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Tumor blood vessels play important roles in tumor progression and metastasis. Targeting tumor endothelial cells (TECs) is one of the strategies for cancer therapy. We previously reported that biglycan, a small leucine-rich proteoglycan, is highly expressed in TECs. TECs utilize biglycan in an autocrine manner for migration and angiogenesis. Furthermore, TEC-derived biglycan stimulates tumor cell migration in a paracrine manner leading to tumor cell intravasation and metastasis. In this study, we explored the therapeutic effect of biglycan inhibition in the TECs of renal cell carcinoma using an in vivo siRNA delivery system known as a multifunctional envelope-type nanodevice (MEND), which contains a unique pH-sensitive cationic lipid. To specifically deliver MEND into TECs, we incorporated cyclo(Arg-Gly-Asp-D-Phe-Lys) (cRGD) into MEND because α V β 3 integrin, a receptor for cRGD, is selective and highly expressed in TECs. We developed RGD-MEND-encapsulating siRNA against biglycan. First, we confirmed that MEND was delivered into OS-RC-2 tumor-derived TECs and induced in vitro RNAi-mediated gene silencing. MEND was then injected intravenously into OS-RC-2 tumor-bearing mice. Flow cytometry analysis demonstrated that MEND was specifically delivered into TECs. Quantitative RT-PCR indicated that biglycan was knocked down by biglycan siRNA-containing MEND. Finally, we analyzed the therapeutic effect of biglycan silencing by MEND in TECs. Tumor growth was inhibited by biglycan siRNA-containing MEND. Tumor microenvironmental factors such as fibrosis were also normalized using biglycan inhibition in TECs. Biglycan in TECs can be a novel target for cancer treatment., (© 2022 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Tumor angiogenesis is necessary for tumor progression and metastasis; therefore, tumor blood vessels are potential therapeutic targets in anticancer therapy. We previously reported that tumor endothelial cells (TECs) exhibit different phenotypes compared with normal endothelial cells (NECs), and microarray analyses of mouse TECs and NECs have shown that several genes are upregulated in TECs compared with NECs. Among these genes, the expression levels of prostaglandin F receptor (PTGFR) mRNA, which encodes the prostaglandin F receptor (FP), were higher in TECs than in NECs. It has been reported that FP and its ligand, prostaglandin F(2α) , are involved in tumor angiogenesis. However, there have been no reports of the expression of PTGFR in the tumor vessels of renal cell carcinoma (RCC). Thus, we isolated human TECs (hTECs) from RCCs. The expression levels of PTGFR mRNA were also upregulated in hTECs. In addition, immunostaining showed that the PTGFR was expressed in human tumor blood vessels in vivo. These findings suggested that PTGFR is a novel TEC marker and that it may be a novel target for antiangiogenic therapy for RCC., (© 2013 The Authors. Pathology International © 2013 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.)

Anti-angiogenic therapy is a potential chemotherapeutic strategy for the treatment of drug resistant cancers. However, a method for delivering such drugs to tumor endothelial cells remains to be a major impediment to the success of anti-angiogenesis therapy. We designed liposomes (LPs) with controlled diameter of around 300 nm, and modified them with a specific ligand and a cell penetrating peptide (CPP) (a dual-ligand LP) for targeting CD13-expressing neovasculature in a renal cell carcinoma (RCC). We modified the LPs with an NGR motif peptide on the top of poly(ethylene glycol) and tetra-arginine (R4) on the surface of the liposome membrane as a specific and CPP ligand, respectively. The large size prevented extravasation of the dual-ligand LP, which allowed it to associate with target vasculature. While a single modification with either the specific or CPP ligand showed no increase in targetability, the dual-ligand enhanced the amount of delivered liposomes after systemic administration to OS-RC-2 xenograft mice. The anti-tumor activity of a dual-ligand LP encapsulating doxorubicin was evaluated and the results were compared with Doxil, which is clinically used to target tumor cells. Even though Doxil showed no anti-tumor activity, the dual-ligand LP suppressed tumor growth because the disruption of tumor vessels was efficiently induced. The comparison showed that tumor endothelial cells (TECs) were more sensitive to doxorubicin by 2 orders than RCC tumor cells, and the disruption of tumor vessels was efficiently induced. Collectively, the dual-ligand LP is promising carrier for the treatment of drug resistant RCC via the disruption of TECs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Tumor angiogenesis is necessary for progression and metastasis of solid tumor. Tumor blood vessels are morphologically different from their normal counterparts. In this study, we isolated tumor endothelial cells (TECs) and revealed their abnormalities. We have compared the gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and found that several genes were upregulated in TECs. Expression of the chemokine receptor CXCR7 mRNA was higher in TECs than in NECs. However, information regarding the expression of CXCR7 in the tumor vessels of renal cell carcinoma is limited. CXCR7 and its ligand CXCL12 have been implicated in tumor cell survival. In this study, the expression of CXCR7 in the tumor vessels of renal cell carcinoma (RCC) was investigated. Real-time PCR revealed higher expression level of CXCR7 in cultured TECs than in cultured NECs. Furthermore, similar to mouse TECs, immunostaining revealed strong expression of CXCR7 in vivo in human tumor vessels. These findings suggest that CXCR7 is a novel TEC marker and a target for antiangiogenic therapy for RCC., (© 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

Adrenomedullin (AM) is a multifunctional 52-amino acid peptide. AM has several effects and acts as a growth factor in several types of cancer cells. Our previous study revealed that an AM antagonist (AMA) suppressed the growth of pancreatic tumors in mice, although its mechanism was not elucidated. In this study, we constructed an AMA expression vector and used it to treat renal cell carcinoma (RCC) in mice. This AMA expression vector significantly reduced tumor growth in mice. In addition, microvessel density was decreased in AMA-treated tumors. To analyze the effect of AMA on tumor angiogenesis in this model, tumor endothelial cells (TECs) were isolated from RCC xenografts. TEC proliferation was stimulated by AM and it was inhibited by AMA significantly. AM induced migration of TECs and it was also blocked by AMA. However, normal ECs (NECs) were not affected by either AM or AMA. These results demonstrate that AMA has inhibitory effects on TECs specifically, not on NEC, thereby inhibiting tumor angiogenesis. Furthermore, we showed that vascular endothelial growth factor-induced mobilization of endothelial progenitor cell (EPC) into circulation was inhibited by AMA. These results suggest that AMA can be considered a good anti-angiogenic reagent that selectively targets TECs and EPC in renal cancer.

Tumor blood vessels are thought to contain genetically normal and stable endothelial cells (ECs), unlike tumor cells, which typically display genetic instability. Yet, chromosomal aberration in human tumor-associated ECs (hTECs) in carcinoma has not yet been investigated. Here we isolated TECs from 20 human renal cell carcinomas and analyzed their cytogenetic abnormalities. The degree of aneuploidy was analyzed by fluorescence in situ hybridization using chromosome 7 and chromosome 8 DNA probes in isolated hTECs. In human renal cell carcinomas, 22-58% (median, 33%) of uncultured hTECs were aneuploid, whereas normal ECs were diploid. The mechanisms governing TEC aneuploidy were then studied using mouse TECs (mTECs) isolated from xenografts of human epithelial tumors. To investigate the contribution of progenitor cells to aneuploidy in mTECs, CD133(+) and CD133(-) mTECs were compared for aneuploidy. CD133(+) mTECs showed aneuploidy more frequently than CD133(-) mTECs. This is the first report showing cytogenetic abnormality of hTECs in carcinoma, contrary to traditional belief. Cytogenetic alterations in tumor vessels of carcinoma therefore can occur and may play a significant role in modifying tumor- stromal interactions.