Human Tumor Vasculature Research Articles

Blood-brain barrier alterations in human brain tumors revealed by genome-wide transcriptomic profiling.

Brain tumors, whether primary or secondary, have limited therapeutic options despite advances in understanding driver gene mutations and heterogeneity within tumor cells. The cellular and molecular composition of brain tumor stroma, an important modifier of tumor growth, has been less investigated to date. Only few studies have focused on the vasculature of human brain tumors despite the fact that the blood-brain barrier (BBB) represents the major obstacle for efficient drug delivery. In this study, we employed RNA sequencing to characterize transcriptional alterations of endothelial cells (EC) isolated from primary and secondary human brain tumors. We used an immunoprecipitation approach to enrich for EC from normal brain, glioblastoma (GBM), and lung cancer brain metastasis (BM). Analysis of the endothelial transcriptome showed deregulation of genes implicated in cell proliferation, angiogenesis, and deposition of extracellular matrix (ECM) in the vasculature of GBM and BM. Deregulation of genes defining the BBB dysfunction module was found in both tumor types. We identified deregulated expression of genes in vessel-associated fibroblasts in GBM. We characterize alterations in BBB genes in GBM and BM vasculature and identify proteins that might be exploited for developing drug delivery platforms. In addition, our analysis on vessel-associated fibroblasts in GBM shows that the cellular composition of brain tumor stroma merits further investigation.

The vascular landscape of human cancer.

Tumors depend on a blood supply to deliver oxygen and nutrients, making tumor vasculature an attractive anticancer target. However, only a fraction of patients with cancer benefit from angiogenesis inhibitors. Whether antiangiogenic therapy would be more effective if targeted to individuals with specific tumor characteristics is unknown. To better characterize the tumor vascular environment both within and between cancer types, we developed a standardized metric - the endothelial index (EI) - to estimate vascular density in over 10,000 human tumors, corresponding to 31 solid tumor types, from transcriptome data. We then used this index to compare hyper- and hypovascular tumors, enabling the classification of human tumors into 6 vascular microenvironment signatures (VMSs) based on the expression of a panel of 24 vascular "hub" genes. The EI and VMS correlated with known tumor vascular features and were independently associated with prognosis in certain cancer types. Retrospective testing of clinical trial data identified VMS2 classification as a powerful biomarker for response to bevacizumab. Thus, we believe our studies provide an unbiased picture of human tumor vasculature that may enable more precise deployment of antiangiogenesis therapy.

Effects of contrast-enhanced ultrasound treatment on neoadjuvant chemotherapy in breast cancer.

Purpose: Preclinical and clinical data indicate that contrast-enhanced ultrasound can enhance tumor perfusion and vessel permeability, thus, improving chemotherapy accumulation and therapeutic outcome. Therefore, we investigated the effects of high mechanical index (MI) contrast-enhanced Doppler ultrasound (CDUS) on tumor perfusion in breast cancer.Methods: In this prospective study, breast cancer patients were randomly assigned to receive either 18 minutes of high MI CDUS during chemotherapy infusion (n = 6) or chemotherapy alone (n = 5). Tumor perfusion was measured before and after at least six chemotherapy cycles using motion-model ultrasound localization microscopy. Additionally, acute effects of CDUS on vessel perfusion and chemotherapy distribution were evaluated in mice bearing triple-negative breast cancer (TNBC).Results: Morphological and functional vascular characteristics of breast cancer in patients were not significantly influenced by high MI CDUS. However, complete clinical tumor response after neoadjuvant chemotherapy was lower in high MI CDUS-treated (1/6) compared to untreated patients (4/5) and size reduction of high MI CDUS treated tumors tended to be delayed at early chemotherapy cycles. In mice with TNBC high MI CDUS decreased the perfused tumor vessel fraction (p < 0.01) without affecting carboplatin accumulation or distribution. Higher vascular immaturity and lower stromal stabilization may explain the stronger vascular response in murine than human tumors.Conclusion: High MI CDUS had no detectable effect on breast cancer vascularization in patients. In mice, the same high MI CDUS setting did not affect chemotherapy accumulation although strong effects on the tumor vasculature were detected histologically. Thus, sonopermeabilization in human breast cancers might not be effective using high MI CDUS protocols and future applications may rather focus on low MI approaches triggering microbubble oscillations instead of destruction. Furthermore, our results show that there are profound differences in the response of mouse and human tumor vasculature to high MI CDUS, which need to be further explored and considered in clinical translation.

Bispecific Targeting of EGFR and Urokinase Receptor (uPAR) Using Ligand-Targeted Toxins in Solid Tumors.

Ligand-targeted toxins (LTTs) are bioengineered molecules which are composed of a targeting component linked to a toxin that induces cell death once the LTT binds its target. Bispecific targeting allows for the simultaneous targeting of two receptors. In this review, we mostly focus on the epidermal growth factor receptor (EGFR) as a target. We discuss the development and testing of a bispecific LTT targeting EGFR and urokinase-type plasminogen activator receptor (uPAR) as two attractive targets implicated in tumor growth and in the regulation of the tumor microvasculature in solid tumors. In vitro and mouse xenograft studies have shown that EGFR-targeted bispecific angiotoxin (eBAT) is effective against human solid tumors. Canine studies have shown that eBAT is both safe and effective against canine hemangiosarcoma, which is physiologically similar to human angiosarcoma. Finding the appropriate dosing strategy and sequencing of eBAT administration, in combination with other therapeutics, are among important factors for future directions. Together, the data indicate that eBAT targets cancer stem cells, it may have a role in inhibiting human tumor vasculature, and its bispecific conformation may have a role in reducing toxicity in comparative oncologic trials in dogs.

Exercise during preoperative therapy increases tumor vascularity in pancreatic tumor patients

The efficacy of chemotherapy is reduced by dysfunctional tumor vasculature, which may limit chemotherapy delivery to tumors. Preclinical studies have shown that moderate aerobic exercise improves tumor vascular function and increases chemotherapy efficacy in mouse models, but the effect of exercise on human tumor vasculature has not yet been determined. Here, we demonstrate that exercise remodels the tumor vasculature, accelerates the regression, and delays the regrowth of pancreatic ductal adenocarcinoma in a patient-derived xenograft mouse model treated with gemcitabine. By evaluating pancreatic adenocarcinoma specimens from patients treated with preoperative chemotherapy or chemoradiation therapy, we also demonstrate for the first time that tumor vascular remodeling occurs in association with exercise in humans. Future studies will evaluate whether exercise-induced vascular remodeling improves gemcitabine or other chemotherapy efficacy in patients, as this study evaluated only changes in tumor vascular structure.

Targeting Tumor Vascular CD99 Inhibits Tumor Growth.

CD99 (MIC2; single-chain type-1 glycoprotein) is a heavily O-glycosylated transmembrane protein (32 kDa) present on leukocytes and activated endothelium. Expression of CD99 on endothelium is important in lymphocyte diapedesis. CD99 is a diagnostic marker for Ewing's Sarcoma (EWS), as it is highly expressed by these tumors. It has been reported that CD99 can affect the migration, invasion and metastasis of tumor cells. Our results show that CD99 is also highly expressed in the tumor vasculature of most solid tumors. Furthermore, we found that in vitro CD99 expression in cultured endothelial cells is induced by starvation. Targeting of murine CD99 by a conjugate vaccine, which induced antibodies against CD99 in mice, resulted in inhibition of tumor growth in both a tumor model with high CD99 (Os-P0109 osteosarcoma) and low CD99 (CT26 colon carcinoma) expression. We demonstrated that vaccination against CD99 is safe, since no toxicity was observed in mice with high antibody titers against CD99 in their sera during a period of almost 11 months. Targeting of CD99 in humans is more complicated due to the fact that the human and mouse CD99 protein are not identical. We are the first to show that growth factor activated endothelial cells express a distinct human CD99 isoform. We conclude that our observations provide an opportunity for specific targeting of CD99 isoforms in human tumor vasculature.

Effect of Tumor Volume on Drug Delivery in Heterogeneous Vasculature of Human Brain Tumors

Drug distribution in tumors is strongly dependent on tumor biological properties such as tumor volume, vasculature, and porosity. An understanding of the drug distribution pattern in tumors can help in enhancing the effectiveness of anticancer treatment. A numerical model is employed to study the distribution of contrast agent in the heterogeneous vasculature of human brain tumors of different volumes. Dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI) has been done for a number of patients with different tumor volumes. Leaky tracer kinetic model (LTKM) is employed to obtain perfusion parameters from the DCE-MRI data. These parameters are used as input in the computational fluid dynamics (CFD) model to predict interstitial fluid pressure (IFP), interstitial fluid velocity (IFV), and distribution of the contrast agent in different tumors. Numerical results demonstrate that the IFP is independent of tumor volume. On the other hand, the IFV increases as the tumor volume increases. Further, the concentration of contrast agent also increases with the tumor volume. The results obtained in this work are in line with the experimental DCE-MRI data. It is observed that large volume tumors tend to retain a higher concentration of contrast agent for a longer duration of time because of large extravasation flux and slow washout as compared to smaller tumors. These results may be qualitatively extrapolated to chemotherapeutic drug delivery, implying faster healing in large volume tumors. This study helps in understanding the effect of tumor volume on the treatment outcome for a wide range of human tumors.

Abstract 5281: An exercise intervention for pancreas cancer patients increases tumor vascularity

Abstract We and others have demonstrated in mice that moderate aerobic exercise remodels tumor vasculature and improves chemotherapy delivery. The sparse, dysfunctional vasculature in pancreatic ductal adenocarcinoma (PDAC) is one major barrier to delivering chemotherapy to the tumor. Improved vascular function and chemotherapy delivery could substantially improve patient survival over the current 7% rate. We first evaluated whether exercise might improve survival for patients with PDAC using a patient-derived xenograft (PDX) mouse model and moderate treadmill running. Treatment of PDX-bearing mice with gemcitabine or gemcitabine plus exercise caused tumor regression. However, gemcitabine was more effective when combined with exercise and excitingly, regrowth of tumors after treatment cessation was significantly delayed in mice treated with exercise combined with gemcitabine compared to gemcitabine alone. Our animal data suggests that exercise may be beneficial for patients with pancreatic cancer. However, it is unclear whether a sufficient level of exercise can be performed by patients with advanced cancers to achieve improved vascular function as predicted by animal studies. Recently, we completed a pilot study delivering a home-based aerobic and strength training exercise program (EP) to patients with potentially operable pancreas cancer. Fifty-eight patients (48% female, median age 66) completed the EP concurrent with chemotherapy or chemoradiation over a mean duration of 15 weeks prior to pancreatectomy. Participants completed a mean of 145.8 minutes of weekly moderate to vigorous physical activity. Surgical tumor specimens were obtained from 28 patients who participated in the EP, and were evaluated for the total number of vessels, vessel density, average number of open lumens, and number of elongated vessels. We found that tumors of patients who participated in the EP had significantly increased vessel density and a significantly increased number of elongated vessels compared to tumors from control patient, similar to our observations in mice. This is the first demonstration that an EP is feasible during the chemotherapy/ chemoradiation phases for pancreatic cancer patients, and importantly, that moderate exercise changes human tumor vascular biology. There was also a trend toward increased immune cell infiltration in tumors from patients who exercised. Cell death in tumors is being evaluated. This data suggests that exercise is a low-cost, low risk way to remodel human tumor vasculature and therefore may be an adjuvant to chemotherapy. Remodeled vasculature may increase chemotherapy delivery to the tumor, improving survival, as predicted in animal models. Further studies evaluating the impact of exercise on patient outcome as well as angiogenic biomarkers are underway. Citation Format: Claudia Alvarez Florez, Nathan Parker, Matthew Katz, An Ngo-Huang, Carol Ferreira Cardoso, Huamin Wang, Maria Petzel, David Fogelman, Keri Schadler. An exercise intervention for pancreas cancer patients increases tumor vascularity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5281.

Hyperthermia-mediated drug delivery induces biological effects at the tumor and molecular levels that improve cisplatin efficacy in triple negative breast cancer

Triple negative breast cancer is an aggressive disease that accounts for at least 15% of breast cancer diagnoses, and a disproportionately high percentage of breast cancer related morbidity. Intensive research efforts are focused on the development of more efficacious treatments for this disease, for which therapeutic options remain limited. The high incidence of mutations in key DNA repair pathways in triple negative breast cancer results in increased sensitivity to DNA damaging agents, such as platinum-based chemotherapies. Hyperthermia has been successfully used in breast cancer treatment to sensitize tumors to radiation therapy and chemotherapy. It has also been used as a mechanism to trigger drug release from thermosensitive liposomes. In this study, mild hyperthermia is used to trigger release of cisplatin from thermosensitive liposomes in the vasculature of human triple negative breast cancer tumors implanted orthotopically in mice. This heat-triggered liposomal formulation of cisplatin resulted in significantly delayed tumor growth and improved overall survival compared to treatment with either non-thermosensitive liposomes containing cisplatin or free cisplatin, as was observed in two independent tumor models (i.e. MDA-MB-231 and MDA-MB-436). The in vitro sensitivity of the cell lines to cisplatin and hyperthermia alone and in combination was characterized extensively using enzymatic assays, clonogenic assays, and spheroid growth assays. Evaluation of correlations between the in vitro and in vivo results served to identify the in vitro approach that is most predictive of the effects of hyperthermia in vivo. Relative expression of several heat shock proteins and the DNA damage repair protein BRCA1 were assayed at baseline and in response to hyperthermia both in vitro and in vivo. Interestingly, delivery of cisplatin in thermosensitive liposomes in combination with hyperthermia resulted in the most significant tumor growth delay, relative to free cisplatin, in the less cisplatin-sensitive cell line (i.e. MDA-MB-231). This work demonstrates that thermosensitive cisplatin liposomes used in combination with hyperthermia offer a novel method for effective treatment of triple negative breast cancer.

Numerical Study of Transport of Anticancer Drugs in Heterogeneous Vasculature of Human Brain Tumors Using Dynamic Contrast Enhanced-Magnetic Resonance Imaging.

Systemic administration of drugs in tumors is a challenging task due to unorganized microvasculature and nonuniform extravasation. There is an imperative need to understand the transport behavior of drugs when administered intravenously. In this study, a transport model is developed to understand the therapeutic efficacy of a free drug and liposome-encapsulated drugs (LED), in heterogeneous vasculature of human brain tumors. Dynamic contrast enhanced-magnetic resonance imaging (DCE-MRI) data is employed to model the heterogeneity in tumor vasculature that is directly mapped onto the computational fluid dynamics (CFD) model. Results indicate that heterogeneous vasculature leads to preferential accumulation of drugs at the tumor position. Higher drug accumulation was found at location of higher interstitial volume, thereby facilitating more tumor cell killing at those areas. Liposome-released drug (LRD) remains inside the tumor for longer time as compared to free drug, which together with higher concentration enhances therapeutic efficacy. The interstitial as well as intracellular concentration of LRD is found to be 2–20 fold higher as compared to free drug, which are in line with experimental data reported in literature. Close agreement between the predicted and experimental data demonstrates the potential of the developed model in modeling the transport of LED and free drugs in heterogeneous vasculature of human tumors.

Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors.

We describe a novel mechanism regulating the tumor endothelial barrier and T cell homing to tumors. Selective expression of the death mediator Fas ligand (FasL/CD95L) was detected in the vasculature of many human and mouse solid tumors but not in normal vasculature, and in these tumors it was associated with scarce CD8+ infiltration and predominance of FoxP3+ T regulatory (Treg) cells. Tumor-derived vascular endothelial growth factor A (VEGF-A), interleukin 10 (IL-10) and prostaglandin E2 (PGE2) cooperatively induced FasL expression on endothelial cells, which acquired the ability to kill effector CD8+ T cells, but not Treg cells, due to higher levels of cFLIP expression in Tregs. In the mouse, genetic or pharmacologic suppression of FasL produced a significant increase in the influx of tumor-rejecting CD8+ over FoxP3+ T cells. Pharmacologic inhibition of VEGF and PGE2 attenuated tumor endothelial FasL expression, produced a significant increase in the influx of tumor-rejecting CD8+ over FoxP3+ T cells, which was FasL-dependent, and led to CD8-dependent tumor growth suppression. Thus, tumor paracrine mechanisms establish a tumor endothelial death barrier, which plays a critical role in establishing immune tolerance and determining the fate of tumors.

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.

Abstract 3891: Hornerin: a novel, non-VEGF mediated human tumor angiogenic protein.

Abstract Angiogenesis is vital to the growth, progression and ultimately metastasis of tumors. As such, many therapies have attempted to eradicate tumors through anti-angiogenic strategies. Most current and past preclinical and clinically approved therapies focus on the vascular endothelial growth factor (VEGF) pathway. Unfortunately, these VEGF therapies have not shown much success, leading oncologists and scientists to become pessimistic about the usefulness of targeting angiogenesis as an anti-tumor strategy. Compensatory pathways may explain the lack of efficacy. Therapies against novel and/or non-VEGF proteins could hold the key to effective anti-angiogenic cancer therapies. Therefore, we used an in vivo phage display screen to find new, non-VEGF mediated angiogenic proteins. After phage selection, non-VEGF binding phage clones were identified by screening on human umbilical vein endothelial cells (HUVEC) treated with tumor conditioned media, VEGF conditioned media or vehicle control. Those that bound tumor conditioned media, but not VEGF conditioned media were selected for binding partner identification via functional proteomics pioneered in our lab. The binding partner of one clone was identified as hornerin, a previously unknown player in tumor angiogenesis. Subsequent validation of hornerin by mRNA expression, immunocytochemistry and immunohistochemistry showed that hornerin was indeed not induced by VEGF. Hornerin expression was 7-fold more in resected human PDAC tumor vasculature than adjacent normal pancreatic vasculature. Interestingly, hornerin expression was also detected in tumor cells of resected human PDAC and 20 other types of tumors, providing a potential new biomarker of pancreatic and other cancers. A functional role in angiogenesis was confirmed by intratumoral injection of short siRNA against endothelial hornerin; treated tumor growth was static and tumor vessels displayed a more physiologic phenotype. Combination therapy consisting of siRNA against hornerin and a VEGF inhibitor (AV-951) produced additive reduction of tumor growth, consistent with the existence of separate and/or compensatory pathways. The discovery of novel, non-VEGF mediated angiogenic proteins provides the potential to develop therapies that synergize with current anti-VEGF therapies. One such protein, hornerin was identified by our in vivo phage screen and shown to be functionally important in angiogenesis. Future studies include identifying the growth factor responsible for endothelial expression, understanding hornerin's mechanism and role in pathologic angiogenesis, and designing new therapeutic strategies. Citation Format: Marc E. Seaman, Kimberly A. Kelly. Hornerin: a novel, non-VEGF mediated human tumor angiogenic protein. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3891. doi:10.1158/1538-7445.AM2013-3891

Abstract 3760: EGFL6 is a tumor vascular regulator of ovarian CSC asymmetric division and a novel therapeutic target.

Abstract Cancer stem cells (CSC) are closely associated with tumor vasculature in the CSC niche. Tumor vascular cells are known to provide critical growth, differentiation, and survival cues for CSC. Unfortunately little is known about the specific factors produced by vascular cells. EGFL6 is a factor specifically secreted by ovarian tumor vascular cells. EGFL6 is reported to regulate the differentiation of normal stem cells. We examined the impact of EGFL6 on ovarian cancer cell growth. We found that EGFL6 significantly promoted ovarian cancer cell growth in vitro. Ovarian cancer cell proliferation was associated with a decrease in the percentage of ALDH+ ovarian CSC. Using a novel single cell microfluidic culture system we found that EGFL6 acts specifically on ALDH+ CSC to promote proliferation via asymmetric division. We used a in vivo novel model of human tumor vasculature to express EGFL6 in vivo and demonstrate that EGFL6 significantly promotes ovarian tumor growth. Finally we generated an EGFL6 blocking antibody and found that anti-EGFL6 antibodies could restrict tumor growth and delay disease recurrences. Thus EGFL6 is an import vascular regulator of ovarian CSC and EGL6 blocking antibodies represent a potentially important ovarian CSC targeting therapeutic. Citation Format: Ronald J. Buckanovich, Shoumei Bai, Ning Deng, Patrick Ingram, Euisik Yoon. EGFL6 is a tumor vascular regulator of ovarian CSC asymmetric division and a novel therapeutic target. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3760. doi:10.1158/1538-7445.AM2013-3760

Notch4 is required for tumor onset and perfusion

BackgroundNotch4 is a member of the Notch family of receptors that is primarily expressed in the vascular endothelial cells. Genetic deletion of Notch4 does not result in an overt phenotype in mice, thus the function of Notch4 remains poorly understood.MethodsWe examined the requirement for Notch4 in the development of breast cancer vasculature. Orthotopic transplantation of mouse mammary tumor cells wild type for Notch4 into Notch4 deficient hosts enabled us to delineate the contribution of host Notch4 independent of its function in the tumor cell compartment.ResultsHere, we show that Notch4 expression is required for tumor onset and early tumor perfusion in a mouse model of breast cancer. We found that Notch4 expression is upregulated in mouse and human mammary tumor vasculature. Moreover, host Notch4 deficiency delayed the onset of MMTV-PyMT tumors, wild type for Notch4, after transplantation. Vessel perfusion was decreased in tumors established in Notch4-deficient hosts. Unlike in inhibition of Notch1 or Dll4, vessel density and branching in tumors developed in Notch4-deficient mice were unchanged. However, final tumor size was similar between tumors grown in wild type and Notch4 null hosts.ConclusionOur results suggest a novel role for Notch4 in the establishment of tumor colonies and vessel perfusion of transplanted mammary tumors.

Sox17 promotes tumor angiogenesis and destabilizes tumor vessels in mice

Little is known about the transcriptional regulation of tumor angiogenesis, and tumor ECs (tECs) remain poorly characterized. Here, we studied the expression pattern of the transcription factor Sox17 in the vasculature of murine and human tumors and investigated the function of Sox17 during tumor angiogenesis using Sox17 genetic mouse models. Sox17 was specifically expressed in tECs in a heterogeneous pattern; in particular, strong Sox17 expression distinguished tECs with high VEGFR2 expression. Whereas overexpression of Sox17 in tECs promoted tumor angiogenesis and vascular abnormalities, Sox17 deletion in tECs reduced tumor angiogenesis and normalized tumor vessels, inhibiting tumor growth. Tumor vessel normalization by Sox17 deletion was long lasting, improved anticancer drug delivery into tumors, and inhibited tumor metastasis. Sox17 promoted endothelial sprouting behavior and upregulated VEGFR2 expression in a cell-intrinsic manner. Moreover, Sox17 increased the percentage of tumor-associated CD11b+Gr-1+ myeloid cells within tumors. The vascular effects of Sox17 persisted throughout tumor growth. Interestingly, Sox17 expression specific to tECs was also observed in highly vascularized human glioblastoma samples. Our findings establish Sox17 as a key regulator of tumor angiogenesis and tumor progression.

In Vivo Evolution of Tumor-Derived Endothelial Cells

The growth of a malignant tumor beyond a certain, limited size requires that it first develop an independent blood supply. In addition to providing metabolic support, this neovasculature also allows tumor cells to access the systemic circulation, thus facilitating metastatic dissemination. The neovasculature may originate either from normal blood vessels in close physical proximity to the tumor and/or from the recruitment of bone marrow-derived endothelial cell (EC) precursors. Recent studies have shown that human tumor vasculature ECs may also arise directly from tumor cells themselves and that the two populations have highly similar or identical karyotypes. We now show that, during the course of serial in vivo passage, these tumor-derived ECs (TDECs) progressively acquire more pronounced EC-like properties. These include higher-level expression of EC-specific genes and proteins, a greater capacity for EC-like behavior in vitro, and a markedly enhanced propensity to incorporate into the tumor vasculature. In addition, both vessel density and size are significantly increased in neoplasms derived from mixtures of tumor cells and serially passaged TDECs. A comparison of early- and late-passage TDECs using whole-genome single nucleotide polymorphism profiling showed the latter cells to have apparently evolved by a process of clonal expansion of a population with a distinct pattern of interstitial chromosomal gains and losses affecting a relatively small number of genes. The majority of these have established roles in vascular development, tumor suppression or epithelial-mesenchymal transition. These studies provide direct evidence that TDECs have a strong evolutionary capacity as a result of their inherent genomic instability. Consequently such cells might be capable of escaping anti-angiogenic cancer therapies by generating resistant populations.

Real-Time Visualization and Quantitation of Vascular Permeability In Vivo: Implications for Drug Delivery

The leaky, heterogeneous vasculature of human tumors prevents the even distribution of systemic drugs within cancer tissues. However, techniques for studying vascular delivery systems in vivo often require complex mammalian models and time-consuming, surgical protocols. The developing chicken embryo is a well-established model for human cancer that is easily accessible for tumor imaging. To assess this model for the in vivo analysis of tumor permeability, human tumors were grown on the chorioallantoic membrane (CAM), a thin vascular membrane which overlays the growing chick embryo. The real-time movement of small fluorescent dextrans through the tumor vasculature and surrounding tissues were used to measure vascular leak within tumor xenografts. Dextran extravasation within tumor sites was selectively enhanced an interleukin-2 (IL-2) peptide fragment or vascular endothelial growth factor (VEGF). VEGF treatment increased vascular leak in the tumor core relative to surrounding normal tissue and increased doxorubicin uptake in human tumor xenografts. This new system easily visualizes vascular permeability changes in vivo and suggests that vascular permeability may be manipulated to improve chemotherapeutic targeting to tumors.

Identification and angiogenic role of the novel tumor endothelial marker CLEC14A

Tumor endothelial markers (TEMs) that are highly expressed in human tumor vasculature compared with vasculature in normal tissue hold clear therapeutic potential. We report that the C-type lectin CLEC14A is a novel TEM. Immunohistochemical and immunofluorescence staining of tissue arrays has shown that CLEC14A is strongly expressed in tumor vasculature when compared with vessels in normal tissue. CLEC14A overexpression in tumor vessels was seen in a wide range of solid tumor types. Functional studies showed that CLEC14A induces filopodia and facilitates endothelial migration, tube formation and vascular development in zebrafish that is, CLEC14A regulates pro-angiogenic phenotypes. CLEC14A antisera inhibited cell migration and tube formation, suggesting that anti-CLEC14A antibodies may have anti-angiogenic activity. Finally, in endothelial cultures, expression of CLEC14A increased at low shear stress, and we hypothesize that low shear stress due to poor blood flow in the disorganized tumor vasculature induces expression of CLEC14A on tumor vessels and pro-angiogenic phenotypes.

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.