Leukocyte Differentiation by Histidine-Rich Glycoprotein/Stanniocalcin-2 Complex Regulates Murine Glioma Growth through Modulation of Antitumor Immunity.

The plasma-protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and antitumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45+ leukocyte and CD8+ T-cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45+ and CD8+ infiltration. Using a novel protein interaction-decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells in vitro and colocalization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14+ differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG-treated gliomas displayed decreased numbers of IL35+ Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control antitumor immunity. Mol Cancer Ther; 17(9); 1961-72. ©2018 AACR.

Histidine-rich glycoprotein uptake and turnover is mediated by mononuclear phagocytes.

Histidine-rich glycoprotein (HRG) is implicated in tumor growth and metastasis by regulation of angiogenesis and inflammation. HRG is produced by hepatocytes and carried to tissues via the circulation. We hypothesized that HRG's tissue distribution and turnover may be mediated by inflammatory cells. Biodistribution parameters were analyzed by injection of radiolabeled, bioactive HRG in the circulation of healthy and tumor-bearing mice. 125I-HRG was cleared rapidly from the blood and taken up in tissues of healthy and tumor-bearing mice, followed by degradation, to an increased extent in the tumor-bearing mice. Steady state levels of HRG in the circulation were unaffected by the tumor disease both in murine tumor models and in colorectal cancer (CRC) patients. Importantly, stromal pools of HRG, detected in human CRC microarrays, were associated with inflammatory cells. In agreement, microautoradiography identified 125I-HRG in blood vessels and on CD45-positive leukocytes in mouse tissues. Moreover, radiolabeled HRG bound in a specific, heparan sulfate-independent manner, to differentiated human monocytic U937 cells in vitro. Suppression of monocyte differentiation by systemic treatment of mice with anti-colony stimulating factor-1 neutralizing antibodies led to reduced blood clearance of radiolabeled HRG and to accumulation of endogenous HRG in the blood. Combined, our data show that mononuclear phagocytes have specific binding sites for HRG and that these cells are essential for uptake of HRG from blood and distribution of HRG in tissues. Thereby, we confirm and extend our previous report that inflammatory cells mediate the effect of HRG on tumor growth and metastatic spread.

Syndecan-2 can promote clearance of T-cell receptor/CD3 from the cell surface.

T cells express the heparan sulphate proteoglycans syndecan-2 and syndecan-4. Syndecan-4 plays a T-cell inhibitory role; however, the function of syndecan-2 is unknown. In an attempt to examine this function, syndecan-2 was expressed constitutively in Jurkat T cells. Interestingly, the expression of syndecan-2 decreased the surface levels of T-cell receptor (TCR)/CD3 complex, concomitant with intracellular retention of CD3ε and partial degradation of the TCR-ζ chain. Immunofluorescence microscopy revealed that intracellular CD3ε co-located with Rab-4 endosomes. However, the intracellular pool of CD3ε did not recycle to the cell surface. The lower TCR/CD3 surface levels caused by syndecan-2 led to reduced TCR/CD3 responsiveness. We show that the cytosolic PDZ-binding domain of syndecan-2 is not necessary to elicit TCR/CD3 down-regulation. These results identify a previously unrecognized means of controlling surface TCR/CD3 expression by syndecan-2.

Genetic deficiency in plasma protein HRG enhances tumor growth and metastasis by exacerbating immune escape and vessel abnormalization.

Histidine-rich glycoprotein (HRG) is a 75-kDa heparin-binding plasma protein implicated in the regulation of tumor growth and vascularization. In this study, we show that hrg(-/-) mice challenged with fibrosarcoma or pancreatic carcinoma grow larger tumors with increased metastatic properties. Compared with wild-type mice, fibrosarcomas in hrg(-/-) mice were more hypoxic, necrotic, and less perfused, indicating enhanced vessel abnormalization. HRG deficiency was associated with a suppressed antitumor immune response, with both increased infiltration of M2 marker-expressing macrophages and decreased infiltration of dendritic cells and cytotoxic T cells. Analysis of transcript expression in tumor-associated as well as peritoneal macrophages from hrg(-/-) mice revealed an increased expression of genes associated with a proangiogenic and immunoinhibitory phenotype. In accordance, expression arrays conducted on HRG-treated peritoneal macrophages showed induction of genes involved in extracellular matrix biology and immune responsiveness. In conclusion, our findings show that macrophages are a direct target of HRG. HRG loss influences macrophage gene regulation, leading to excessive stimulation of tumor angiogenesis, suppression of tumor immune response, and increased tumor growth and metastatic spread.

Syndecan-2 downregulation impairs angiogenesis in human microvascular endothelial cells.

The formation of new blood vessels, or angiogenesis, is a necessary process during development but also for tumour growth and other pathologies. It is promoted by different growth factors that stimulate endothelial cells to proliferate, migrate, and generate new tubular structures. Syndecans, transmembrane heparan sulphate proteoglycans, bind such growth factors through their glycosaminoglycan chains and could transduce the signal to the cytoskeleton, thus regulating cell behaviour. We demonstrated that syndecan-2, the major syndecan expressed by human microvascular endothelial cells, is regulated by growth factors and extracellular matrix proteins, in both bidimensional and tridimensional culture conditions. The role of syndecan-2 in "in vitro" tumour angiogenesis was also examined by inhibiting its core protein expression with antisense phosphorothioate oligonucleotides. Downregulation of syndecan-2 reduces spreading and adhesion of endothelial cells, enhances their migration, but also impairs the formation of capillary-like structures. These results suggest that syndecan-2 has an important function in some of the necessary steps that make up the angiogenic process. We therefore propose a pivotal role of this heparan sulphate proteoglycan in the formation of new blood vessels.

Syndecan-2 expression increases serum-withdrawal-induced apoptosis, mediated by re-distribution of Fas into lipid rafts, in stably transfected Swiss 3T3 cells.

To examine the function of syndecan-2, one of the most abundant heparan sulfate proteoglycans in fibroblasts, we obtained stably transfected Swiss 3T3 clones. We examined the effects of stable syndecan-2 overexpression on programmed cell death, finding that syndecan-2 transfected cells were more sensitive to apoptosis induced by serum-withdrawal than control cells. In addition, overexpression of syndecan-2 correlates with increased membrane levels of the Fas/CD95 receptor, suggesting that the increased serum-withdrawal apoptosis observed in Swiss 3T3 cells might be Fas receptor-dependent. Differences in Fas membrane levels between both control and syndecan-2 transfected cells result from a redistribution of the Fas receptor. Our data clearly demonstrate that increased Fas levels are primarily related to lipid rafts and that this increase is a key factor in Fas/CD95-mediated apoptosis. Moreover, disruption of lipid rafts with methyl-beta-cyclodextrin or filipin significantly reduced apoptosis in response to serum withdrawal. The differences in Fas/CD95 membrane distribution could explain why syndecan-2 transfected cells have a higher susceptibility to serum-withdrawal-induced apoptosis.