Miyako Kondoh

Tumor Endothelial Cells Acquire Drug Resistance by MDR1 Up-Regulation via VEGF Signaling in Tumor Microenvironment

Tumor endothelial cells (TECs) are therapeutic targets in anti-angiogenic therapy. Contrary to the traditional assumption, TECs can be genetically abnormal and might also acquire drug resistance. In this study, mouse TECs and normal ECs were isolated to investigate the drug resistance of TECs and the mechanism by which it is acquired. TECs were more resistant to paclitaxel with the up-regulation of multidrug resistance (MDR) 1 mRNA, which encodes the P-glycoprotein, compared with normal ECs. Normal human microvascular ECs were cultured in tumor-conditioned medium (CM) and became more resistant to paclitaxel through MDR1 mRNA up-regulation and nuclear translocation of Y-box-binding protein 1, which is an MDR1 transcription factor. Vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Akt were activated in human microvascular ECs by tumor CM. We observed that tumor CM contained a significantly high level of VEGF. A VEGFR kinase inhibitor, Ki8751, and a phosphatidylinositol 3-kinase-Akt inhibitor, LY294002, blocked tumor CM-induced MDR1 up-regulation. MDR1 up-regulation, via the VEGF-VEGFR pathway in the tumor microenvironment, is one of the mechanisms of drug resistance acquired by TECs. We observed that VEGF secreted from tumors up-regulated MDR1 through the activation of VEGFR2 and Akt. This process is a novel mechanism of the acquisition of drug resistance by TECs in the tumor microenvironment.

Heterogeneity of Tumor Endothelial Cells: Comparison between Tumor Endothelial Cells Isolated from High- and Low-Metastatic Tumors

An important concept in tumor angiogenesis is that tumor endothelial cells (TECs) are genetically normal and homogeneous. However, we previously reported that TECs differ from normal ECs. Whether the characteristics of TECs derived from different tumors differ remains unknown. To elucidate this, in this study, we isolated two types of TECs from high-metastatic (HM) and low-metastatic (LM) tumors and compared their characteristics. HM tumor-derived TECs (HM-TECs) showed higher proliferative activity and invasive activity than LM tumor-derived TECs (LM-TECs). Moreover, the mRNA expression levels of pro-angiogenic genes, such as vascular endothelial growth factor (VEGF) receptors 1 and 2, VEGF, and hypoxia-inducible factor-1α, were higher in HM-TECs than in LM-TECs. The tumor blood vessels themselves and the surrounding area in HM tumors were exposed to hypoxia. Furthermore, HM-TECs showed higher mRNA expression levels of the stemness-related gene stem cell antigen and the mesenchymal marker CD90 compared with LM-TECs. HM-TECs were spheroid, with a smoother surface and higher circularity in the stem cell spheroid assay. HM-TECs differentiated into osteogenic cells, expressing activated alkaline phosphatase in an osteogenic medium at a higher rate than either LM-TECs or normal ECs. Furthermore, HM-TECs contained more aneuploid cells than LM-TECs. These results indicate that TECs from HM tumors have a more pro-angiogenic phenotype than those from LM tumors.

Tumor-Derived Microvesicles Induce Proangiogenic Phenotype in Endothelial Cells via Endocytosis

Background Increasing evidence indicates that tumor endothelial cells (TEC) differ from normal endothelial cells (NEC). Our previous reports also showed that TEC were different from NEC. For example, TEC have chromosomal abnormality and proangiogenic properties such as high motility and proliferative activity. However, the mechanism by which TEC acquire a specific character remains unclear. To investigate this mechanism, we focused on tumor-derived microvesicles (TMV). Recent studies have shown that TMV contain numerous types of bioactive molecules and affect normal stromal cells in the tumor microenvironment. However, most of the functional mechanisms of TMV remain unclear. Methodology/Principal Findings Here we showed that TMV isolated from tumor cells were taken up by NEC through endocytosis. In addition, we found that TMV promoted random motility and tube formation through the activation of the phosphoinositide 3-kinase/Akt pathway in NEC. Moreover, the effects induced by TMV were inhibited by the endocytosis inhibitor dynasore. Our results indicate that TMV could confer proangiogenic properties to NEC partly via endocytosis. Conclusion We for the first time showed that endocytosis of TMV contributes to tumor angiogenesis. These findings offer new insights into cancer therapies and the crosstalk between tumor and endothelial cells mediated by TMV in the tumor microenvironment.

Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.

Lysyl oxidase secreted by tumour endothelial cells promotes angiogenesis and metastasis

Background:Molecules that are highly expressed in tumour endothelial cells (TECs) may be candidates for specifically targeting TECs. Using DNA microarray analysis, we found that the lysyl oxidase (LOX) gene was upregulated in TECs compared with its expression in normal endothelial cells (NECs). LOX is an enzyme that enhances invasion and metastasis of tumour cells. However, there are no reports on the function of LOX in isolated TECs.Methods:TECs and NECs were isolated to investigate LOX function in TECs. LOX inhibition of in vivo tumour growth was also assessed using β-aminopropionitrile (BAPN).Results:LOX expression was higher in TECs than in NECs. LOX knockdown inhibited cell migration and tube formation by TECs, which was associated with decreased phosphorylation of focal adhesion kinase (Tyr 397). Immunostaining showed high LOX expression in human tumour vessels in vivo. Tumour angiogenesis and micrometastasis were inhibited by BAPN in an in vivo tumour model.Conclusion:LOX may be a TEC marker and a possible therapeutic target for novel antiangiogenic therapy.

Abstract 5275: Biglycan is a specific marker and an autocrine angiogenic factor of tumor endothelial cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels are morphologically different from their normal counterparts. We isolated tumor endothelial cells (TECs), demonstrated their abnormalities, compared gene expression profiles of TECs and normal endothelial cells (NECs) by microarray analysis and identified several genes upregulated in TECs. We focused on the gene encoding biglycan, a small leucine-rich repeat proteoglycan. Biglycan is overexpressed in inflammation and fibrosis. However, there has been no report about the expression or the function of biglycan in TEC. In this study, we investigated the expression and the role of biglycan in TECs. Real-time PCR, western blotting and immunocytochemistry revealed higher biglycan expression levels in TECs than in NECs. Furthermore, we confirmed that biglycan was secreted from TECs. Biglycan knockdown inhibited cell migration and tube formation in TECs. TLR2 and TLR4 are the biglycan receptors. TLR2 and TLR4 blocking antibodies suppressed biglycan mediated cell migration and tube formation. We isolated TECs from human renal cell carcinoma tissue and NECs from normal renal tissue in the same patients. TECs and NECs were obtained from six patients. Real-time RT-PCR revealed that the biglycan expression levels were significantly higher in four of the six TEC samples than in the corresponding NEC samples. Furthermore, immunostaining revealed strong biglycan expression in vivo in several human tumor vessels, as in mouse TECs. Biglycan was detected in the sera of cancer patients but was hardly detected in those of healthy volunteers. Biglycan is an autocrine angiogenic factor stimulating tumor endothelial cell migration and tube formation. These findings suggested that biglycan is a novel TEC marker and a target for anti-angiogenic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5275. doi:1538-7445.AM2012-5275

Abstract 906: Gene expression analysis of circulating endothelial cells in cancer patients

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL It has been reported that the number of circulating endothelial cells (CECs) is higher in cancer patients compared to healthy controls. CECs may be acting as possible markers of vascular turnover or damage. Elevated level of CEC number has been reported to be correlated with tumor progression or response to anti-angiogenic therapy. Tumor endothelial cells (TECs) could be shed into circulation and being included in CECs. We have reported that primary cultured mouse TECs express reported TEC-specific markers, such as CD13, TEMs, Dickkopf-3 (Dkk-3) and others. In this study, we analyzed the expression of these TEC markers in CECs in lung cancer patients. Before and/or after dissecting tumor tissue, blood was collected and RNAs from peripheral blood mononuclear cells (PBMCs) were extracted from the patients. And mRNA expression of EC markers and several TEC markers, including both reported and novel candidate, were analyzed. We detected CD31 and CD105 expression in both lung cancer patients’ and healthy controls’ PBMCs. The expression levels of TEC markers and several novel TEC candidate genes were analyzed and compared with healthy controls. Expression levels of several TEC markers including novel TEC marker X and Y, which were expressed in human cultured TECs, were higher in cancer patients than in healthy controls. However, it was difficult for us to detect TEC marker gene expressions in PBMC from cancer patients’ blood since TECs are very small population in PBMC (0.2%). In order to obtain more concentrated TECs from lung cancer patients’ blood, we used blood from lung tumor specimen when lung cancer specimens were dissected under video-assisted thoracic surgery. This specimen (lung tumor specimen blood) contains pulmonary vein blood and it was thought to contain more TECs shed from lung tumor. So we isolated mononuclear cells form this specimen and analyzed TEC marker gene expressions. We sorted mononuclear cells by flow cytometry using anti-CD31 and anti-CD45. TECs are supposed to be included in CD31(+) CD45(−) (Q4) fraction. Novel TEC candidate marker gene X and Y expressions were detected, in crude mononuclear cells from the lung tumor specimen blood at high levels, but very low levels in CD31(−))CD45(+) (Q1) fraction or CD31(+)CD45(+) (Q2) fraction, leading a possibility that the gene X and Y were highly expressed in Q4 fraction. These results suggest that the lung tumor specimen blood can be one of useful materials to discover novel CTEC marker genes in lung cancer patients, and that these markers could be surrogate markers or predicted markers for anti-angiogenic therapy in lung cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 906. doi:10.1158/1538-7445.AM2011-906

Abstract 1509: Analysis of interaction between tumor endothelial cells and tumor cells

Interaction between tumor endothelial cells (TECs) and tumor cells plays a key role in the early stage of hematogenous metastasis. TECs provide the principal route by which tumor cells exit the primary tumor site and enter the circulation. We have reported TECs are different from normal endothelial cells (NECs) in various aspects, such as chromosomal abnormality and gene expression profiles. In this study, we isolated two types of TECs from different human tumor xenografts in nude mice to analyze interaction between TECs and tumor cells. One is HMTEC isolated from highly metastatic tumor and the other is LMTEC from low metastatic tumor. We also isolated NEC from dermis of normal nude mouse as normal control. HMTEC expressed higher levels of mRNA of angiogenesis-related genes than LMTEC or NEC. We hypothesized HMTECs may promote metastasis, in particular, intravasation at the primary site. We investigated the roles of TECs in tumor metastasis; 1) migration of tumor cells towards TECs, 2) adhesion to endothelial layer, 3) crossing the endothelium. Tumor cells migrated towards conditioned-media (CM) from HMTEC more than LMTEC-CM or NEC-CM in vitro. Tumor cells were more adhesive to HMTEC than to LMTEC or NEC. They migrated through the HMTEC-monolayer most among all ECs in transendothelial migration assay. These results suggested that HMTEC may help tumor cells to metastasize. Finally, we analyzed the effects of CM from highly metastatic tumor cells on NECs. Tumor CM induced mRNA expressions of several genes, such as CXCL12, VEGFA and extracellular matrix in NEC. Tumor cells more migrated towards tumor CM- treated NEC than non-treated NEC. Tumor cells adhered more to the tumor CM- treated NEC than to non-treated NEC. These results suggest that TECs “educated” in tumor microenvironment which have highly metastatic potential, may be collaborating with tumor cells for metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1509. doi:10.1158/1538-7445.AM2011-1509

Abstract 3432: A green tea polyphenol epigallocatechin-3 gallate specifically targets tumor-associated endothelial cells

A green tea polyphenol, epigallocatechin-3 gallate (EGCG) suppresses tumor growth in vivo. We investigated mechanisms of specific tumor angiogenesis inhibition using tumor-associated endothelial cells (TECs), peripheral blood-derived ECs, and normal endothelial cells (NECs). TECs were isolated and cultured from human tumor xenografts in nude mice. Peripheral blood-derived ECs were isolated from peripheral blood of nude mice. EGCG suppressed migration of TECs and peripheral blood-derived ECs. EGCG also inhibited the phosphorylation of Akt in TECs and peripheral blood-derived ECs. The PI3K inhibitor, LY294002 blocked the migration of TECs induced by VEGF. Furthermore, VEGF-induced mobilization of CD133/VEGFR-2 double-positive cells into circulation was inhibited by EGCG. MMP-9 in the bone marrow is involved in mobilizing bone marrow-derived VEGFR-2 positive cells into peripheral circulation. Expression of MMP-9 mRNA was suppressed in bone marrow stromal cells by EGCG. In vivo model, EGCG reduced melanoma growth. Our study showed that EGCG specifically inhibits TEC and circulating EC through PI3K in EC and MMP-9 expression in bone marrow stroma. EGCG is a promising angiogenesis inhibitor for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3432.