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Identification of novel targets for antiangiogenic therapy by comparing the gene expressions of tumor and normal endothelial cells.

Otsubo T, Hida Y, Ohga N, Sato H, Kai T, Matsuki Y, Takasu H, Akiyama K, Maishi N, Kawamoto T, Shinohara N, Nonomura K, Hida K - Cancer Sci. (2014)

Bottom Line: Targeting tumor angiogenesis is an established strategy for cancer therapy.We identified 131 genes that were differentially upregulated in mTEC.The expression of DEF6 and TMEM176B was upregulated in tumor vessels of human renal cell carcinoma specimens, suggesting that they are potential targets for antiangiogenic intervention for renal cell carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery II, DSP Cancer Institute, Dainippon Sumitomo Pharma Co., Ltd, Osaka, Japan.

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Analysis of TMEM176B and DEF6 expression in vitro and in vivo. (a) Verification of endothelial cells (EC) from a human sample. The binding of ulex europaeus agglutinin 1 (UEA-1 lectin), expression of CD31, CD105 and lack of expression of CD45 (white area) indicates high purity of the isolated human tumor endothelial cells (hTEC) and human normal endothelial cells (hNEC). The isotype control is shown in gray. (b) Upregulated expression of TMEM176B and DEF6 in hTEC. qRT-PCR analysis detected high levels of expression of both genes in hTEC compared with the corresponding hNEC in all three cases. Expression levels of the mRNA were normalized to that of GAPDH (**P < 0.01). (c) Both TMEM176B and DEF6 were strongly stained in tumor vessels using an anti-CD31 antibody in combination with an antibody against either TMEM176B or DEF6. In contrast, normal vessels (glomerular) of normal renal tissue were weakly stained. All samples were counterstained with DAPI. Profiles of immunofluorescence intensities along the dashed lines are shown in Figure S3(a,b). The signal intensities of TMEM176B or DEF6 in the CD31-positive area of whole sections were analyzed by ImageJ (NIH, Bethesda, MD, USA) quantitatively (Fig. S3c,d). Bar, 20 μm.
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fig05: Analysis of TMEM176B and DEF6 expression in vitro and in vivo. (a) Verification of endothelial cells (EC) from a human sample. The binding of ulex europaeus agglutinin 1 (UEA-1 lectin), expression of CD31, CD105 and lack of expression of CD45 (white area) indicates high purity of the isolated human tumor endothelial cells (hTEC) and human normal endothelial cells (hNEC). The isotype control is shown in gray. (b) Upregulated expression of TMEM176B and DEF6 in hTEC. qRT-PCR analysis detected high levels of expression of both genes in hTEC compared with the corresponding hNEC in all three cases. Expression levels of the mRNA were normalized to that of GAPDH (**P < 0.01). (c) Both TMEM176B and DEF6 were strongly stained in tumor vessels using an anti-CD31 antibody in combination with an antibody against either TMEM176B or DEF6. In contrast, normal vessels (glomerular) of normal renal tissue were weakly stained. All samples were counterstained with DAPI. Profiles of immunofluorescence intensities along the dashed lines are shown in Figure S3(a,b). The signal intensities of TMEM176B or DEF6 in the CD31-positive area of whole sections were analyzed by ImageJ (NIH, Bethesda, MD, USA) quantitatively (Fig. S3c,d). Bar, 20 μm.

Mentions: The hTEC and hNEC were obtained from three patients. The clinical backgrounds of patients with RCC who donated tissue specimens are shown in Table2. The binding of ulex europaeus agglutinin 1 (UEA-1 lectin), the expression of CD31 and CD105, and lack of expression of CD45 determined by flow cytometric analysis confirmed the high purity of the isolated human EC (hEC). Representative data are shown in Figure5(a). The expression levels of TMEM176B and DEF6 revealed by qRT-PCR analysis were significantly higher in hTEC than in hNEC for all paired samples (Fig.5b). In contrast, we were unable to detect upregulation of NSG1, ENAH or PCDHB15 (human ortholog of Pcdhb22) in hTEC (Fig. S1). These results indicate that two out of five TEC markers that we identified in mice were upregulated in mTEC (Fig. S2) and hTEC (Fig.5b). Furthermore, to determine the expression levels of TMEM176B and DEF6 in tumor blood vessels in RCC in vivo, we performed immunofluorescence double staining of the frozen sections of human renal tumors and normal kidney tissues (glomerulus) using anti-CD31 with either anti-TMEM176B or anti-DEF6 antibodies. TMEM176B and DEF6 were expressed in tumor blood vessels in renal cancer, but at much lower levels in normal blood vessels (Figs5c, S3). These results suggest that the transcription of these two genes was upregulated in hTEC in vivo and might be involved in tumor angiogenesis in cancer patients.


Identification of novel targets for antiangiogenic therapy by comparing the gene expressions of tumor and normal endothelial cells.

Otsubo T, Hida Y, Ohga N, Sato H, Kai T, Matsuki Y, Takasu H, Akiyama K, Maishi N, Kawamoto T, Shinohara N, Nonomura K, Hida K - Cancer Sci. (2014)

Analysis of TMEM176B and DEF6 expression in vitro and in vivo. (a) Verification of endothelial cells (EC) from a human sample. The binding of ulex europaeus agglutinin 1 (UEA-1 lectin), expression of CD31, CD105 and lack of expression of CD45 (white area) indicates high purity of the isolated human tumor endothelial cells (hTEC) and human normal endothelial cells (hNEC). The isotype control is shown in gray. (b) Upregulated expression of TMEM176B and DEF6 in hTEC. qRT-PCR analysis detected high levels of expression of both genes in hTEC compared with the corresponding hNEC in all three cases. Expression levels of the mRNA were normalized to that of GAPDH (**P < 0.01). (c) Both TMEM176B and DEF6 were strongly stained in tumor vessels using an anti-CD31 antibody in combination with an antibody against either TMEM176B or DEF6. In contrast, normal vessels (glomerular) of normal renal tissue were weakly stained. All samples were counterstained with DAPI. Profiles of immunofluorescence intensities along the dashed lines are shown in Figure S3(a,b). The signal intensities of TMEM176B or DEF6 in the CD31-positive area of whole sections were analyzed by ImageJ (NIH, Bethesda, MD, USA) quantitatively (Fig. S3c,d). Bar, 20 μm.
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fig05: Analysis of TMEM176B and DEF6 expression in vitro and in vivo. (a) Verification of endothelial cells (EC) from a human sample. The binding of ulex europaeus agglutinin 1 (UEA-1 lectin), expression of CD31, CD105 and lack of expression of CD45 (white area) indicates high purity of the isolated human tumor endothelial cells (hTEC) and human normal endothelial cells (hNEC). The isotype control is shown in gray. (b) Upregulated expression of TMEM176B and DEF6 in hTEC. qRT-PCR analysis detected high levels of expression of both genes in hTEC compared with the corresponding hNEC in all three cases. Expression levels of the mRNA were normalized to that of GAPDH (**P < 0.01). (c) Both TMEM176B and DEF6 were strongly stained in tumor vessels using an anti-CD31 antibody in combination with an antibody against either TMEM176B or DEF6. In contrast, normal vessels (glomerular) of normal renal tissue were weakly stained. All samples were counterstained with DAPI. Profiles of immunofluorescence intensities along the dashed lines are shown in Figure S3(a,b). The signal intensities of TMEM176B or DEF6 in the CD31-positive area of whole sections were analyzed by ImageJ (NIH, Bethesda, MD, USA) quantitatively (Fig. S3c,d). Bar, 20 μm.
Mentions: The hTEC and hNEC were obtained from three patients. The clinical backgrounds of patients with RCC who donated tissue specimens are shown in Table2. The binding of ulex europaeus agglutinin 1 (UEA-1 lectin), the expression of CD31 and CD105, and lack of expression of CD45 determined by flow cytometric analysis confirmed the high purity of the isolated human EC (hEC). Representative data are shown in Figure5(a). The expression levels of TMEM176B and DEF6 revealed by qRT-PCR analysis were significantly higher in hTEC than in hNEC for all paired samples (Fig.5b). In contrast, we were unable to detect upregulation of NSG1, ENAH or PCDHB15 (human ortholog of Pcdhb22) in hTEC (Fig. S1). These results indicate that two out of five TEC markers that we identified in mice were upregulated in mTEC (Fig. S2) and hTEC (Fig.5b). Furthermore, to determine the expression levels of TMEM176B and DEF6 in tumor blood vessels in RCC in vivo, we performed immunofluorescence double staining of the frozen sections of human renal tumors and normal kidney tissues (glomerulus) using anti-CD31 with either anti-TMEM176B or anti-DEF6 antibodies. TMEM176B and DEF6 were expressed in tumor blood vessels in renal cancer, but at much lower levels in normal blood vessels (Figs5c, S3). These results suggest that the transcription of these two genes was upregulated in hTEC in vivo and might be involved in tumor angiogenesis in cancer patients.

Bottom Line: Targeting tumor angiogenesis is an established strategy for cancer therapy.We identified 131 genes that were differentially upregulated in mTEC.The expression of DEF6 and TMEM176B was upregulated in tumor vessels of human renal cell carcinoma specimens, suggesting that they are potential targets for antiangiogenic intervention for renal cell carcinoma.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery II, DSP Cancer Institute, Dainippon Sumitomo Pharma Co., Ltd, Osaka, Japan.

Show MeSH
Related in: MedlinePlus