Characterization of TEM1/endosialin in human and murine brain tumors

Department of Neurosurgery, The University of Rochester, Rochester, NY, USA.
BMC Cancer (Impact Factor: 3.36). 11/2009; 9(1):417. DOI: 10.1186/1471-2407-9-417
Source: PubMed


TEM1/endosialin is an emerging microvascular marker of tumor angiogenesis. We characterized the expression pattern of TEM1/endosialin in astrocytic and metastatic brain tumors and investigated its role as a therapeutic target in human endothelial cells and mouse xenograft models.
In situ hybridization (ISH), immunohistochemistry (IH) and immunofluorescence (IF) were used to localize TEM1/endosialin expression in grade II-IV astrocytomas and metastatic brain tumors on tissue microarrays. Changes in TEM1/endosialin expression in response to pro-angiogenic conditions were assessed in human endothelial cells grown in vitro. Intracranial U87MG glioblastoma (GBM) xenografts were analyzed in nude TEM1/endosialin knockout (KO) and wildtype (WT) mice.
TEM1/endosialin was upregulated in primary and metastatic human brain tumors, where it localized primarily to the tumor vasculature and a subset of tumor stromal cells. Analysis of 275 arrayed grade II-IV astrocytomas demonstrated TEM1/endosialin expression in 79% of tumors. Robust TEM1/endosialin expression occurred in 31% of glioblastomas (grade IV astroctyomas). TEM1/endosialin expression was inversely correlated with patient age. TEM1/endosialin showed limited co-localization with CD31, alphaSMA and fibronectin in clinical specimens. In vitro, TEM1/endosialin was upregulated in human endothelial cells cultured in matrigel. Vascular Tem1/endosialin was induced in intracranial U87MG GBM xenografts grown in mice. Tem1/endosialin KO vs WT mice demonstrated equivalent survival and tumor growth when implanted with intracranial GBM xenografts, although Tem1/endosialin KO tumors were significantly more vascular than the WT counterparts.
TEM1/endosialin was induced in the vasculature of high-grade brain tumors where its expression was inversely correlated with patient age. Although lack of TEM1/endosialin did not suppress growth of intracranial GBM xenografts, it did increase tumor vascularity. The cellular localization of TEM1/endosialin and its expression profile in primary and metastatic brain tumors support efforts to therapeutically target this protein, potentially via antibody mediated drug delivery strategies.

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Available from: Melissa C Whiteman, Jul 08, 2014
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    • "Thus, to exploit both passive and active targeting potentials, we engineered SHK-loaded PEGylated PLGA NPs decorated with anti-TEM1 Ab/scFv. TEM1 has been reported to be expressed by the endothelial cells of ovarian tumor vasculature25,44–47 and other malignancies such as brain and abdominal tumors.48,49 Based on the release profile, less than 20% of drug can be released within 2 hours, during which the engineered NPs can safely pass the blood stream and reach the target site with negligible inadvertent detrimental impacts on blood-circulating immune system cells. "
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    ABSTRACT: Conventional chemotherapy of ovarian cancer often fails because of initiation of drug resistance and/or side effects and trace of untouched remaining cancerous cells. This highlights an urgent need for advanced targeted therapies for effective remediation of the disease using a cytotoxic agent with immunomodulatory effects, such as shikonin (SHK). Based on preliminary experiments, we found SHK to be profoundly toxic in ovarian epithelial cancer cells (OVCAR-5 and ID8 cells) as well as in normal ovarian IOSE-398 cells, endothelial MS1 cells, and lymphocytes. To limit its cytotoxic impact solely to tumor cells within the tumor microenvironment (TME), we aimed to engineer SHK as polymeric nanoparticles (NPs) with targeting moiety toward tumor microvasculature. To this end, using single/double emulsion solvent evaporation/diffusion technique with sonication, we formulated biodegradable NPs of poly(lactic-co-glycolic acid) (PLGA) loaded with SHK. The surface of NPs was further decorated with solubilizing agent polyethylene glycol (PEG) and tumor endothelial marker 1 (TEM1)/endosialin-targeting antibody (Ab) through carbodiimide/N-hydroxysuccinimide chemistry. Having characterized the physicochemical and morphological properties of NPs, we studied their drug-release profiles using various kinetic models. The biological impact of NPs was also evaluated in tumor-associated endothelial MS1 cells, primary lymphocytes, and epithelial ovarian cancer OVCAR-5 cells. Based on particle size analysis and electron microscopy, the engineered NPs showed a smooth spherical shape with size range of 120 to 250 nm and zeta potential value of -30 to -40 mV. Drug entrapment efficiency was ~80%-90%, which was reduced to ~50%-60% upon surface decoration with PEG and Ab. The liberation of SHK from NPs showed a sustained-release profile that was best fitted with Wagner log-probability model. Fluorescence microscopy and flow cytometry analysis showed active interaction of Ab-armed NPs with TEM1-positive MS1 cells, but not with TEM1-negative MS1 cells. While exposure of the PEGylated NPs for 2 hours was not toxic to lymphocytes, long-term exposure of the Ab-armed and PEGylated NPs was significantly toxic to TEM1-positive MS1 cells and OVCAR-5 cells. Based on these findings, we propose SHK-loaded Ab-armed PEGylated PLGA NPs as a novel nanomedicine for targeted therapy of solid tumors.
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    • "While largely absent in normal tissues, CD248 is markedly upregulated in almost all cancers. Highest expression is found in neuroblastomas and in subsets of carcinomas, such as breast and colon cancers, and in addition, in glioblastomas and mesenchymal tumors, such as fibrosarcomas and synovial sarcomas [8,14,15,17,19,20], where it is mostly detected in perivascular and tumor stromal cells, but also in the tumor cells themselves [21,22]. CD248 is also expressed in placenta and during wound healing and in wounds such as ulcers. "
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    • "This is relevant to CD248, as two groups have reported a CD248-dependent inverse relationship between microvessel density and size of tumor. Tumors of the large intestine derived from LLC cells [25] and intracranial glioblastomas derived from U87MG cells [21] were reportedly smaller in size and weight in CD248-deficient mice, yet these tumors had higher vessel density than those in wild-type mice. We assessed the role of the cytoplasmic tail of CD248 in modulating the relationship between tumor growth and angiogenesis by first examining vessel density in the s.c. "
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