Glioblastoma-derived tumorospheres identify a population of tumor stem-like cells with angiogenic potential and enhanced multidrug resistance phenotype.

We investigated in vitro the properties of selected populations of cancer stem-like cells defined as tumorospheres that were obtained from human glioblastoma. We also assessed their potential and capability of differentiating into mature cells of the central nervous system. In vivo, their tumorigenicity was confirmed after transplantation into the brain of non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice. The angiogenic potential of tumorospheres and glioblastoma-derived cells grown as adherent cells was revealed by evaluating the release of angiogenic factors such as vascular endothelial growth factor and CXCL12 by ELISA, as well as by rat aortic ring assay. The proliferative response of tumorospheres in the presence of CXCL12 was observed for the first time. Multidrug resistance-associated proteins 1 and 3 as well as other molecules conferring multidrug resistance were higher when compared with primary adherent cells derived from the same tumor. Finally, we obtained cells from the tumor developing after grafting that clearly expressed the putative neural stem cell marker CD133 as shown by FACS analysis and also nestin and CXCR4. The cells' positivity for glial fibrillary acidic protein was very low. Moreover these cells preserved their angiogenic potential. We conclude that human glioblastoma could contain tumor cell subsets with angiogenic and chemoresistance properties and that this chemoresistance potential is highly preserved by immature cells whereas the angiogenic potential is, to a higher extent, a property of mature cells. A better understanding of the features of these cell subsets may favor the development of more specifically targeted therapies.

CXCL12 in malignant glial tumors: a possible role in angiogenesis and cross-talk between endothelial and tumoral cells.

CXCL12 (stromal cell-derived factor-1/CXCL12) regulates leukocyte, endothelial and hematopoietic precursor migration, bone-marrow myelopoiesis and angiogenesis. CXCL12 and its receptor CXCR4 are over-expressed in malignant gliomas, which are highly vascularized tumors with a poor prognosis. We studied the expression of CXCL12 and CXCR4 in glioma cell lines, endothelial cells, tissue sections and endocavitary fluids from patients with gliomas. We then analyzed the proliferative and the apoptotic effect of CXCL12 in endothelial cells and glioma primary cultures. We observed the release of CXCL12 in supernatants of human brain microvascular endothelial cells and at variable levels, in post-surgical endocavitary fluids. CXCL12 was expressed in both glioma and endothelial cells as assessed by immunostaining of surgical brain sections. CXCR4 was found in cells lines and primary cultures from malignant gliomas as well as in endothelial cells and was increased by vascular endothelial growth factor and basic fibroblast growth factor (bFGF). CXCL12 inhibited bFGF-induced proliferation of endothelial cells and increased the survival of endothelial cells. The survival of primary cells obtained from glioma specimens was also enhanced in the presence of CXCL12. We point out the presence and the release of CXCL12 in tumor microenvironment and we observed a modulating effect of CXCL12 on proliferation and survival of both endothelial and tumoral cells. Our data support in vivo studies suggesting a role in angiogenesis played by CXCL12, which could represent a possible prognostic factor.

Expression and modulation of IFN-gamma-inducible chemokines (IP-10, Mig, and I-TAC) in human brain endothelium and astrocytes: possible relevance for the immune invasion of the central nervous system and the pathogenesis of multiple sclerosis.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) mediated by blood-derived immune cells invading the CNS. This invasion could be determined by chemokines, and their role within the MS-affected brain is still poorly defined. We investigated the expression by RT-PCR and protein release by ELISA of the interferon-gamma (IFN-gamma)-inducible chemokines in human brain microvascular endothelial cells (HBMECs) and astrocytes. The monokine induced by IFN-gamma (Mig) behaves as a homing chemokine constitutively expressed in HBMECs and astrocytes, whereas the IFN-gamma-inducible 10-kDa protein (IP-10) and IFN-inducible T cell alpha-chemoattractant (I-TAC) are induced only after inflammatory stimuli. The biologic activity of IFN-gamma-inducible chemokines from an endothelial source was analyzed, and the transendothelial migration of activated lymphocytes was partly antagonized by specific antibodies, especially anti-Mig antibody. Our data highlight the capability of cells of the CNS to activate the chemoattractant machinery in a proinflammatory environment and in MS.

Methylprednisolone acts on peripheral blood mononuclear cells and endothelium in inhibiting migration phenomena in patients with multiple sclerosis.

BACKGROUND: Intravenous methylprednisolone hemisuccinate is administered to patients with multiple sclerosis (MS) during exacerbations to improve the rate of recovery. Corticosteroids could be beneficial in MS exacerbations also by decreasing transmigration of peripheral blood mononuclear cells (PBMNCs) through the blood-brain barrier. OBJECTIVES: To evaluate how in vivo intravenous methylprednisolone treatment in patients with MS could influence transmigration of PBMNCs in an in vitro model; to perform transmigration experiments through a methylprednisolone-treated endothelium with PBMNCs from untreated healthy control subjects to evaluate putative selective effects of corticosteroids on endothelium; concomitantly, to quantify the concentration of matrix metalloproteinases 2 and 9 in supernatants of PBMNCs and in serum samples from methylprednisolone-treated patients with MS; to evaluate monokine induced by interferon-gamma release in the supernatants of human umbilical vein endothelial cells treated with interferon-gamma alone or interferon-gamma and methylprednisolone; and to perform gene expression studies of matrix metalloproteinases 2 and 9 in human umbilical vein endothelial cells and PBMNCs from methylprednisolone-treated patients with MS. PATIENTS: Eight patients with MS in exacerbation were studied before and 3 and 24 hours after intravenous methylprednisolone treatment, 1 g. RESULTS: The absolute number of transmigrated PBMNCs from methylprednisolone-treated patients with MS significantly (P<.01) decreased at 3 hours and increased again at 24 hours, reaching values higher than those before treatment onset. Methylprednisolone was also able to significantly (P<.03) reduce the number of PBMNCs from healthy controls migrating through interferon-gamma-stimulated or unstimulated endothelium. In vitro methylprednisolone treatment decreased monokine induced by interferon-gamma production in human umbilical vein endothelial cells. CONCLUSIONS: Methylprednisolone may be able to decrease transmigration of PBMNCs through the blood-brain barrier, exerting its inhibitory effects on PBMNCs and endothelium. A "rebound" of transmigration at 24 hours suggests that a single infusion is not optimal for achieving a persistent reduction in transmigration.