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1.
bioRxiv ; 2024 May 18.
Article in English | MEDLINE | ID: mdl-38798370

ABSTRACT

Understanding pancreatic cancer biology is fundamental for identifying new targets and for developing more effective therapies. In particular, the contribution of the stromal microenvironment to pancreatic cancer tumorigenesis requires further exploration. Here, we report the stromal roles of the synaptic protein Netrin G1 Ligand (NGL-1) in pancreatic cancer, uncovering its pro-tumor functions in cancer-associated fibroblasts and in immune cells. We observed that the stromal expression of NGL-1 inversely correlated with patients' overall survival. Moreover, germline knockout (KO) mice for NGL-1 presented decreased tumor burden, with a microenvironment that is less supportive of tumor growth. Of note, tumors from NGL-1 KO mice produced less immunosuppressive cytokines and displayed an increased percentage of CD8 + T cells than those from control mice, while preserving the physical structure of the tumor microenvironment. These effects were shown to be mediated by NGL-1 in both immune cells and in the local stroma, in a TGF-ß-dependent manner. While myeloid cells lacking NGL-1 decreased the production of immunosuppressive cytokines, NGL-1 KO T cells showed increased proliferation rates and overall polyfunctionality compared to control T cells. CAFs lacking NGL-1 were less immunosuppressive than controls, with overall decreased production of pro-tumor cytokines and compromised ability to inhibit CD8 + T cells activation. Mechanistically, these CAFs downregulated components of the TGF-ß pathway, AP-1 and NFAT transcription factor families, resulting in a less tumor-supportive phenotype. Finally, targeting NGL-1 genetically or using a functionally antagonistic small peptide phenocopied the effects of chemotherapy, while modulating the immunosuppressive tumor microenvironment (TME), rather than eliminating it. We propose NGL-1 as a new local stroma and immunomodulatory molecule, with pro-tumor roles in pancreatic cancer. Statement of Significance: Here we uncovered the pro-tumor roles of the synaptic protein NGL-1 in the tumor microenvironment of pancreatic cancer, defining a new target that simultaneously modulates tumor cell, fibroblast, and immune cell functions. This study reports a new pathway where NGL-1 controls TGF-ß, AP-1 transcription factor members and NFAT1, modulating the immunosuppressive microenvironment in pancreatic cancer. Our findings highlight NGL-1 as a new stromal immunomodulator in pancreatic cancer.

2.
Cell Mol Gastroenterol Hepatol ; 17(5): 737-743, 2024.
Article in English | MEDLINE | ID: mdl-38316215

ABSTRACT

Bearing a dismal 5-year survival rate, pancreatic ductal adenocarcinoma (PDAC) is a challenging disease that features a unique fibroinflammatory tumor microenvironment. As major components of the PDAC tumor microenvironment, cancer-associated fibroblasts are still poorly understood and their contribution to the several hallmarks of PDAC, such as resistance to therapies, immunosuppression, and high incidence of metastasis, is likely underestimated. There have been encouraging advances in the understanding of these fascinating cells, but many controversies remain, leaving the field still actively exploring the full scope of their contributions in PDAC progression. Here we pose several important considerations regarding PDAC cancer-associated fibroblast functions. We posit that transcriptomic analyses be interpreted with caution, when aiming to uncover the functional contributions of these cells. Moreover, we propose that normalizing these functions, rather than eliminating them, will provide the opportunity to enhance therapeutic response. Finally, we propose that cancer-associated fibroblasts should not be studied in isolation, but in conjunction with its extracellular matrix, because their respective functions are coordinated and concordant.


Subject(s)
Cancer-Associated Fibroblasts , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Humans , Cancer-Associated Fibroblasts/pathology , Pancreatic Neoplasms/pathology , Carcinoma, Pancreatic Ductal/pathology , Stromal Cells , Gene Expression Profiling , Tumor Microenvironment
3.
bioRxiv ; 2024 Jan 15.
Article in English | MEDLINE | ID: mdl-38293200

ABSTRACT

Pancreatic cancer is becoming increasingly deadly, with treatment options limited due to, among others, the complex tumor microenvironment (TME). This short communications study investigates pulsed low-dose-rate radiation (PLDR) as a potential alternative to conventional radiotherapy for pancreatic cancer neoadjuvant treatment. Our ex vivo research demonstrates that PLDR, in combination with chemotherapy, promotes a shift from tumor-promoting to tumor-suppressing properties in a key component of the pancreatic cancer microenvironment we called CAFu (cancer-associated fibroblasts and selfgenerated extracellular matrix functional units). This beneficial effect translates to reduced desmoplasia (fibrous tumor expansion) and suggests PLDR's potential to improve total neoadjuvant therapy effectiveness. To comprehensively assess this functional shift, we developed the HOST-Factor, a single score integrating multiple biomarkers. This tool provides a more accurate picture of CAFu function compared to individual biomarkers and could be valuable for guiding and monitoring future therapeutic strategies. Our findings support the ongoing NCT04452357 clinical trial testing PLDR safety and TME normalization potential in pancreatic cancer patients. The HOST-Factor will be used in samples collected from this trial to validate its potential as a key tool for personalized medicine in this aggressive disease.

4.
bioRxiv ; 2023 Sep 17.
Article in English | MEDLINE | ID: mdl-37745612

ABSTRACT

In pancreatic ductal adenocarcinoma (PDAC), the fibroblastic stroma constitutes most of the tumor mass and is remarkably devoid of functional blood vessels. This raises an unresolved question of how PDAC cells obtain essential metabolites and water-insoluble lipids. We have found a critical role for cancer-associated fibroblasts (CAFs) in obtaining and transferring lipids from blood-borne particles to PDAC cells via trogocytosis of CAF plasma membranes. We have also determined that CAF-expressed phospholipid scramblase anoctamin 6 (ANO6) is an essential CAF trogocytosis regulator required to promote PDAC cell survival. During trogocytosis, cancer cells and CAFs form synapse-like plasma membranes contacts that induce cytosolic calcium influx in CAFs via Orai channels. This influx activates ANO6 and results in phosphatidylserine exposure on CAF plasma membrane initiating trogocytosis and transfer of membrane lipids, including cholesterol, to PDAC cells. Importantly, ANO6-dependent trogocytosis also supports the immunosuppressive function of pancreatic CAFs towards cytotoxic T cells by promoting transfer of excessive amounts of cholesterol. Further, blockade of ANO6 antagonizes tumor growth via disruption of delivery of exogenous cholesterol to cancer cells and reverses immune suppression suggesting a potential new strategy for PDAC therapy.

5.
Cancer Res Commun ; 2(9): 1017-1036, 2022 09.
Article in English | MEDLINE | ID: mdl-36310768

ABSTRACT

It is projected that in 5 years, pancreatic cancer will become the second deadliest cancer in the United States. A unique aspect of pancreatic ductal adenocarcinoma (PDAC) is its stroma; rich in cancer-associated fibroblasts (CAFs) and a dense CAF-generated extracellular matrix (ECM). These pathogenic stroma CAF/ECM units cause the collapse of local blood vessels rendering the tumor microenvironment nutrient-poor. PDAC cells are able to survive this state of nutrient stress via support from CAF-secreted material, which includes small extracellular vesicles (sEVs). The tumor-supportive CAFs possess a distinct phenotypic profile, compared to normal-like fibroblasts, expressing NetrinG1 (NetG1) at the plasma membrane, and active Integrin α5ß1 localized to the multivesicular bodies; traits indicative of poor patient survival. We herein report that NetG1+ CAFs secrete sEVs that stimulate Akt-mediated survival in nutrient-deprived PDAC cells, protecting them from undergoing apoptosis. Further, we show that NetG1 expression in CAFs is required for the pro-survival properties of sEVs. Additionally, we report that the above-mentioned CAF markers are secreted in distinct subpopulations of EVs; with NetG1 being enriched in exomeres, and Integrin α5ß1 being enriched in exosomes. Finally, we found that NetG1 and Integrin α5ß1 were detected in sEVs collected from plasma of PDAC patients, while their levels were significantly lower in plasma-derived sEVs of sex/age-matched healthy donors. The discovery of these tumor-supporting CAF-EVs elucidates novel avenues in tumor-stroma interactions and pathogenic stroma detection.


Subject(s)
Cancer-Associated Fibroblasts , Carcinoma, Pancreatic Ductal , Extracellular Vesicles , Pancreatic Neoplasms , Humans , Cancer-Associated Fibroblasts/metabolism , Pancreatic Neoplasms/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Integrin alpha5beta1/metabolism , Extracellular Vesicles/metabolism , Tumor Microenvironment , Pancreatic Neoplasms
6.
Methods Mol Biol ; 2514: 15-28, 2022.
Article in English | MEDLINE | ID: mdl-35771414

ABSTRACT

The development of vasculature in vivo is an extremely complex process that requires temporal and spatial coordination between multiple cell types to produce an effective vessel. The formation of vasculature from preexisting blood vessels, known as angiogenesis, plays important roles in several physiological and pathological processes, including wound healing, organ development and growth, ischemia, inflammatory disorders, fibrosis, and cancer. Means to deconstruct these complicated biological systems are necessary to gain mechanistic insight into their development, function, and modulation that can be tested in in vivo models and ultimately the clinic. In this chapter, we will first review the classical in vitro techniques to study angiogenesis. Next, we will explore the exciting recent advances that rely on 3D multicellular systems to more accurately mimic vasculature development in vitro. Finally, we will discuss the applications of in vitro angiogenic methods to study related vasculature phenomena, such as vasculogenic mimicry.


Subject(s)
Neoplasms , Neovascularization, Physiologic , Cardiovascular Physiological Phenomena , Humans , Neoplasms/blood supply , Neovascularization, Pathologic , Wound Healing
7.
Methods Mol Biol ; 2514: 31-38, 2022.
Article in English | MEDLINE | ID: mdl-35771415

ABSTRACT

Vasculature development is a combination of complex processes that require precise coordination of multiple cell types, through time and space, to generate functional blood-carrying vessels. Moreover, vasculature development can be altered when normal physiological conditions are disrupted, such as in cancer, and means to study blood vessels are of great importance. While the gold standard to explore these processes is the use of in vivo animal models, they are costly and time-consuming, and it is often difficult to dissect the molecular mechanisms involved. Thus, there are several ways to deconstruct vasculature development in vitro, in order to produce tunable systems that lead to a better understanding of cellular and molecular communication between different cell types involved, such as endothelial cells and supporting mesenchymal cells. In this method chapter, we will go into detail for one of the most popular ways of studying vasculature development in the context of cancer, which is the application of Matrigel to study tube formation of various cell types involved with vasculature development. We will provide step-by-step instructions to perform mono- and co-cultures of the major cells involved with the production of vasculature, how the results of these assays can be interpreted, and some advice to avoid common mistakes associated with Matrigel tube formation assays.


Subject(s)
Endothelial Cells , Neoplasms , Animals , Collagen , Drug Combinations , Laminin , Neovascularization, Pathologic , Proteoglycans
8.
Cancer Discov ; 11(2): 446-479, 2021 02.
Article in English | MEDLINE | ID: mdl-33127842

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell-mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. SIGNIFICANCE: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.See related commentary by Sherman, p. 230.This article is highlighted in the In This Issue feature, p. 211.


Subject(s)
Adenocarcinoma/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Netrins/metabolism , Pancreatic Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Humans , Immunosuppression Therapy , Nutritional Support , Tumor Microenvironment
9.
Cancer Cell ; 38(4): 567-583.e11, 2020 10 12.
Article in English | MEDLINE | ID: mdl-32976774

ABSTRACT

Oncogenic transformation alters lipid metabolism to sustain tumor growth. We define a mechanism by which cholesterol metabolism controls the development and differentiation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by conditional inactivation of the rate-limiting enzyme Nsdhl or treatment with cholesterol-lowering statins switches glandular pancreatic carcinomas to a basal (mesenchymal) phenotype in mouse models driven by KrasG12D expression and homozygous Trp53 loss. Consistently, PDACs in patients receiving statins show enhanced mesenchymal features. Mechanistically, statins and NSDHL loss induce SREBP1 activation, which promotes the expression of Tgfb1, enabling epithelial-mesenchymal transition. Evidence from patient samples in this study suggests that activation of transforming growth factor ß signaling and epithelial-mesenchymal transition by cholesterol-lowering statins may promote the basal type of PDAC, conferring poor outcomes in patients.


Subject(s)
Biosynthetic Pathways/genetics , Carcinoma, Pancreatic Ductal/genetics , Cholesterol, LDL/biosynthesis , Pancreatic Neoplasms/genetics , Transforming Growth Factor beta/genetics , 3-Hydroxysteroid Dehydrogenases/genetics , 3-Hydroxysteroid Dehydrogenases/metabolism , Animals , Atorvastatin/pharmacology , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/metabolism , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line, Tumor , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/genetics , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Kaplan-Meier Estimate , Mice, Inbred C57BL , Mice, Knockout , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Signal Transduction/genetics , Transforming Growth Factor beta/metabolism , Xenograft Model Antitumor Assays/methods
10.
Elife ; 62017 01 31.
Article in English | MEDLINE | ID: mdl-28139197

ABSTRACT

Desmoplasia, a fibrotic mass including cancer-associated fibroblasts (CAFs) and self-sustaining extracellular matrix (D-ECM), is a puzzling feature of pancreatic ductal adenocarcinoma (PDACs). Conflicting studies have identified tumor-restricting and tumor-promoting roles of PDAC-associated desmoplasia, suggesting that individual CAF/D-ECM protein constituents have distinguishable tumorigenic and tumor-repressive functions. Using 3D culture of normal pancreatic versus PDAC-associated human fibroblasts, we identified a CAF/D-ECM phenotype that correlates with improved patient outcomes, and that includes CAFs enriched in plasma membrane-localized, active α5ß1-integrin. Mechanistically, we established that TGFß is required for D-ECM production but dispensable for D-ECM-induced naïve fibroblast-to-CAF activation, which depends on αvß5-integrin redistribution of pFAK-independent active α5ß1-integrin to assorted endosomes. Importantly, the development of a simultaneous multi-channel immunofluorescence approach and new algorithms for computational batch-analysis and their application to a human PDAC panel, indicated that stromal localization and levels of active SMAD2/3 and α5ß1-integrin distinguish patient-protective from patient-detrimental desmoplasia and foretell tumor recurrences, suggesting a useful new prognostic tool.


Subject(s)
Cancer-Associated Fibroblasts/chemistry , Carcinoma, Pancreatic Ductal/complications , Carcinoma, Pancreatic Ductal/pathology , Cell Membrane/chemistry , Fibroma, Desmoplastic/complications , Fibroma, Desmoplastic/pathology , Integrin alpha5beta1/analysis , Biomarkers, Tumor/analysis , Extracellular Matrix/metabolism , Humans , Transforming Growth Factor beta/metabolism
11.
Carcinogenesis ; 38(1): 51-63, 2017 01.
Article in English | MEDLINE | ID: mdl-27797827

ABSTRACT

The tumor microenvironment offers multiple targets for cancer therapy, including pro-tumorigenic inflammation. Natural compounds represent an enormous source of new anti-inflammatory and anticancer agents. We previously showed that the styryl lactone goniothalamin (GTN) has promising antiproliferative and anti-inflammatory activities. Because inflammation is a major driver of colorectal cancer (CRC), we therefore evaluated the therapeutic and preventive potentials of GTN in colitis, colitis-associated cancer (CAC) and spontaneous CRC. First, in a simplistic model of inflammation in vitro, GTN was able to inhibit cytokine production in bone marrow-derived macrophages induced by lipopolysaccharide. Next, in dextran sulfate sodium (DSS) induced-colitis model, mice treated with GTN displayed restored tissue architecture, increased cell proliferation in the colonic crypts and reduced epithelial damage. Moreover, colon tissue from GTN-treated mice had significantly less expression of the inflammatory genes interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), S100A9, interleukin 23A (IL-23A), IL-22 and IL-17A In the azoxymethane/DSS model of CAC, GTN reduced tumor multiplicity, load and size. Additionally, GTN suppressed production of IL-6, IL-17 and TNF-α in tumor tissue, as well as abrogated stromal immune cell activation and nuclear translocation of NF-κB. Finally, in a tamoxifen inducible model of sporadic CRC, GTN-treated mice had significantly fewer tumors and decreased levels of IL-17A, IL-6, S100A9 and TNF-α protein within the tumors. These results suggest that GTN possesses anti-inflammatory and antitumor activities and represents a preventive and therapeutic agent modulating the inflammatory environment in the colon during colitis as well as CAC and CRC development.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Cell Transformation, Neoplastic/drug effects , Colitis/complications , Colorectal Neoplasms/prevention & control , Inflammation Mediators/antagonists & inhibitors , Pyrones/pharmacology , Animals , Azoxymethane/toxicity , Biological Products/pharmacology , Carcinogens/toxicity , Cell Transformation, Neoplastic/metabolism , Cell Transformation, Neoplastic/pathology , Cells, Cultured , Colitis/chemically induced , Colorectal Neoplasms/etiology , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Cytokines/antagonists & inhibitors , Cytokines/genetics , Cytokines/metabolism , Dextran Sulfate/toxicity , Inflammation/chemically induced , Inflammation/complications , Inflammation Mediators/metabolism , Macrophages/drug effects , Macrophages/metabolism , Macrophages/pathology , Mice , Mice, Inbred C57BL
12.
Inflamm Bowel Dis ; 21(2): 409-18, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25563695

ABSTRACT

Inflammatory bowel diseases (IBDs) are debilitating conditions that result in intestinal damage due to chronic inflammation. In addition, the perpetual state of inflammation predisposes individuals to the development of colitis-associated cancer. Because of the immense immune cell infiltration into colon, cytokines produced by immune cells are major players in the initiation and progression of IBD and colitis-associated cancer. In this review, we will explore the functions of many key cytokines and their roles in IBD and colitis-associated cancer, as well as their influences on the immune system and stromal cells. Finally, we will briefly discuss current therapies and current clinical trials targeting cytokines in IBD.


Subject(s)
Colitis/complications , Cytokines/immunology , Immune System/immunology , Inflammatory Bowel Diseases/physiopathology , Neoplasms/etiology , Animals , Humans , Inflammatory Bowel Diseases/immunology , Neoplasms/pathology
13.
Cancer J ; 20(3): 181-9, 2014.
Article in English | MEDLINE | ID: mdl-24855005

ABSTRACT

Inflammation has long been suspected to play a major role in the pathogenesis of cancer. Only recently, however, have some mechanisms of its tumor promoting effects become known. Microbes, both commensal and pathogenic, are critical regulators of the host immune system and, ultimately, of inflammation. Consequently, microbes have the potential power to influence tumor progression as well, through a wide variety of routes, including chronic activation of inflammation, alteration of tumor microenvironment, induction of genotoxic responses, and metabolism. In this review, we will provide a general overview of commensal microbiota, inflammation, and cancer, as well as how microbes fit into this emerging field.


Subject(s)
Inflammation/microbiology , Microbiota , Neoplasms/microbiology , Animals , Humans , Inflammation/immunology , Inflammation/pathology , Neoplasms/immunology , Neoplasms/pathology
14.
Int J Biochem Cell Biol ; 51: 45-52, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24691276

ABSTRACT

Elevated serum levels of a secreted glycoprotein chitinase 3 like 1 (CHI3L1) are associated with poor prognosis and short survival time of patients with cervical cancer (CxCa). Our previous microarray data showed the increased expression of CHI3L1 in invasive CxCa compared to normal tissue, implicating a potential role of CHI3L1 in CxCa. To establish the pathological role of CHI3L1 in the development of CxCa, this study focused on its expression in CxCa and angiogenic impacts in tumor vessel formation. CHI3L1 activated angiogenesis by promoting endothelial cell migration and tube formation in vitro but failed to protect CxCa cell lines, CaSki and HeLa against apoptosis induced by γ-irradiation. In addition, the capability of CHI3L1 to induce proliferation and migration of CaSki and HeLa cells was cell type specific. In an analysis of 103 specimens from CxCa patients, increased expression levels of CHI3L1 mRNA and protein in invasive CxCa were 4-fold (P<0.05) and 2-fold (P<0.01), respectively, stronger than those in normal subjects. The immunostaining of CHI3L1 was positively correlated with VEGF expression (P=0.0019) and microvessel density (P=0.0110). Moreover, CHI3L1 expression was also positively associated with cancer metastasis (P=0.011). The data suggest the crucial role of CHI3L1 by promoting angiogenesis, which may contribute to the development and progression of CxCa. The findings help establish CHI3L1 as a prognostic biomarker and therapeutic target for CxCa patients.


Subject(s)
Adipokines/metabolism , Lectins/metabolism , Uterine Cervical Neoplasms/blood supply , Uterine Cervical Neoplasms/metabolism , Adipokines/biosynthesis , Adipokines/genetics , Apoptosis/physiology , Cell Growth Processes/physiology , Cell Line, Tumor , Chitinase-3-Like Protein 1 , Disease Progression , Female , HeLa Cells , Humans , Lectins/biosynthesis , Lectins/genetics , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/pathology , Prognosis , Uterine Cervical Neoplasms/pathology
15.
Carcinogenesis ; 35(2): 373-82, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24282289

ABSTRACT

Chemo/radiotherapies are the most common adjuvant modality treated for patients with glioblastoma (GBM) following surgery. However, the overall therapeutic benefits are still uncertain, as the mortality remains high. Elevated expression of YKL-40 in GBM was correlated with increases in mural cell-associated vessel coverage, stability and density, and decreases in vessel permeability and disease survival. To explore the potential role of YKL-40 in mural cell-mediated tumor vascularization, we employed an anti-YKL-40 neutralizing antibody (mAY) and ionizing irradiation (IR) in xenografted brain tumor models. Although single treatment with mAY or IR partially increased mouse survival, their combination led to dramatic inhibition in tumor growth and increases in mouse survival. mAY blocked mural cell-mediated vascular stability, integrity and angiogenesis; whereas IR merely promoted tumor cell and vascular cell apoptosis. Vascular radioresistance is at least partially attributed to expression of YKL-40 in mural cells. These divergent effects were also recapitulated in cultured systems using endothelial cells and mural cells differentiated from glioblastoma stem-like cells (GSCs). Dysfunction of intercellular contact N-cadherin was found to mediate mAY-inhibited vascularization. Collectively, the data suggest that the conjunction therapy with mAY and IR synergistically inhibit tumor vascularization and progression. The evidence may shed light on a new adjuvant therapy in clinic.


Subject(s)
Adipokines/immunology , Antibodies, Monoclonal/therapeutic use , Brain Neoplasms/prevention & control , Glioblastoma/prevention & control , Lectins/immunology , Neoplastic Stem Cells/pathology , Neovascularization, Pathologic/prevention & control , Radiation, Ionizing , Animals , Apoptosis , Blotting, Western , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Differentiation , Cell Proliferation , Chitinase-3-Like Protein 1 , Combined Modality Therapy , Drug Synergism , Fluorescent Antibody Technique , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Immunoenzyme Techniques , Immunoprecipitation , Mice , Mice, SCID , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/radiation effects
16.
J Neurosci ; 32(37): 12950-60, 2012 Sep 12.
Article in English | MEDLINE | ID: mdl-22973019

ABSTRACT

Recent evidence has shown that glioblastoma stem-like cells (GSCs) can transdifferentiate into endothelial cells and vascular-like tumor cells. The latter pattern of vascularization indicates an alternative microvascular circulation known as vasculogenic mimicry (VM). However, it remains to be clarified how the GSC-driven VM makes a significant contribution to tumor vasculature. Here, we investigated 11 cases of glioblastomas and found that most of them consisted of blood-perfused vascular channels that coexpress mural cell markers smooth muscle α-actin and platelet-derived growth factor receptor ß, epidermal growth factor receptor, and vascular endothelial growth factor receptor 2 (Flk-1), but not CD31 or VE-cadherin. This microvasculature coexisted with endothelial cell-associated vessels. GSCs derived from patients with glioblastomas developed vigorous mural cell-associated vascular channels but few endothelial cell vessels in orthotopic animal models. Suppression of Flk-1 activity and gene expression abrogated GSC transdifferentiation and vascularization in vitro, and inhibited VM in animal models. This study establishes mural-like tumor cells differentiated from GSCs as a significant contributor to microvasculature of glioblastoma and points to Flk-1 as a potential target for therapeutic intervention that could complement current anti-angiogenic treatment.


Subject(s)
Endothelial Cells/pathology , Glioblastoma/pathology , Neoplastic Stem Cells/pathology , Neovascularization, Pathologic/pathology , Cell Differentiation , Humans , Tumor Cells, Cultured
17.
J Biol Chem ; 287(29): 24821-31, 2012 Jul 13.
Article in English | MEDLINE | ID: mdl-22654102

ABSTRACT

Glioblastoma (GBM) is extremely aggressive and essentially incurable. Its malignancy is characterized by vigorous microvascular proliferations. Recent evidence has shown that tumor cells display the ability to drive blood-perfused vasculogenic mimicry (VM), an alternative microvascular circulation independent of endothelial cell angiogenesis. However, molecular mechanisms underlying this vascular pathogenesis are poorly understood. Here, we found that vascular channels of VM in GBM were composed of mural-like tumor cells that strongly express VEGF receptor 2 (Flk-1). To explore a potential role of Flk-1 in the vasculogenesis, we investigated two glioblastoma cell lines U87 and GSDC, both of which express Flk-1 and exhibit a vascular phenotype on Matrigel. Treatment of both cell lines with either Flk-1 gene knockdown or Flk-1 kinase inhibitor SU1498 abrogated Flk-1 activity and impaired vascular function. Furthermore, inhibition of Flk-1 activity suppressed intracellular signaling cascades, including focal adhesion kinase and mitogen-activated protein kinase ERK1/2. In contrast, blockade of VEGF activity by the neutralizing antibody Bevacizumab failed to recapitulate the impact of SU1498, suggesting that Flk-1-mediated VM is independent of VEGF. Xenotransplantation of SCID/Beige mice with U87 cells and GSDCs gave rise to tumors harboring robust mural cell-associated vascular channels. Flk-1 shRNA restrained VM in tumors and subsequently inhibited tumor development. Collectively, all the data demonstrate a central role of Flk-1 in the formation of VM in GBM. This study has shed light on molecular mechanisms mediating tumor aggressiveness and also provided a therapeutic target for patient treatment.


Subject(s)
Glioblastoma/metabolism , Neovascularization, Pathologic/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism , Animals , Cell Line, Tumor , Cinnamates/pharmacology , Fluorescent Antibody Technique , Humans , Immunoblotting , Immunohistochemistry , Mice , Neovascularization, Pathologic/genetics , RNA, Small Interfering , Vascular Endothelial Growth Factor Receptor-2/genetics , Xenograft Model Antitumor Assays
18.
J Vis Exp ; (55)2011 Sep 07.
Article in English | MEDLINE | ID: mdl-21931289

ABSTRACT

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM). There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.


Subject(s)
Collagen/chemistry , Endothelial Cells/cytology , Laminin/chemistry , Neoplasms/blood supply , Neoplasms/pathology , Proteoglycans/chemistry , Animals , Breast Neoplasms/blood supply , Breast Neoplasms/pathology , Cell Line, Tumor , Colonic Neoplasms/blood supply , Colonic Neoplasms/pathology , Drug Combinations , Female , HCT116 Cells , Humans , Melanoma, Experimental/blood supply , Melanoma, Experimental/pathology , Mice , Neovascularization, Pathologic/pathology
19.
J Biol Chem ; 286(17): 15332-43, 2011 Apr 29.
Article in English | MEDLINE | ID: mdl-21385870

ABSTRACT

Glioblastoma is one of the most fatal cancers, characterized by a strong vascularized phenotype. YKL-40, a secreted glycoprotein, is overexpressed in patients with glioblastomas and has potential as a novel tumor biomarker. The molecular mechanisms of YKL-40 in glioblastoma development, however, are poorly understood. Here, we aimed to elucidate the role YKL-40 plays in the regulation of VEGF expression, tumor angiogenesis, and radioresistance. YKL-40 up-regulated VEGF expression in glioblastoma cell line U87, and both YKL-40 and VEGF synergistically promote endothelial cell angiogenesis. Interestingly, long term inhibition of VEGF up-regulated YKL-40. YKL-40 induced coordination of membrane receptor syndecan-1 and integrin αvß5, and triggered a signaling cascade through FAK(397) to ERK-1 and ERK-2, leading to elevated VEGF and enhanced angiogenesis. In addition, γ-irradiation of U87 cells increased YKL-40 expression that protects cell death through AKT activation and also enhances endothelial cell angiogenesis. Blockade of YKL-40 activity or expression decreased tumor growth, angiogenesis, and metastasis in xenografted animals. Immunohistochemical analysis of human glioblastomas revealed a correlation between YKL-40, VEGF, and patient survival. These findings have shed light on the mechanisms by which YKL-40 promotes tumor angiogenesis and malignancy, and thus provide a therapeutic target for tumor treatment.


Subject(s)
Glioblastoma/pathology , Glycoproteins/physiology , Lectins/physiology , Neovascularization, Pathologic/etiology , Vascular Endothelial Growth Factor A/genetics , Adipokines , Animals , Cell Line, Tumor , Chitinase-3-Like Protein 1 , Disease Progression , Gamma Rays , Glioblastoma/blood supply , Glioblastoma/radiotherapy , Glycoproteins/analysis , Glycoproteins/antagonists & inhibitors , Humans , Lectins/analysis , Lectins/antagonists & inhibitors , Neoplasm Metastasis , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Survival Rate , Transplantation, Heterologous , Vascular Endothelial Growth Factor A/analysis
20.
Mol Cancer Ther ; 10(5): 742-51, 2011 May.
Article in English | MEDLINE | ID: mdl-21357475

ABSTRACT

Accumulating evidence has indicated that expression levels of YKL-40, a secreted glycoprotein, were elevated in multiple advanced human cancers. Recently, we have identified an angiogenic role of YKL-40 in cancer development. However, blockade of the function of YKL-40, which implicates therapeutic value, has not been explored yet. Our current study sought to establish a monoclonal anti-YKL-40 antibody as a neutralizing antibody for the purpose of blocking tumor angiogenesis and metastasis. A mouse monoclonal anti-YKL-40 antibody (mAY) exhibited specific binding with recombinant YKL-40 and with YKL-40 secreted from osteoblastoma cells MG-63 and brain tumor cells U87. In the functional analysis, we found that mAY inhibited tube formation of microvascular endothelial cells in Matrigel induced by conditioned medium of MG-63 and U87 cells, as well as recombinant YKL-40. mAY also abolished YKL-40-induced activation of the membrane receptor VEGF receptor 2 (Flk-1/KDR) and intracellular signaling mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (Erk) 1 and Erk 2. In addition, mAY enhanced cell death response of U87 line to γ-irradiation through decreased expression of pAKT and AKT and accordingly, abrogated angiogenesis induced by the conditioned medium of U87 cells in which YKL-40 levels were elevated by treatment with γ-irradiation. Furthermore, treatment of xenografted tumor mice with mAY restrained tumor growth, angiogenesis, and progression. Taken together, this study has shown the therapeutic use for the mAY in treatment of tumor angiogenesis and metastasis.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Antibodies, Neutralizing/pharmacology , Antineoplastic Agents/pharmacology , Glycoproteins/metabolism , Lectins/metabolism , Adipokines , Animals , Antibodies, Neutralizing/metabolism , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Chitinase-3-Like Protein 1 , Disease Progression , Endothelial Cells/metabolism , Gene Expression Regulation, Neoplastic , Glycoproteins/genetics , Glycoproteins/immunology , Humans , Lectins/genetics , Lectins/immunology , Mice , Mice, SCID , Neoplasms/pathology , Neoplasms/therapy , Neovascularization, Pathologic/pathology , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Receptors, Vascular Endothelial Growth Factor/metabolism , Signal Transduction/drug effects , Xenograft Model Antitumor Assays
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