the association of nucleolin with importin α1β1 via the nuclear localization sequence. Taken together, this study reveals the pathway by which endostatin translocates to the nucleus and the importance of nucleolin in this process, providing a new perspective for the functional investigation of the nuclear-translocated endostatin in endothelial cells.

Peptide Assembly Integration of Fibroblast‐Targeting and Cell‐Penetration Features for Enhanced Antitumor Drug Delivery

T. Ji, Dr. Y. Ding, Dr. Y. Zhao, J. Wang, H. Qin, J. Lang, R. Zhao, Y. Zhang, J. Shi, Prof. G. Nie CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST) 11 Beiyitiao, Zhongguancun Beijing 100190 , China E-mail: zhaoying@nanoctr.cn; niegj@nanoctr.cn X. Liu, Dr. N. Tao, Prof. Z. Qin CAS Key Laboratory of Protein and Peptide Pharmaceuticals Institute of Biophysics 15 Datun Road , Beijing 100101 , China E-mail: zhihai@ibp.ac.cn

The CXCL12-CXCR4 Chemokine Pathway: A Novel Axis Regulates Lymphangiogenesis

Purpose: Lymphangiogenesis, the growth of lymphatic vessels, contributes to lymphatic metastasis. However, the precise mechanism underlying lymphangiogenesis remains poorly understood. This study aimed to examine chemokine/chemokine receptors that directly contribute to chemoattraction of activated lymphatic endothelial cells (LEC) and tumor lymphangiogenesis. Experimental Design: We used quantitative RT-PCR to analyze specifically expressed chemokine receptors in activated LECs upon stimulation of vascular endothelial growth factor-C (VEGF-C). Subsequently, we established in vitro and in vivo models to show lymphangiogenic functions of the chemokine axis. Effects of targeting the chemokine axis on tumor lymphangiogenesis and lymphatic metastasis were determined in an orthotopic breast cancer model. Results: VEGF-C specifically upregulates CXCR4 expression on lymphangiogenic endothelial cells. Moreover, hypoxia-inducible factor-1α (HIF-1α) mediates the CXCR4 expression induced by VEGF-C. Subsequent analyses identify the ligand CXCL12 as a chemoattractant for LECs. CXCL12 induces migration, tubule formation of LECs in vitro, and lymphangiogenesis in vivo. CXCL12 also stimulates the phosphorylation of intracellular signaling Akt and Erk, and their specific antagonists impede CXCL12-induced chemotaxis. In addition, its level is correlated with lymphatic vessel density in multiple cancer tissues microarray. Furthermore, the CXCL12–CXCR4 axis is independent of the VEGFR-3 pathway in promoting lymphangiogenesis. Intriguingly, combined treatment with anti-CXCL12 and anti-VEGF-C antibodies results in additive inhibiting effects on tumor lymphangiogenesis and lymphatic metastasis. Conclusions: These results show the role of the CXCL12–CXCR4 axis as a novel chemoattractant for LECs in promoting lymphangiogenesis, and support the potential application of combined targeting of both chemokines and lymphangiogenic factors in inhibiting lymphatic metastasis. Clin Cancer Res; 18(19); 5387–98. ©2012 AACR.

Field-Free Isolation of Exosomes from Extracellular Vesicles by Microfluidic Viscoelastic Flows

Exosomes, molecular cargos secreted by almost all mammalian cells, are considered as promising biomarkers to identify many diseases including cancers. However, the small size of exosomes (30-200 nm) poses serious challenges in their isolation from complex media containing a variety of extracellular vesicles (EVs) of different sizes, especially in small sample volumes. Here we present a viscoelasticity-based microfluidic system to directly separate exosomes from cell culture media or serum in a continuous, size-dependent, and label-free manner. Using a small amount of biocompatible polymer as the additive in the media to control the viscoelastic forces exerted on EVs, we are able to achieve a high separation purity (>90%) and recovery (>80%) of exosomes. The proposed technique may serve as a versatile platform to facilitate exosome analyses in diverse biochemical applications.

An MMP-2 Responsive Liposome Integrating Antifibrosis and Chemotherapeutic Drugs for Enhanced Drug Perfusion and Efficacy in Pancreatic Cancer.

Fibrotic stroma, a critical character of pancreatic tumor microenvironment, provides a critical barrier against the penetration and efficacy of various antitumor drugs. Therefore, new strategies are urgently needed to alleviate the fibrotic mass and increase the drug perfusion within pancreatic cancer tissue. In our current work, we developed a β-cyclodextrin (β-CD) modified matrix metalloproteinase-2 (MMP-2) responsive liposome, integrating antifibrosis and chemotherapeutic drugs for regulation of pancreatic stellate cells (PSCs), a key source of the fibrosis, and targeted delivery of cytotoxic drugs for pancreatic cancer therapy. These liposomes disassembed into two functional parts upon MMP-2 cleavage at the tumor site. One part was constituted by the β-CDs and the antifibrosis drug pirfenidone, which was kept in the stroma and inhibited the expression of collagen I and TGF-β in PSCs, down-regulating the fibrosis and decreasing the stromal barrier. The other segment, the RGD peptide-modified-liposome loading the chemotherapeutic drug gemcitabine, targeted and killed pancreatic tumor cells. This integrated nanomedicine, showing an increased drug perfusion without any overt side effects, may provide a potential strategy for improvement of the pancreatic cancer therapy.