Malignant gliomas are uniformly lethal tumors whose morbidity is mediated in large part by the angiogenic response of the brain to the invading tumor. This profound angiogenic response leads to aggressive tumor invasion and destruction of surrounding brain tissue as well as blood-brain barrier breakdown and life-threatening cerebral edema. To investigate the molecular mechanisms governing the proliferation of abnormal microvasculature in malignant brain tumor patients, we have undertaken a cell-specific transcriptome analysis from surgically harvested nonneoplastic and tumor-associated endothelial cells. SAGE-derived endothelial cell gene expression patterns from glioma and nonneoplastic brain tissue reveal distinct gene expression patterns and consistent up-regulation of certain glioma endothelial marker genes across patient samples. We define the G-protein-coupled receptor RDC1 as a tumor endothelial marker whose expression is distinctly induced in tumor endothelial cells of both brain and peripheral vasculature. Further, we demonstrate that the glioma-induced gene, PV1, shows expression both restricted to endothelial cells and coincident with endothelial cell tube formation. As PV1 provides a framework for endothelial cell caveolar diaphragms, this protein may serve to enhance glioma-induced disruption of the blood-brain barrier and transendothelial exchange. Additional characterization of this extensive brain endothelial cell gene expression database will provide unique molecular insights into vascular gene expression.

Purpose: Endosialin/CD248/tumor endothelial marker 1is expressed in stromal cells, endothelial cells, and pericytes in various tumors; however, few studies have focused on expression in malignant cells. Experimental Design: We studied expression of endosialin in clinical specimens, cell culture, and animal models and designed an anti-endosialin therapeutic prototype. Results: Fifty human tumor cell lines and 6 normal cell types in culture were assayed by reverse transcription-PCR and/or flow cytometry for endosialin. Cell surface protein was found on 7 sarcoma lines, 1neuroblastoma, and 4 normal cell types in culture. A fully human anti-endosialin antibody bound to human A-673 Ewing's sarcoma cells and SK-N-AS neuroblastoma cells but not HT-1080 cells. Exposure of cells to an anti-human IgG conjugated to saporin resulted in growth inhibition only of endosialin-expressing cells. Endosialin expression was assessed by immunohistochemistry in 250 clinical specimens of human cancer including 20 cancer subtypes. Endosialin is frequently found in human cancers. Endosialin expression is mainly a perivascular feature in carcinomas, with some expression in stromal cells. In sarcomas, endosialin is expressed by malignant cells, perivascular cells, and stromal cells. Development and characterization of experimental models for studying endosialin biology in sarcomas and evaluating anti-endosialin therapies is presented. Conclusions: Findings suggest that an anti-endosialin immunotoxin might be a promising therapeutic approach for endosialin-positive neoplasia, especially synovial sarcoma, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, and osteosarcoma. Thus, a diagnostic/therapeutic targeted therapeutic approach to treatment of endosialin-expressing tumors may be possible.

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Tumor vasculature is irregular, abnormal, and essential for tumor growth. Pericytes and endothelial precursor cells (EPC) contribute to the formation of blood vessels under angiogenic conditions. As primary cells in culture, pericytes and EPC share many properties such as tube/network formation and response to kinase inhibitors selective for angiogenic pathways. Expression of cell surface proteins including plateletderived growth factor receptor, vascular cell adhesion molecule, intercellular adhesion molecule, CD105, desmin, and neural growth proteoglycan 2 was similar between pericytes and EPC, whereas expression of P1H12 and lymphocyte function-associated antigen-1 clearly differentiates the cell types. Further distinction was observed in the molecular profiles for expression of angiogenic genes. Pericytes or EPC enhanced the invasion of MDA-MB-231 breast cancer cells in a coculture assay system. The s.c. coinjection of live pericytes or EPC along with MDA-MB-231 cells resulted in an increased rate of tumor growth compared with coinjection of irradiated pericytes or EPC. Microvessel density analysis indicated there was no difference in MDA-MB-231 tumors with or without EPC or pericytes. However, immunohistochemical staining of vasculature suggested that EPC and pericytes may stabilize or normalize vasculature rather than initiate vasculogenesis. In addition, tumors arising from the coinjection of EPC and cancer cells were more likely to develop lymphatic vessels. These results support the notion that pericytes and EPC contribute to malignancy and that these cell types can be useful as cellbased models for tumor vascular development and selection of agents that may provide therapeutic benefit. (Cancer Res 2005; 65(21): 9741-50)

Abstract. Tumor development is a complex and dynamic process that involves malignant, vascular, and stromal cells. Endosialin is a tumor endothelial marker (TEM) present in the microvasculature and stroma of human tumors. Cancerassociated fibroblasts (CAF) have been implicated in promoting tumor development and have been associated with mesenchymal stem cells (MSC). Since stem/progenitor cells recruited either from bone marrow or residing in nearby tissues can contribute to pathological processes we investigated endosialin in MSC using a novel monoclonal antibody. Endosialin is highly expressed by CAF and human bone marrow-derived MSC. MSC can form networks in a tube formation assay that is inhibited by an anti-endosialin antibody. Immunohistochemistry for human endosialin in xenograft tumors following co-injection of MSC and cancer cells identified MSC in tumor stroma. MSC are a potential target for anticancer therapeutic intervention and endosialin expression offers a new tool for the identification of MSC. Endosialin expression by both CAF and MSC further implies the potential contribution of MSC to tumor stroma via differentiation into tumor stromal fibroblasts.