NG2 proteoglycan-dependent recruitment of tumor macrophages promotes pericyte-endothelial cell interactions required for brain tumor vascularization

Early stage growth of intracranial B16F10 tumors is reduced by 87% in myeloid-specific NG2 null (Mac-NG2ko) mice and by 77% in pericyte-specific NG2 null (PC-NG2ko) mice, demonstrating the importance of the NG2 proteoglycan in each of these stromal compartments. In both genotypes, loss of pericyte-endothelial cell interaction results in numerous structural defects in tumor blood vessels, including decreased formation of endothelial cell junctions and decreased assembly of the vascular basal lamina. All vascular deficits are larger in Mac-NG2ko mice than in PC-NG2ko mice, correlating with the greater decrease in pericyte-endothelial cell interaction in Mac-NG2ko animals. Accordingly, tumor vessels in Mac-NG2ko mice have a smaller diameter, lower degree of patency, and higher degree of leakiness than tumor vessels in PC-NG2ko mice, leading to less efficient tumor blood flow and to increased intratumoral hypoxia. While reduced pericyte interaction with endothelial cells in PC-NG2ko mice is caused by loss of NG2-dependent pericyte activation of β1 integrin signaling in endothelial cells, reduced pericyte-endothelial cell interaction in Mac-NG2ko mice is due to a 90% reduction in NG2-dependent macrophage recruitment to tumors. The absence of a macrophage-derived signal(s) in Mac-NG2ko mice results in the loss of pericyte ability to associate with endothelial cells, possibly due to reduced expression of N-cadherin by both pericytes and endothelial cells.

[1]  Jeffrey W. Pollard,et al.  Macrophage Diversity Enhances Tumor Progression and Metastasis , 2010, Cell.

[2]  C. Betsholtz,et al.  A Two-Way Communication between Microglial Cells and Angiogenic Sprouts Regulates Angiogenesis in Aortic Ring Cultures , 2011, PloS one.

[3]  Fusanori Yotsumoto,et al.  NG2 proteoglycan promotes tumor vascularization via integrin-dependent effects on pericyte function , 2013, Angiogenesis.

[4]  W. You,et al.  Tumorigenesis and Neoplastic Progression Collagen VI Ablation Retards Brain Tumor Progression Due to Deficits in Assembly of the Vascular Basal Lamina , 2012 .

[5]  Russell Hughes,et al.  Tumor-associated macrophages: effectors of angiogenesis and tumor progression. , 2009, Biochimica et biophysica acta.

[6]  J. Rubin.  Only in Congenial Soil: The Microenvironment in Brain Tumorigenesis , 2009, Brain pathology.

[7]  V. Yong,et al.  Reduced inflammation accompanies diminished myelin damage and repair in the NG2 null mouse spinal cord , 2011, Journal of Neuroinflammation.

[8]  T. Mustelin,et al.  Differential phosphorylation of NG2 proteoglycan by ERK and PKCα helps balance cell proliferation and migration , 2007, The Journal of cell biology.

[9]  W. Stallcup,et al.  Expression of the NG2 proteoglycan enhances the growth and metastatic properties of melanoma cells , 1998, Journal of cellular physiology.

[10]  Zena Werb,et al.  Stromal Effects on Mammary Gland Development and Breast Cancer , 2002, Science.

[11]  W. Stallcup,et al.  FGF2-dependent neovascularization of subcutaneous Matrigel plugs is initiated by bone marrow-derived pericytes and macrophages , 2008, Development.

[12]  B. Garmy-Susini,et al.  Integrin α4β1 Promotes Monocyte Trafficking and Angiogenesis in Tumors , 2006 .

[13]  A. Roberts,et al.  Ablation of NG2 Proteoglycan Leads to Deficits in Brown Fat Function and to Adult Onset Obesity , 2012, PloS one.

[14]  Lu Gao,et al.  Tumor-associated macrophages promote cancer stem cell-like properties via transforming growth factor-beta1-induced epithelial-mesenchymal transition in hepatocellular carcinoma. , 2014, Cancer letters.

[15]  C. Miller,et al.  Transglutaminase 2 inhibitor, KCC009, disrupts fibronectin assembly in the extracellular matrix and sensitizes orthotopic glioblastomas to chemotherapy , 2007, Oncogene.

[16]  Douglas Hanahan,et al.  Accessories to the Crime: Functions of Cells Recruited to the Tumor Microenvironment Prospects and Obstacles for Therapeutic Targeting of Function-enabling Stromal Cell Types , 2022 .

[17]  C. Betsholtz,et al.  Endothelial/Pericyte Interactions , 2005, Circulation research.

[18]  Na Zhang,et al.  Deletion of Vascular Endothelial Growth Factor in myeloid cells accelerates tumorigenesis , 2008, Nature.

[19]  V. P. Collins,et al.  NG2 expression in glioblastoma identifies an actively proliferating population with an aggressive molecular signature. , 2011, Neuro-oncology.

[20]  R. Adams,et al.  Ephrin-B2 Controls Cell Motility and Adhesion during Blood-Vessel-Wall Assembly , 2006, Cell.

[21]  Yang Luo,et al.  N-cadherin acts upstream of VE-cadherin in controlling vascular morphogenesis , 2005, The Journal of cell biology.

[22]  Robert Pless,et al.  Capillary and arteriolar pericytes attract innate leukocytes exiting through venules and 'instruct' them with pattern-recognition and motility programs , 2012, Nature Immunology.

[23]  Elisabet Englund,et al.  Phagocytic properties in tumor astrocytes , 2012, Neuropathology : official journal of the Japanese Society of Neuropathology.

[24]  J. Pollard,et al.  Macrophages regulate the angiogenic switch in a mouse model of breast cancer. , 2006, Cancer research.

[25]  Jean-Loup Duband,et al.  Cross Talk between Adhesion Molecules: Control of N-cadherin Activity by Intracellular Signals Elicited by β1 and β3 Integrins in Migrating Neural Crest Cells , 1997, The Journal of cell biology.

[26]  S. Ferrone,et al.  Cancer immunotherapy targeting the high molecular weight melanoma-associated antigen protein results in a broad antitumor response and reduction of pericytes in the tumor vasculature. , 2008, Cancer research.

[27]  J. Fukushi,et al.  NG2 proteoglycan promotes endothelial cell motility and angiogenesis via engagement of galectin-3 and alpha3beta1 integrin. , 2004, Molecular biology of the cell.

[28]  Jeffrey W Pollard,et al.  Tumor-associated macrophages: from mechanisms to therapy. , 2014, Immunity.

[29]  N. Ortéga,et al.  Extracellular matrix‐bound vascular endothelial growth factor promotes endothelial cell adhesion, migration, and survival through integrin ligation , 2003, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[30]  J. Lilien,et al.  The Nonreceptor Tyrosine Kinase Fer Mediates Cross-Talk between N-Cadherin and β1-Integrins , 2000, The Journal of cell biology.

[31]  Gui-yuan Li,et al.  CSPG4, a potential therapeutic target, facilitates malignant progression of melanoma , 2011, Pigment cell & melanoma research.

[32]  W. Stallcup,et al.  NG2 Proteoglycan Promotes Endothelial Cell Motility and Angiogenesis via Engagement of Galectin-3 and α3β1 Integrin , 2004 .

[33]  H. Immervoll,et al.  Targeting the NG2/CSPG4 Proteoglycan Retards Tumour Growth and Angiogenesis in Preclinical Models of GBM and Melanoma , 2011, PloS one.

[34]  W. Reith,et al.  Conditional gene targeting in macrophages and granulocytes using LysMcre mice , 1999, Transgenic Research.

[35]  W. Barry,et al.  CSPG4 protein as a new target for the antibody-based immunotherapy of triple-negative breast cancer. , 2010, Journal of the National Cancer Institute.

[36]  W. Stallcup,et al.  A role for the NG2 proteoglycan in glioma progression , 2008, Cell adhesion & migration.

[37]  K. Plate,et al.  Angiogenesis in malignant gliomas , 1995, Glia.

[38]  Youliang Wang,et al.  Endothelial Smad4 maintains cerebrovascular integrity by activating N-cadherin through cooperation with Notch. , 2011, Developmental cell.

[39]  T. Papayannopoulou,et al.  Unique and redundant roles of alpha4 and beta2 integrins in kinetics of recruitment of lymphoid vs myeloid cell subsets to the inflamed peritoneum revealed by studies of genetically deficient mice. , 2007, Experimental hematology.

[40]  P. Lønning,et al.  The progenitor cell marker NG2/MPG promotes chemoresistance by activation of integrin-dependent PI3K/Akt signaling , 2008, Oncogene.

[41]  B. Garmy-Susini,et al.  Integrin alpha4beta1 promotes monocyte trafficking and angiogenesis in tumors. , 2006, Cancer research.

[42]  M. Bissell,et al.  The dominance of the microenvironment in breast and ovarian cancer. , 2002, Seminars in cancer biology.

[43]  R. Schwendener,et al.  Intratumoral macrophages contribute to epithelial-mesenchymal transition in solid tumors , 2012, BMC Cancer.

[44]  Pier Andrea Nicolosi,et al.  NG2/CSPG4-collagen type VI interplays putatively involved in the microenvironmental control of tumour engraftment and local expansion. , 2013, Journal of molecular cell biology.

[45]  A. Beaudet,et al.  Severe inflammatory defect and reduced viability in CD18 and E-selectin double-mutant mice. , 2000, The Journal of clinical investigation.

[46]  Luigi Naldini,et al.  Elusive identities and overlapping phenotypes of proangiogenic myeloid cells in tumors. , 2010, The American journal of pathology.

[47]  L. Naldini,et al.  Tie2-expressing monocytes (TEMs): novel targets and vehicles of anticancer therapy? , 2009, Biochimica et biophysica acta.

[48]  W. You,et al.  Pericyte deficiencies lead to aberrant tumor vascularizaton in the brain of the NG2 null mouse. , 2010, Developmental biology.

[49]  I. Bernstein,et al.  The human homologue of rat NG2, a chondroitin sulfate proteoglycan, is not expressed on the cell surface of normal hematopoietic cells but is expressed by acute myeloid leukemia blasts from poor-prognosis patients with abnormalities of chromosome band 11q23. , 1996, Blood.

[50]  E. Crivellato,et al.  Immune cells and angiogenesis , 2009, Journal of cellular and molecular medicine.

[51]  R. Cardiff,et al.  Early vascular deficits are correlated with delayed mammary tumorigenesis in the MMTV-PyMT transgenic mouse following genetic ablation of the NG2 proteoglycan , 2012, Breast Cancer Research.

[52]  Luigi Naldini,et al.  Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. , 2005, Cancer cell.

[53]  Jeffrey W Pollard,et al.  Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases. , 2003, The American journal of pathology.