Isolated Tumor Endothelial Cells Research Articles

ROS enhance angiogenic properties via regulation of NRF2 in tumor endothelial cells.

Reactive oxygen species (ROS) are unstable molecules that activate oxidative stress. Because of the insufficient blood flow in tumors, the tumor microenvironment is often exposed to hypoxic condition and nutrient deprivation, which induces ROS accumulation. We isolated tumor endothelial cells (TECs) and found that they have various abnormalities, although the underlying mechanisms are not fully understood. Here we showed that ROS were accumulated in tumor blood vessels and ROS enhanced TEC migration with upregulation of several angiogenesis related gene expressions. It was also demonstrated that these genes were upregulated by regulation of Nuclear factor erythroid 2-related factor 2 (NRF2). Among these genes, we focused on Biglycan, a small leucine-rich proteoglycan. Inhibition of Toll-like receptors 2 and 4, known BIGLYCAN (BGN) receptors, cancelled the TEC motility stimulated by ROS. ROS inhibited NRF2 expression in TECs but not in NECs, and NRF2 inhibited phosphorylation of SMAD2/3, which activates transcription of BGN. These results indicated that ROS-induced BGN caused the pro-angiogenic phenotype in TECs via NRF2 dysregulation.

Abstract B51: Targeting galectin-1 to improve therapeutic efficacy in head and neck cancers

Abstract Head and neck cancers (HNC) are associated with significant changes in the immune profile, with lower systemic absolute lymphocyte count and reduced tumor-infiltrating lymphocytes (TILs), which are inversely correlated with overall survival. Galectin-1(Gal-1), which is a carbohydrate binding protein, is secreted at high level by cancer cells, including HNC, and contributes to tumor-immune escape and disease progression. Our group has shown that both hypoxia and radiotherapy (RT) can individually stimulate tumor Gal-1 secretion, which is detrimental to therapeutic efficacy. Targeting Gal-1 in HNC therefore may synergize with radiotherapy and/or immunotherapy. Using the CRISPR/Cas9 deletion approach, we developed an orthotopic model of HNC with/without Gal-1 expression. We found that Gal-1 KO tumors showed a marked decrease in tumor growth and nodal metastases. Gal-1 WT tumors had very low infiltration of CD4+/CD8+ T cells (TILs) compared to Gal-1 null tumors. The decrease in TILs was due to both Gal-1-induced apoptosis of activated T cells and Gal-1 inhibition of trans-endothelial T-cell migration, which was confirmed by in vitro assays. Endothelial cells within Gal-1 WT tumors showed uniformed staining of PD-L1 expression by immunohistochemistry, that was not present in Gal-1 null tumors. Similarly, isolated tumor endothelial cells (TECs) from Gal-1 WT tumors showed significantly higher expression of PD-L1 (PD1 ligand) and Gal-9 (Tim-3 ligand) than TECs from Gal-1 null tumor. Concurrently, there were more PD1+ and Tim3+ intra-tumoral T cells within the Gal-1 WT than Gal-1 null tumors. Our study therefore suggest that in addition to T-cell apoptosis, tumor Gal-1 can impede transendothelial T cell migration, leading to less TIL, resulting in immune escape and possible resistance to immune check point therapy. Therefore, a combined approach of targeting Gal-1 with immune checkpoint inhibitors may lead to better therapeutic efficacy in HNC patients. (Acknowlegement: NIH Grant-R01CA161585-03) Citation Format: Dhanya K. Nambiar, Hongbin Cao, Quynh Thu Le. Targeting galectin-1 to improve therapeutic efficacy in head and neck cancers. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B51.

Solid tumor therapy by selectively targeting stromal endothelial cells

Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors.

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

Abstract 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

CXCR7: A novel tumor endothelial marker in renal cell carcinoma

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.

Altered angiogenesis in the tumor microenvironment

Tumor blood vessels play an important role in tumor progression and metastasis. Thus, targeting the tumor blood vessels is an important strategy in cancer therapy. Tumor blood vessels generally arise from pre-existing vessels and have been thought to be genetically normal. However, they have been shown to differ from their normal counterparts, e.g. with regard to the morphological changes. We isolated tumor endothelial cells (TEC) from mouse tumor xenografts and showed that they were abnormal. TEC up-regulate many genes, proliferate more rapidly and migrate more than normal endothelial cells (NEC). Furthermore, the TEC in our study were cytogenetically abnormal. We concluded that TEC can acquire cytogenetic abnormalities while in the tumor microenvironment. In order to develop ideal antiangiogenic therapies, understanding the crosstalk between blood vessels and the tumor microenvironment is important. This review considers the current studies on TEC abnormalities and discusses the possible mechanism by which the tumor microenvironment produces abnormal TEC.

Cyclooxygenase‐2 inhibition causes antiangiogenic effects on tumor endothelial and vascular progenitor cells

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Cyclooxygenase (COX)-2 is known to play an important role in cancer growth and invasion, and it activates the signaling pathways controlling cell proliferation, migration, apoptosis, and angiogenesis. COX-2 is reported to be expressed in many cancer cells. Several studies have reported successful treatment of cancer cells with COX-2 inhibitors (COX-2is). However, the effect of COX-2 inhibition on the tumor endothelium remains to be elucidated. Our study shows that COX-2 is expressed in the vasculature of surgically resected human tumors. To investigate the effects of COX-2 inhibition on the tumor endothelium in vitro, we isolated tumor endothelial cells (TECs) from human melanoma and oral carcinoma xenografts in mice, in which we confirmed that tumor growth was suppressed by inhibiting angiogenesis with the COX-2is NS398. COX-2 mRNA was upregulated in TECs compared to normal endothelial cells (NECs). Cell migration and proliferation were suppressed by NS398 in TECs but not in NECs. The effects of NS398 in vivo were consistent with the in vitro results. The number of CD133+ /vascular endothelial growth factor receptor-2+ cells in circulation was significantly suppressed by COX-2 inhibition. In addition, the number of progenitor marker-positive cells decreased in the tumor blood vessels after COX-2i treatment, which suggests that the homing of progenitor cells into the tumor was also blocked. We conclude that NS398 specifically targets both TECs and vascular progenitor cells without affecting NECs.

Crosstalk between Blood Vessels and Tumor Microenvironment

Tumor blood vessels play an important role in tumor progression and metastasis. Thus, targeting tumor blood vessels is an important strategy for cancer therapy, especially for head and neck cancer patients. Tumor blood vessels generally sprout from pre-existing vessels and have been thought to be genetically normal. However, tumor blood vessels have been shown to differ from their normal counterparts, for example, by changes in morphology. The authors isolated tumor endothelial cells (TECs) from mouse tumor xenografts and have shown that the TECs are abnormal. TECs up-regulate many genes and proliferate more rapidly and migrate more than normal endothelial cells (NECs). Furthermore, TECs were found to be cytogenetically abnormal. We conclude that TECs can acquire cytogenetic abnormalities while in a tumor microenvironment.To develop ideal antiangiogenic therapies, understanding the crosstalk between blood vessels and the tumor microenvironment is important.Here, we provide an overview of the current studies on TEC abnormalities and a discussion about possible mechanisms for how tumor the microenvironment makes TECs abnormal.

Isolated tumor endothelial cells maintain specific character during long-term culture

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Increasing evidence indicates that tumor endothelial cells (TECs) are more relevant to the study of tumor angiogenesis than normal endothelial cells (NECs) because their morphologies and gene expression are different from NECs. However, it is challenging to isolate and culture large numbers of pure ECs from tumor tissue since the percentage of ECs is only about 1–2% and tumor cells and fibroblasts easily overgrow them. In addition, there has been concern that isolated TECs may lose their special phenotype once they are dissociated from tumor cells. In this study, we have successfully purified murine TECs from four different human tumor xenografts and NECs from murine dermal tissue. Isolated ECs expressed endothelial markers, such as CD31, VE-cadherin (CD144), and endoglin (CD105), for more than 3 months after isolation. TECs maintained tumor endothelial-specific markers, such as tumor endothelial marker 8 (TEM8) and aminopeptidase N (APN), as in tumor blood vessels in vivo. In addition, TECs were more proliferative and motile than NECs. TECs showed a higher response to VEGF and higher expression of VEGF receptors-1 and -2 than NECs did. Stem cell antigen-1 was up-regulated in all four TECs, suggesting that they have a kind of stemness. Cultured TECs maintain distinct biological differences from NECs as in vivo. In conclusion, it was suggested that TECs are relevant material for tumor angiogenesis research.

Calcification of Multipotent Prostate Tumor Endothelium

Solid tumors require new blood vessels for growth and metastasis, yet the biology of tumor-specific endothelial cells is poorly understood. We have isolated tumor endothelial cells from mice that spontaneously develop prostate tumors. Clonal populations of tumor endothelial cells expressed hematopoietic and mesenchymal stem cell markers and differentiated to form cartilage- and bone-like tissues. Chondrogenic differentiation was accompanied by an upregulation of cartilage-specific col2a1 and sox9, whereas osteocalcin and the metastasis marker osteopontin were upregulated during osteogenic differentiation. In human and mouse prostate tumors, ectopic vascular calcification was predominately luminal and colocalized with the endothelial marker CD31. Thus, prostate tumor endothelial cells are atypically multipotent and can undergo a mesenchymal-like transition.

Understanding tumor endothelial cell abnormalities to develop ideal anti‐angiogenic therapies

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Tumor blood vessels have been shown to differ from their normal counterparts, for example, by changes in morphology. An important concept in tumor angiogenesis is that tumor endothelial cells are assumed to be genetically normal, even though these endothelial cells are structurally and functionally abnormal. To date, many anti-angiogenic drugs have been developed, but, their therapeutic efficacy is not dramatic and they have also been reported to cause toxic side effects. To develop ideal antiangiogenic therapies, understanding tumor endothelial cell abnormalities is important. We have isolated tumor endothelial cells from mouse tumor xenografts and have shown that tumor-associated endothelial cells are abnormal. Tumor-associated endothelial cells upregulate many genes, such as epidermal growth factor receptor (EGFR). Tumor-associated endothelial cells are also more sensitive to EGF. They also have relatively large, heterogeneous nuclei. Unexpectedly, tumor endothelial cells are cytogenetically abnormal. Fluorescence in situ hybridization (FISH) analysis showed that freshly isolated uncultured tumor endothelial cells were aneuploid and had abnormal multiple centrosomes. The degree of aneuploidy was exacerbated by passage in culture. In marked contrast, freshly isolated normal skin and adipose endothelial cells were diploid. They had normal centrosomes and remained cytogenetically stable in culture even up to 20 passages. We conclude that tumor endothelial cells can acquire cytogenetic abnormalities while in the tumor microenvironment. Questions as to whether or not tumor endothelial cells become resistant to antiangiogenic drugs are thus raised. Our preliminary data show that tumor endothelial cells are more resistant to certain chemotherapeutic drugs. Studies to evaluate the mechanism for cytogenetic abnormalities in tumor endothelial cells are underway. It is becoming quite clear that the tumor vasculature is much more complex and unpredictable than initially perceived. Here, we provide an overview of the current studies on tumor endothelial cell abnormalities.

Angiogenesis modulates the tumour immune response.

Outgrowth of solid tumors and metastases is dependent on the process of angiogenesis. Tumors escape from the formation of an effective infiltrate by downregulation of endothelial adhesion molecules. This downregulation of adhesion receptors is governed by the exposure to angiogenic factors. In recent years proof for this has been provided by demonstrating that freshly isolated tumor endothelial cells exhibit a decreased expression of ICAM-1 and -2 as compared to endothelial cells in normal tissue. In addition, adhesion molecules are downregulated on normal tissue endothelial cells when cultured with angiogenesis stimulators such as basic fibroblast growth factor and vascular endothelial cell growth factor, while under these conditions endothelial cells become less responsive to cytokines such as tumor necrosis factor-alpha with respect to the upregulation of endothelial adhesion molecules. Very recently it has been demonstrated that this harmful endothelial cell anergy can be counteracted by inhibitors of angiogenesis.