Human Tumor Endothelial Cells Research Articles

The vascular gene Apold1 is dispensable for normal development but controls angiogenesis under pathological conditions

The molecular mechanisms of angiogenesis have been intensely studied, but many genes that control endothelial behavior and fate still need to be described. Here, we characterize the role of Apold1 (Apolipoprotein L domain containing 1) in angiogenesis in vivo and in vitro. Single-cell analyses reveal that - across tissues - the expression of Apold1 is restricted to the vasculature and that Apold1 expression in endothelial cells (ECs) is highly sensitive to environmental factors. Using Apold1−/− mice, we find that Apold1 is dispensable for development and does not affect postnatal retinal angiogenesis nor alters the vascular network in adult brain and muscle. However, when exposed to ischemic conditions following photothrombotic stroke as well as femoral artery ligation, Apold1−/− mice display dramatic impairments in recovery and revascularization. We also find that human tumor endothelial cells express strikingly higher levels of Apold1 and that Apold1 deletion in mice stunts the growth of subcutaneous B16 melanoma tumors, which have smaller and poorly perfused vessels. Mechanistically, Apold1 is activated in ECs upon growth factor stimulation as well as in hypoxia, and Apold1 intrinsically controls EC proliferation but not migration. Our data demonstrate that Apold1 is a key regulator of angiogenesis in pathological settings, whereas it does not affect developmental angiogenesis, thus making it a promising candidate for clinical investigation.

Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop.

The lack of Tcell infiltrates is a major obstacle to effective immunotherapy in cancer. Conversely, the formation of tumor-associated tertiary-lymphoid-like structures (TA-TLLSs), which are the local site of humoral and cellular immune responses against cancers, is associated with good prognosis, and they have recently been detected in immune checkpoint blockade (ICB)-responding patients. However, how these lymphoid aggregates develop remains poorly understood. By employing single-cell transcriptomics, endothelial fate mapping, and functional multiplex immune profiling, we demonstrate that antiangiogenic immune-modulating therapies evoke transdifferentiation of postcapillary venules into inflamed high-endothelial venules (HEVs) via lymphotoxin/lymphotoxin beta receptor (LT/LTβR) signaling. In turn, tumor HEVs boost intratumoral lymphocyte influx and foster permissive lymphocyte niches for PD1- and PD1+TCF1+ CD8 Tcell progenitors that differentiate into GrzB+PD1+ CD8T effector cells. Tumor-HEVs require continuous CD8 and NK cell-derived signals revealing that tumor HEV maintenance is actively sculpted by the adaptive immune system through a feed-forward loop.

Tumor-Treating Fields Inhibit the Metastatic Potential of Osteosarcoma Cells.

The prognosis of metastatic osteosarcoma (OS) remains poor with a <20% survival rate, particularly in cases of distant (non-lung) metastases. Tumor-treating field (TTF) therapy is a novel electric field-based treatment that causes metaphase arrest and tumor cell death, with the advantage of reduced side effects compared to radiation and chemotherapy. TTF shows promise in glioblastoma and other solid tumors; however, few studies have examined its potential in the treatment of osteosarcoma. Therefore, we explored the mechanism of TTF-induced metastasis inhibition and cell death using in vitro models. TTF (1.5 V/cm, 150 kHz) was applied to U2OS and KHOS/NP OS cell lines. In addition, a 3-dimensional culture system was established using these OS cell lines. Cell migration and invasion (i.e., metastatic potential) were examined using a wound-healing scratch assay and transwell assay, respectively. Western blotting of metastasis- and angiogenesis-related proteins was performed. TTF suppressed the migration of and invasion by OS cells and inhibited the expression of epithelial markers, thereby preventing epithelial-mesenchymal transition (EMT), a hallmark of metastasis. Moreover, TTF prevented angiogenesis in human tumor endothelial cells and downregulated matrix metalloproteinase-2 (MMP2) and vascular endothelial growth factor (VEGF) expression. Therefore, TTF shows potential as an improved treatment for osteosarcoma, warranting further preclinical studies in animal models to support clinical trials.

Development of Immortalized Human Tumor Endothelial Cells from Renal Cancer.

Tumor angiogenesis research and antiangiogenic drug development make use of cultured endothelial cells (ECs) including the human microvascular ECs among others. However, it has been reported that tumor ECs (TECs) are different from normal ECs (NECs). To functionally validate antiangiogenic drugs, cultured TECs are indispensable tools, but are not commercially available. Primary human TECs are available only in small quantities from surgical specimens and have a short life span in vitro due to their cellular senescence. We established immortalized human TECs (h-imTECs) and their normal counterparts (h-imNECs) by infection with lentivirus producing simian virus 40 large T antigen and human telomerase reverse transcriptase to overcome the replication barriers. These ECs exhibited an extended life span and retained their characteristic endothelial morphology, expression of endothelial marker, and ability of tube formation. Furthermore, h-imTECs showed their specific characteristics as TECs, such as increased proliferation and upregulation of TEC markers. Treatment with bevacizumab, an antiangiogenic drug, dramatically decreased h-imTEC survival, whereas the same treatment failed to alter immortalized NEC survival. Hence, these h-imTECs could be a valuable tool for drug screening to develop novel therapeutic agents specific to TECs or functional biological assays in tumor angiogenesis research.

Phenotypic and functional characterization of tumor-derived endothelial cells isolated from primary human hepatocellular carcinoma.

Tumor endothelial cells (TECs) have been investigated using human tumor xenografts in mice models. In order to provide pure human TECs for the updating of clinical anti-angiogenic cancer therapy, in the present study we established a protocol of purification of TECs derived from clinical hepatocellular carcinoma (HCC) and revealed the TEC features by in vitro and in vivo assays. We isolated TECs from fresh surgical resections of HCC by magnetic-activated cell sorting and purified by flow cytometry sorting upon CD31 expression, referred to as ECDHCCs. Next, we identified cultured ECDHCCs by morphology, phenotype, genotype, and functional assays. The ECDHCCs appeared as Weibel-Palade bodies under electron microscopy. They expressed endothelial markers, such as CD31, CD105, and vascular endothelial growth factor receptor 2, and expressed the genes that are associated with pro-angiogenesis, especially vascular endothelial growth factor, epiregulin, and programmed cell death 10. Functionally, ECDHCCs were capable of tube formation, wound healing, and Transwell migration in vitro. These in vitro behaviors were validated by in vivo Matrigel plug assay in mice. Finally, comparison of ECDHCC with the Hep-G2 liver cancer cell line showed there was no similarity of phenotype or function between these two types of cells. Tumor endothelial cells derived from human HCC can be isolated and purified from clinical samples by flow cytometer. They have the endothelial phenotype and morphologic features and are capable of tube formation and migration. This study provides a useful model for researchers to study tumor angiogenesis and screening of candidate targets.

CXCR7 silencing inhibits the migration and invasion of human tumor endothelial cells derived from hepatocellular carcinoma by suppressing STAT3.

C-X-C chemokine receptor type 7 (CXCR7) is reported to be overexpressed in tumor endothelial cells (TECs), which are the primary target cells of antivascular chemotherapy. However, the role of CXCR7 in TECs is not fully understood. In the present study, CXCR7 expression was inhibited in TECs derived from hepatocellular carcinoma (HCC) using short hairpin (sh)RNA plasmids to investigate the role of CXCR7 in the regulation of migration and invasion of TECs as well as its underlying mechanisms. The data showed that the downregulation of CXCR7 significantly inhibited the migration and invasion of TECs. Further study showed that silencing CXCR7 resulted in decreased phosphorylated signal transducer and activator of transcription 3 (STAT3) at Tyr705 and its downstream target genes in TECs, including matrix metalloproteinase‑2 (MMP2) and vascular endothelial growth factor (VEGF). However, restoring STAT3 phosphorylation abolished the CXCR7‑shRNA‑induced decrease in TECs migration and invasion, as well as the downregulation of MMP2 and VEGF in TECs. These findings indicate that CXCR7 may regulate the migration and invasion of TECs derived from HCC via the STAT3 signaling pathway and that CXCR7 could be a potential target for the antivascular therapy of HCC.

Tumor endothelial cells with high aldehyde dehydrogenase activity show drug resistance.

Tumor blood vessels play an important role in tumor progression and metastasis. We previously reported that tumor endothelial cells (TEC) exhibit several altered phenotypes compared with normal endothelial cells (NEC). For example, TEC have chromosomal abnormalities and are resistant to several anticancer drugs. Furthermore, TEC contain stem cell‐like populations with high aldehyde dehydrogenase (ALDH) activity (ALDH high TEC). ALDH high TEC have proangiogenic properties compared with ALDH low TEC. However, the association between ALDH high TEC and drug resistance remains unclear. In the present study, we found that ALDH mRNA expression and activity were higher in both human and mouse TEC than in NEC. Human NEC:human microvascular endothelial cells (HMVEC) were treated with tumor‐conditioned medium (tumor CM). The ALDH high population increased along with upregulation of stem‐related genes such as multidrug resistance 1, CD90, ALP, and Oct‐4. Tumor CM also induced sphere‐forming ability in HMVEC. Platelet‐derived growth factor (PDGF)‐A in tumor CM was shown to induce ALDH expression in HMVEC. Finally, ALDH high TEC were resistant to fluorouracil (5‐FU) in vitro and in vivo. ALDH high TEC showed a higher grade of aneuploidy compared with that in ALDH low TEC. These results suggested that tumor‐secreting factor increases ALDH high TEC populations that are resistant to 5‐FU. Therefore, ALDH high TEC in tumor blood vessels might be an important target to overcome or prevent drug resistance.

Dangerous Liaisons: Deviant Endothelium NOTCHes toward Tumor Metastasis

In this issue of Cancer Cell, Wieland etal. uncover a feedback loop in which tumor cells, by augmenting Notch signaling, provoke a senescent and pro-inflammatory state in endothelial cells, promoting neutrophil infiltration, tumor cell adhesion, and metastasis. Interfering with this Notch-dependent crosstalk may be a therapeutic approach to block metastasis.

Tumor endothelial cells express high pentraxin 3 levels.

It has been described that tumor progression has many similarities to inflammation and wound healing in terms of the signaling processes involved. Among biological responses, angiogenesis, which is necessary for tumor progression and metastasis, is a common hallmark; therefore, tumor blood vessels have been considered as important therapeutic targets in anticancer therapy. We focused on pentraxin 3 (PTX3), which is a marker of cancer-related inflammation, but we found no reports on its expression and function in tumor blood vessels. Here we showed that PTX3 is expressed in mouse and human tumor blood vessels based on immunohistochemical analysis. We found that PTX3 is upregulated in primary mouse and human tumor endothelial cells compared to normal endothelial cells. We also showed that PTX3 plays an important role in the proliferation of the tumor endothelial cells. These results suggest that PTX3 is an important target for antiangiogenic therapy.

Abstract B23: Neuroblastoma patient-derived orthotopic xenografts: Clinically relevant models for drug testing

Abstract Background: We previously established neuroblastoma patient-derived orthotopic xenografts (PDXs) by implanting patient neuroblastoma fragments into immunodeficient NSG mice. SNP array analysis confirmed that PDXs maintain patient-specific chromosomal aberrations 1p del, MYCN amp and 17q gain. Immunohistochemistry showed that PDXs retain neuroblastoma markers and a highly infiltrative growth pattern. Importantly, we found spontaneous distant metastasis to lungs, liver and bone marrow. In vitro cultures established from the PDXs express neuroblastoma markers and retain their tumorigenic and metastatic ability in vivo after orthotopic injection. Methods and Results: Given the important role of the tumor stroma for tumor progression and treatment response, we examined PDX stroma by immunohistochemistry. PDXs were highly vascularized with mouse endothelial cells and two PDX models also formed tumor vasculature by co-engrafted human tumor endothelial cells. Tumors contained pericytes, cancer-associated fibroblasts, macrophages and extracellular components resembling the patient disease. PDXs established in athymic nude mice additionally developed intratumoral lymphatic vessels and contained mouse-derived CD45+ lymphoid cells. Thus, PDXs reflect important tumor microenvironment hallmarks for high-stage neuroblastoma. Furthermore, we demonstrate the feasibility of using short-term in vitro cultured PDX-derived cells as a drug-testing model, and show that the HIF2A oncogene is transcriptionally regulated at hypoxia via the PI3K/mTORC2 pathway in these cells. The results establish the PI3K and mTORC2 pathways as potential therapeutic targets of aggressive neuroblastoma. Conclusion: Neuroblastoma orthotopic PDXs reflect clinical aspects of high-risk disease including spontaneous metastasis, copy number changes and microenvironmental hallmarks. Neuroblastoma PDXs are relevant models for in vitro and in vivo preclinical drug testing. Citation Format: Daniel Bexell, Noémie Braekeveldt, Sofie Mohlin, Caroline Wigerup, David Gisselsson, Irene Tadeo, Ana P. Berbegall, Sam Navarro, Rosa Noguera, Sven Påhlman. Neuroblastoma patient-derived orthotopic xenografts: Clinically relevant models for drug testing. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B23.

Abstract A02: Neuroblastoma patient-derived orthotopic xenografts: Clinically relevant models for drug testing

Abstract Widespread metastasis is a major problem for the treatment of high-risk neuroblastoma. Relevant neuroblastoma animal models are hence needed to study and target high-risk metastatic neuroblastoma. We developed neuroblastoma patient-derived orthotopic xenografts (PDXs) using viably cryopreserved or fresh patient neuroblastoma fragments which were implanted orthotopically into immunodeficient NSG mice. Immunohistochemistry showed that PDXs retain neuroblastoma markers and a highly infiltrative growth pattern. Importantly, we found distant metastasis to lungs, liver and bone marrow. Single nucleotide polymorphism array analysis confirmed that PDXs maintain patient-specific chromosomal aberrations 1p del, MYCN amp and 17q gain. In vitro cultures established from the PDXs express neuroblastoma markers and retain their tumorigenic and metastatic ability in vivo after orthotopic injection. PDXs were highly vascularized with mouse endothelial cells and one PDX model formed tumor vasculature through co-engrafted human tumor endothelial cells. Tumors also contained pericytes, cancer-associated fibroblasts, macrophages and extracellular components resembling the patient disease. PDXs established in athymic nude mice additionally developed intratumoral lymphatic vessels and contained mouse-derived CD45+ lymphoid cells. Thus, PDXs maintain important tumor microenvironment hallmarks for high stage neuroblastoma. Furthermore, we demonstrate the feasibility of using short-term in vitro cultured PDX-derived cells as a drug-testing model, and show that the HIF2A oncogene is transcriptionally regulated at hypoxia via the PI3K/mTORC2 pathway in these cells. The results establish the PI3K and mTORC2 pathways as potential therapeutic targets of aggressive neuroblastoma. In conclusion, neuroblastoma orthotopic PDXs resemble many clinical aspects of the patient disease, e.g., distant metastases, and PDXs are valuable models for preclinical drug testing. Citation Format: Noémie Braekeveldt, Caroline Wigerup, David Gisselsson, Sofie Mohlin, My Merselius, Siv Beckman, Irene Tadeo, Anna P. Berbegall, Ingrid Öra, Samuel Navarro, Rosa Noguera, Sven Påhlman, Daniel Bexell. Neuroblastoma patient-derived orthotopic xenografts: Clinically relevant models for drug testing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A02.

Abstract 2375: Endothelial cells isolated from colorectal carcinoma exhibit tumor microenvironment-dependent plasticity allowing the identification of SPARCL1 as a novel endothelial cell quiescence factor

Abstract It is well known that tumor cell heterogeneity and the tumor microenvironment (TME) contribute to tumorigenesis. In contrast, stromal cell heterogeneity and plasticity in response to different TMEs and its impact on tumorigenesis has not been conclusively investigated in human tumors, as yet. Here we analyzed TME-induced heterogeneity of tumor endothelial cells (TECs) in colorectal carcinoma (CRC). To this goal vital and highly pure TEC cultures were isolated from CRC with a Th1-like TME (Th1-TME) and from CRC with a non-Th1-like TME (Ctrl-TME). Tumors with a Th1-TME exhibited a significantly reduced angiogenic activity and were associated with an improved prognosis of CRC patients. Comparative transcriptome analyses of the established TEC cultures identified a cluster of genes which was homogeneously expressed in TECs from Th1-TME but significantly different in TECs from Ctrl-TME. The gene encoding secreted protein, acidic and rich in cysteine-like 1 (SPARCL1) was identified to be most strongly up-regulated in TECs derived from a Th1-TME. We could confirm with in situ hybridization and immunohistochemistry that in normal colon and CRC tissues SPARCL1 expression is associated with endothelial cells (EC). Of note, SPARCL1 was consistently and highly expressed in normal colon, to a lower extent in CRC with a Th1-TME and low or absent in CRC tissues with a Ctrl.-TME. In cell culture SPARCL1 expression was absent in actively proliferating EC, but highly induced in EC reaching a confluent quiescent state. The SPARCL1 induction in confluent cells was reversible after the reduction of cell density. In functional studies SPARCL1 inhibited proliferation, migration and 3D sprouting of cultivated EC indicating potent anti-angiogenic activity. At the morphological level vessels in normal colon and in CRC with a Th1-TME were larger as compared to vessels in tumors with Ctrl-TME. Accordingly, SPARCL1 characterizes quiescent mature vessels that are larger as compared to rapidly sprouting microvessels. Altogether our results show that TECs isolated from CRC with different TMEs exhibit specific and differential long-term gene expression profiles. Characterization of the respective genes identified SPARCL1 as an anti-angiogenic gene associated with EC quiescence. These results demonstrate for the first time that human tumor endothelial cells retain differential TMEs by cellular memory allowing the identification of genes functionally imprinting the TME in these cellular sensors. Citation Format: Andrea Liebl, Elisabeth Naschberger, Vera S. Schellerer, Ute Schaal, Patrick Kölbel, Sandra Grenz, Nathalie Britzen-Laurent, Tilman T. Rau, Susanne Merkel, Barbara Dietel, Ludger Klein-Hitpass, Werner Hohenberger, Roland S. Croner, Michael Stürzl. Endothelial cells isolated from colorectal carcinoma exhibit tumor microenvironment-dependent plasticity allowing the identification of SPARCL1 as a novel endothelial cell quiescence factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2375. doi:10.1158/1538-7445.AM2015-2375

Suprabasin as a novel tumor endothelial cell marker

Recent studies have reported that stromal cells contribute to tumor progression. We previously demonstrated that tumor endothelial cells (TEC) characteristics were different from those of normal endothelial cells (NEC). Furthermore, we performed gene profile analysis in TEC and NEC, revealing that suprabasin (SBSN) was upregulated in TEC compared with NEC. However, its role in TEC is still unknown. Here we showed that SBSN expression was higher in isolated human and mouse TEC than in NEC. SBSN knockdown inhibited the migration and tube formation ability of TEC. We also showed that the AKT pathway was a downstream factor of SBSN. These findings suggest that SBSN is involved in the angiogenic potential of TEC and may be a novel TEC marker.

Vasculature analysis of patient derived tumor xenografts using species-specific PCR assays: evidence of tumor endothelial cells and atypical VEGFA-VEGFR1/2 signalings

BackgroundTumor endothelial transdifferentiation and VEGFR1/2 expression by cancer cells have been reported in glioblastoma but remain poorly documented for many other cancer types.MethodsTo characterize vasculature of patient-derived tumor xenografts (PDXs), largely used in preclinical anti-angiogenic assays, we designed here species-specific real-time quantitative RT-PCR assays. Human and mouse PECAM1/CD31, ENG/CD105, FLT1/VEGFR1, KDR/VEGFR2 and VEGFA transcripts were analyzed in a large series of 150 PDXs established from 8 different tumor types (53 colorectal, 14 ovarian, 39 breast and 15 renal cell cancers, 6 small cell and 5 non small cell lung carcinomas, 13 cutaneous melanomas and 5 glioblastomas) and in two bevacizumab-treated non small cell lung carcinomas xenografts.ResultsAs expected, mouse cell proportion in PDXs -evaluated by quantifying expression of the housekeeping gene TBP- correlated with all mouse endothelial markers and human VEGFA RNA levels. More interestingly, we observed human PECAM1/CD31 and ENG/CD105 expression in all tumor types, with higher rate in glioblastoma and renal cancer xenografts. Human VEGFR expression profile varied widely depending on tumor types with particularly high levels of human FLT1/VEGFR1 transcripts in colon cancers and non small cell lung carcinomas, and upper levels of human KDR/VEGFR2 transcripts in non small cell lung carcinomas. Bevacizumab treatment induced significant low expression of mouse Pecam1/Cd31, Eng/Cd105, Flt1/Vegfr1 and Kdr/Vefr2 while the human PECAM1/CD31 and VEGFA were upregulated.ConclusionsTaken together, our results strongly suggest existence of human tumor endothelial cells in all tumor types tested and of both stromal and tumoral autocrine VEGFA-VEGFR1/2 signalings. These findings should be considered when evaluating molecular mechanisms of preclinical response and resistance to tumor anti-angiogenic strategies.

An Active Learning Approach for Rapid Characterization of Endothelial Cells in Human Tumors

Currently, no available pathological or molecular measures of tumor angiogenesis predict response to antiangiogenic therapies used in clinical practice. Recognizing that tumor endothelial cells (EC) and EC activation and survival signaling are the direct targets of these therapies, we sought to develop an automated platform for quantifying activity of critical signaling pathways and other biological events in EC of patient tumors by histopathology. Computer image analysis of EC in highly heterogeneous human tumors by a statistical classifier trained using examples selected by human experts performed poorly due to subjectivity and selection bias. We hypothesized that the analysis can be optimized by a more active process to aid experts in identifying informative training examples. To test this hypothesis, we incorporated a novel active learning (AL) algorithm into FARSIGHT image analysis software that aids the expert by seeking out informative examples for the operator to label. The resulting FARSIGHT-AL system identified EC with specificity and sensitivity consistently greater than 0.9 and outperformed traditional supervised classification algorithms. The system modeled individual operator preferences and generated reproducible results. Using the results of EC classification, we also quantified proliferation (Ki67) and activity in important signal transduction pathways (MAP kinase, STAT3) in immunostained human clear cell renal cell carcinoma and other tumors. FARSIGHT-AL enables characterization of EC in conventionally preserved human tumors in a more automated process suitable for testing and validating in clinical trials. The results of our study support a unique opportunity for quantifying angiogenesis in a manner that can now be tested for its ability to identify novel predictive and response biomarkers.

A Novel Model for Evaluating Therapies Targeting Human Tumor Vasculature and Human Cancer Stem–like Cells

Human tumor vessels express tumor vascular markers (TVM), proteins that are not expressed in normal blood vessels. Antibodies targeting TVMs could act as potent therapeutics. Unfortunately, preclinical in vivo studies testing anti-human TVM therapies have been difficult to do due to a lack of in vivo models with confirmed expression of human TVMs. We therefore evaluated TVM expression in a human embryonic stem cell-derived teratoma (hESCT) tumor model previously shown to have human vessels. We now report that in the presence of tumor cells, hESCT tumor vessels express human TVMs. The addition of mouse embryonic fibroblasts and human tumor endothelial cells significantly increases the number of human tumor vessels. TVM induction is mostly tumor-type-specific with ovarian cancer cells inducing primarily ovarian TVMs, whereas breast cancer cells induce breast cancer specific TVMs. We show the use of this model to test an anti-human specific TVM immunotherapeutics; anti-human Thy1 TVM immunotherapy results in central tumor necrosis and a three-fold reduction in human tumor vascular density. Finally, we tested the ability of the hESCT model, with human tumor vascular niche, to enhance the engraftment rate of primary human ovarian cancer stem-like cells (CSC). ALDH(+) CSC from patients (n = 6) engrafted in hESCT within 4 to 12 weeks whereas none engrafted in the flank. ALDH(-) ovarian cancer cells showed no engraftment in the hESCT or flank (n = 3). Thus, this model represents a useful tool to test anti-human TVM therapy and evaluate in vivo human CSC tumor biology.

The F‐prostaglandin receptor is a novel marker for tumor endothelial cells in renal cell carcinoma

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.

Development of a Novel DNA Aptamer Ligand Targeting to Primary Cultured Tumor Endothelial Cells by a Cell-Based SELEX Method

The present study used a spontaneous cell-based SELEX method (Systemic Evolution of Ligands by EXponential Enrichment) to produce DNA aptamers that specifically bind to cell surface proteins or biomarkers produced by primary cultured mouse tumor endothelial cells (mTECs). In solid tumors, new blood vessels are formed through an angiogenesis process, and this plays a critical role in cancer development as well as metastasis. To combat angiogenesis, an appropriate diagnosis and a molecular-level understanding of the different cancer types are now a high priority. The novel DNA aptamer AraHH001, developed in this study, binds specifically to mTECs with high affinity in the nano-molar range, but does not bind to normal skin endothelial cells (skin-ECs). The selected DNA aptamer was also found to bind to cultured human tumor endothelial cells (hTECs), isolated from a clinical patient with a renal carcinoma. The aptamer AraHH001 showed significant anti-angiogenesis activity by inhibiting tube formation by mTECs on matrigel. Interestingly, a confocal laser scanning microscopy examination of in vitro cellular uptake revealed that AraHH001 was assimilated by mTECs, and became co-localized in acidic compartments, as detected by labeling with Lysotracker Red. Therefore, the development of a specific DNA aptamer that binds to mTECs, as reported here for the first time, holds great promise not only as a therapeutic aptamer but also as a targeted molecular probe that appears to play a major role in angiogenesis, and for the development of a targeted new drug delivery system.

Abstract 3886: Development of a new DNA aptamer against primary cultured tumor endothelial cells by a cell-based SELEX method to discover a new drug delivery system

Abstract The purpose of the present study was to develop DNA aptamers through a spontaneous cell-based SELEX method (systemic evolution of ligands by exponential enrichment) that specifically binding to cell surface proteins or biomarkers produced by primary cultured tumor endothelial cells (TECs). In solid tumors, new blood vessels are formed through an angiogenesis process, and this plays a critical role in cancer development as well as the development of metastasis. To combat angiogenesis, an appropriate diagnosis and understanding at the molecular level of different cancer types is now a high priority. Our initial study explored the use of the cell-based SELEX method, which has been applied in TECs to generate high affinity DNA aptamers. Our selected DNA aptamer AraHH001 bound specifically and with a high affinity to TECs in the nanomolar range but did not bind to normal skin endothelial cells (skin-ECs) and tumor parenchymal OSRC-II cells. The selected DNA aptamers also bounded to primary cultured human tumor endothelial cells (hTECs) isolated from a clinical patient with a renal carcinoma. Therefore, the development of specific DNA aptamers that bind to TECs is reported here for the first time. We conclude that such new DNA aptamers promise to hold great promise as targeted molecular probes that appear to play a major role in angiogenesis, drug delivery, identification of biomarker as well as in cancer 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 3886. doi:1538-7445.AM2012-3886

F3-Targeted Cisplatin-Hydrogel Nanoparticles as an Effective Therapeutic That Targets Both Murine and Human Ovarian Tumor Endothelial Cells In vivo

Recent studies indicate that ovarian cancer may be highly responsive to antivascular therapeutics. We have developed an antivascular tumor therapeutic using the F3 peptide to target cisplatin-loaded nanoparticles (F3-Cis-Np) to tumor vessels. We show that although F3-Cis-Np bind with high specificity to both human ovarian tumor cells and tumor endothelial cells in vitro, they only show cytotoxic activity against the tumor endothelial cells. In vivo these nanoparticles bind primarily to tumor endothelial cells. Therapeutic studies in both flank and orthotopic i.p. murine ovarian tumor models, as well as human tumor xenograft models, show rapid tumor regression with treatment. Treatment was associated with significant vascular necrosis consistent with an antivascular effect. Furthermore, treatment was active in both platinum-sensitive and platinum-resistant cell lines. Importantly, we show that F3-Cis-Np bind to human tumor endothelial cells in vitro and to human tumor vessels in vivo. Therapy targeting human vasculature in vivo with F3-Cis-Np led to near complete loss of all human tumor vessels in a murine model of human tumor vasculature. Our studies indicate that F3-targeted vascular therapeutics may be an effective treatment modality in human ovarian cancer.