Characterization of TEM1/endosialin in human and murine brain tumors - PubMed (original) (raw)

Characterization of TEM1/endosialin in human and murine brain tumors

Eleanor B Carson-Walter et al. BMC Cancer. 2009.

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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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Figures

Figure 1

Figure 1

Localization of TEM1/endosialin expression. A. Representative RT-PCR for TEM1/endosialin (TEM1) in whole cell lysates from two GBMs (T1, T2), two metastatic brain tumors (T3, T4) and two non-neoplastic controls (N1, N2). Expression was normalized to von Willebrand Factor (vWF), an endothelial positive control. B. Combined quantitative RT-PCR results for TEM1/endosialin in two controls (N), five GBMs (GBM) and five metastatic tumors (Met). Error bars, standard error of the mean. *p = 0.05. C. TEM1/endosialin was visualized by immunohistochemistry (IHC) using the TEM1/33 custom antibody or in situ hybridization (ISH) in glioblastoma (GBM), metastatic brain tumor (MET) and non-neoplastic brain tissues (Normal). Staining for vWF was used as an endothelial positive control. Original magnification for whole spot, 40×; detail, 100×.

Figure 2

Figure 2

Immunofluorescence for TEM1/endosialin: co-localization in tumor specimens. TEM1 and CD31 (top panel), αSMA (middle panel) and fibronectin (FN, bottom panel) co-localize to microvessels in human GBMs, but demonstrate limited cellular overlap. Anti-TEM1 (TEM1/33), red. Anti-CD31, anti-αSMA and anti-FN, green. Nuclei, blue. Scale bar, 20 μm.

Figure 3

Figure 3

Expression of TEM1/endosialin by cultured endothelial cells. A. Expression of TEM1/endosialin after treatment of endothelial cells with pro-angiogenic growth factors. Cells were treated for 48 hours (see Methods) and analyzed by quantitative RT-PCR. Error bars, standard error of the mean. No significant change in TEM1/endosialin was detected. B. Induction of TEM1/endosialin after growth of endothelial cells in matrigel. Cells were grown on plastic (Control) or in matrigel (Matrigel) for 6 hours then analyzed by quantitative RT-PCR. Error bars, standard error of the mean. *p = 0.05. After 6 hours on matrigel and prior to harvest, HMVEC cells demonstrate branching tubulogenesis (lower right) while cells grown on plastic do not (lower left).

Figure 4

Figure 4

Tem1/endosialin expression in mouse brain tumor models. A. Induction of Tem1/endosialin in intracranial U87MG xenografts grown in nude mice. ISH with mouse specific riboprobes detected Tem1/endosialin (Tem1) in tumor (T) but not normal mouse brain (NL). Probes for mouse vWF were used as a positive endothelial control (vWF). Original magnification, 200×. B. Survival of nude TEM1/endosialin WT and KO mice after intracranial injection with 105 U87MG glioblastoma cells. Animals sacrificed after 100 days showed no evidence of tumor take. C. Tumor vessels stained for vWF in intracranial xenografts from nude TEM1/endosialin WT or KO mice. Size bar, 50 μm. D. Comparison of vessel numbers from nude TEM1/endosialin WT and KO tumors. Ten non-overlapping high power fields were counted per animal. Error bars, standard error of the mean. *p = 0.001.

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