Endothelial Cells Of Tumor Vessels Research Articles

Non-invasive monitoring of tumor-vessel infarction by retargeted truncated tissue factor tTF–NGR using multi-modal imaging

The fusion protein tTF-NGR consists of the extracellular domain of the thrombogenic human tissue factor (truncated tissue factor, tTF) and the peptide GNGRAHA (NGR), a ligand of the surface protein CD13 (aminopeptidase N), upregulated on endothelial cells of tumor vessels. tTF-NGR preferentially activates blood coagulation within tumor vasculature, resulting in tumor vessel infarction and subsequent tumor growth retardation/regression. The anti-vascular mechanism of the tTF-NGR therapy approach was verified by quantifying the reduced tumor blood-perfusion with contrast-enhanced ultrasound, the reduced relative tumor blood volume by ultrasmallsuperparamagnetic iron oxide-enhanced magnetic resonance imaging, and by in vivo-evaluation of hemorrhagic bleeding with fluorescent biomarkers (AngioSense(680)) in fluorescence reflectance imaging. The accumulation of tTF-NGR within the tumor was proven by visualizing the distribution of the iodine-123-labelled protein by single-photon emission computed tomography. Use of these multi-modal vascular and molecular imaging tools helped to assess the therapeutic effect even at real time and to detect non-responding tumors directly after the first tTF-NGR treatment. This emphasizes the importance of imaging within clinical studies with tTF-NGR. The imaging techniques as used here have applicability within a wider scope of therapeutic regimes interfering with tumor vasculature. Some even are useful to obtain predictive biosignals in personalized cancer treatment.

Abstract 2841: Widespread endothelial cell infection and tumor cell apoptosis after intravenous injection of oncolytic vaccinia virus JX-594 into RIP-Tag2 mice.

Abstract Replication-competent oncolytic viruses are being developed as a promising strategy for treating certain types of cancer. JX-594 is an oncolytic vaccinia virus that lacks thymidine kinase and expresses human granulocyte-macrophage colony stimulating factor (hGM-CSF). Replication of JX-594 is promoted by EGFR/Ras pathway signaling in cancer cells. In addition to having direct oncolytic effects on tumor cells and recruiting an immune response, JX-594 has been found to cause rapid reduction of tumor blood flow in preclinical models and in clinical trials. To understand the mechanism of this vascular effect, we injected mouse-adapted JX-594 vaccinia virus (mJX-594, on Western Reserve backbone and expressing hGM-CSF) intravenously into RIP-Tag2 transgenic mice and examined the pancreatic neuroendocrine tumors at 6 hours or 1, 2, or 5 days after injection. Dot-like vaccinia immunoreactivity was widespread in endothelial cells of tumor vessels at 6 hours. Scattered endothelial cells had intense staining. Vaccinia immunoreactivity in tumor blood vessels was still widespread at 1 day, but at 5 days, most tumor vessels were narrowed, some appeared fragmented, and tumor vascularity was reduced by 40%. Normalization of tumor vessels, as observed after inhibition of VEGF signaling, was not found at any time examined. Intratumoral hypoxia was evident as diffuse regions of pimonidazole staining at 6 hours. Pimonidazole staining increased from 1 to 5 days, and was especially strong in regions of vascular pruning. Extravascular vaccinia immunoreactivity was located in scattered cells at 1 day and increased with time. At 5 days, most tumors had large patches of vaccinia-positive cells. This staining was not present in the surrounding normal acinar pancreas. Apoptotic cells stained for activated caspase-3 were scattered throughout tumors at 6 hours. Most of these apoptotic cells were endothelial cells. Apoptotic tumor cells were sparse at 6 hours, but at 5 days apoptotic cells were 23-fold more numerous than at baseline and even more widespread than vaccinia immunoreactivity, and tumors tended to be smaller than corresponding controls. These findings are consistent with oncolytic virus mJX-594 causing rapid infection of tumor vascular endothelial cells in RIP-Tag2 mice. Endothelial cell infection is followed by regression of tumor blood vessels, exaggeration of intratumoral hypoxia, and oncolytic actions on tumor cells that lead to widespread tumor cell apoptosis. Citation Format: Barbara Sennino, Brian J. Schriver, Caroline J. Breitbach, Naomi De Silva, John C. Bell, David H. Kirn, Donald M. McDonald. Widespread endothelial cell infection and tumor cell apoptosis after intravenous injection of oncolytic vaccinia virus JX-594 into RIP-Tag2 mice. [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 2841. doi:10.1158/1538-7445.AM2013-2841

Building blood vessels in development and disease

This review will examine developmental angiogenesis and tumor-related changes to endothelial cells. Processes that govern developmental angiogenesis become dysfunctional in the tumor environment, leading to abnormal tumor endothelial cells and blood vessels. Recent findings suggest that tumor endothelial cells are permanently modified compared with normal counterparts. Coordination of numerous intracellular and extracellular programs promotes the formation of new blood vessels that are necessary for both development and certain diseases. Developmental angiogenesis uses canonical signaling modalities to effectively assemble endothelial cells into predictable vessel structures, and disruption of critical signaling factors has dramatic effects on blood vessel development. Solid tumors co-opt developmental cues to promote formation of tumor vessels that sustain their growth, but these angiogenic signals are not well regulated and produce endothelial cell dysfunction. Aberrant growth factor signaling contributes to phenotypic changes and acquired irreversible intracellular signaling, cytoskeletal and genetic modifications in endothelial cells of tumor vessels. Permanently altered tumor endothelial cells may represent a significant population.

Anticancer Therapy by Tumor Vessel Infarction with Polyethylene Glycol Conjugated Retargeted Tissue Factor

tTF-NGR consists of the extracellular domain of tissue factor and the peptide GNGRAHA, a ligand of the surface protein aminopeptidase N and of integrin αvβ3. Both surface proteins are upregulated on endothelial cells of tumor vessels. tTF-NGR shows antitumor activity in xenografts and inhibition of tumor blood flow in cancer patients. We performed random TMS(PEG)12 PEGylation of tTF-NGR to improve the antitumor profile of the molecule. PEGylation resulted in an approximately 2-log step decreased procoagulatory activity of the molecule. Pharmacokinetic studies in mice showed a more than 1-log step higher mean area under the curve. Comparison of the LD10 values for both compounds and their lowest effective antitumor dose against human tumor xenografts showed an improved therapeutic range (active/toxic dose in mg/kg body weight) of 1/5 mg/kg for tTF-NGR and 3/>160 mg/kg for TMS(PEG)12 tTF-NGR. Results demonstrate that PEGylation can significantly improve the therapeutic range of tTF-NGR.

Differences in the Angiogenesis of Benign and Malignant Surface Epithelial Ovarian Tumors Demonstrated by Microvessel Density and Immunohistochemistry

OBJECTIVES: Angiogenesis plays a key role in tumor growth and metastasis. The analysis of tumor vascularization by microvessel density (MVD) and its prognostic significance has been evaluated in many tumors including ovary. However, very few studies have tried to evaluate the characteristics of these vessels. This study aims to quantitatively assess the characteristics of tumor vessels with the aid of immunohistochemistry and thus, establish its role in difference in biological behavior of benign and malignant surface epithelial ovarian tumors. METHODS: We examined 42 cases of surface epithelial ovarian tumors and divided them in two groups (14 malignant and 28 benign tumors). Both study groups were compared for smooth muscle and endothelial cells in tumor vessels using monoclonal antibodies against smooth muscle actin (SMA) and CD34 (endothelial cell marker) respectively. MVD, SMA expression index and CD34 intensity index was calculated in both groups. RESULTS: The malignant surface epithelial ovarian tumors showed higher MVD (34.48±12.87) as compared to benign ovarian tumors (16.49±6.17) (p

NF-κB Activity Regulates Mesenchymal Stem Cell Accumulation at Tumor Sites

Mesenchymal stem cells (MSC) accumulate at tumor sites when injected into tumor-bearing mice, perhaps offering cellular vectors for cancer-targeted gene therapy. However, the molecular mechanisms involved in MSC targeting the tumors are presently little understood. We focused on MSC-endothelial cell (EC) adhesion following TNF-α stimulation in an attempt to elucidate these mechanisms. Interestingly, stimulation of MSCs with TNF-α enhanced the adhesion of MSCs to endothelial cells in vitro. This adhesion was partially inhibited by blocking antibodies against vascular cell adhesion molecule-1 (VCAM-1) and very late antigen-4 (VLA-4). It is well known that TNF-α induces VCAM-1 expression via the NF-κB signaling pathway. Parthenolide has an anti-inflammatory activity and suppressed NF-κB activity by inhibition of IκBα phosphorylation after TNF-α stimulation and strongly inhibited TNF-α-induced VCAM-1 expression on MSCs. In vivo imaging using luciferase-expressing MSCs revealed that the bioluminescent signal gradually increased at tumor sites in mice injected with untreated MSCs. In contrast, we observed very weak signals at tumor sites in mice injected with parthenolide-treated MSCs. Our results suggest that NF-κB activity regulates MSC accumulation at tumors, by inducing VCAM-1 and thereby its interaction with tumor vessel endothelial cells. These findings have implications for the ongoing development of efficient MSC-based gene therapies for cancer treatment.

Tissue factor regulates tumor angiogenesis of retinoblastoma via the extracellular signal-regulated kinase pathway

Retinoblastoma, a well-vascularized tumor that is dependent on a very robust angiogenic response, is the most common intraocular malignancy in children. Tissue factor (TF) is known to regulate tumor progression and in the present study we demonstrated that TF regulates tumor angiogenesis of retinoblastoma. In an orthotopic transplantation model of retinoblastoma, TF was selectively expressed in the proliferative area of retinoblastoma including tumor vessels as well as tumor cells, where TF expression was co-localized with endothelial cells of tumor vessels. TF expression progressively increased with fibroblast growth factor-2 (FGF-2)-induced proliferation of human umbilical vein endothelial cells (HUVECs), which was effectively inhibited by blockade of the TF pathway by TF pathway inhibitor (TFPI). In addition, FGF-2-induced angiogenic processes of migration and tube formation of vascular endothelial cells were also effectively suppressed by TFPI, which would be mediated by inhibition of extracellular signal-regulated kinase activation. Therefore, further to our previous report that TF is involved in tumor cell proliferation of retinoblastoma, our current data suggest that blockade of the TF pathway by TFPI could effectively inhibit tumor growth by suppressing tumor cell proliferation and tumor angiogenesis at the same time.

P3-17-06: Final Results of a Controlled, Randomized 3-Arm Phase II Trial of EndoTAG”-1, a Cationic Liposomal Formulation of Paclitaxel Targeting Tumor Endothelial Cells, in Advanced Triple-Negative Breast Cancer (TNBC).

Abstract Background EndoTAG™-1 (ET) is an innovative therapy of paclitaxel (P) embedded in cationic liposomes displaying antitumor activity by targeting negatively charged activated endothelial cells of tumor vessels. Methods: 140 patients (pts) with locally relapsed (r) or metastatic (m) centrally verified TNBC were randomized to i.v. weekly ET (22mg/m2 ) plus P (70mg/m2) (ET+P), biweekly ET alone (44mg/m2) or weekly P (90mg/m2) in a 2:2:1 ratio. Pts had ≤ 1 prior chemotherapy (CT) for r or m disease and ≥ 6 months (mo) after taxane. A cycle (cy) comprised 3 weeks(w) of therapy and 1 w rest. Pts were treated for a minimum of 4 cy or until disease progression/unacceptable toxicity. Primary endpoint was progression-free survival (PFS) rate at week 16 based on blinded central imaging and local assessment. Secondary endpoints included median overall survival (mOS), tumor response (RECIST), Quality of Life (QoL) and safety. The study was not powered for intergroup comparisons. Results: Pts baseline characteristics were overall well distributed. Appr. 80% of all pts were treated 1st line for r or m TNBC. Results of PFS rates at week 16 are shown in table. Based on central review, median PFS at week 16 was 4.2 mo for ET+P [95% CI: 3.5−9.1], 3.4 on ET [2.0−3.8] and 3.7 on P [1.9−6.7]. Antitumor activity was highest for ET+P with a clinical benefit rate (complete response (CR) + partial response (PR) + stable disease (SD) ≥6 mo at data cut off date of week 41) of 53% (26/49 evaluable pts), 31% (15/49) on ET and 36% (9/25) on P. The best overall response (CR, PR, SD at any time) on ET+P was 80% (39/49), on ET 65% (32/49) and 68% (17/25) on P. Analysis of median OS is shown in table. Analysis of the not predefined subgroup (centrally TNBC + ECOG 0/1 + first line population) showed an impressive median OS of 17.8 mo for ET+P. No differences were reported for QoL. No additional toxicities to those known for ET and P were observed except uncomplicated grade 3/4 neutropenia in the ET + P arm. Conclusions: At cut-off date week 41, the final analysis demonstrated ET+P as a promising combination compared to single agent arms. ET+P was well tolerated. These data will be the basis for a confirmatory phase III study. Acknowlegments: B. Glasschroeder, U. Elsasser Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-17-06.

Abstract LB-56: LGR5 is differentially expressed in hepato-gastrointestinal tumors

Abstract Background: The orphan G-protein-coupled receptor (GPCR) and Wnt-target protein LGR5 was recently identified as a stem cell marker for cells with intestinal differentiation. In this study we generated a polyclonal anti-LGR5 antibody to investigate protein expression in various hepato-gastrointestinal carcinomas and its correlation with clinico-pathological patient characteristics. Materials and Methods: Differential expression of LGR5 was studied on transcriptional (real time-polymerase chain reaction) and translational level (immunohistochemistry) in carcinomas and corresponding normal mucosal specimens comprising seven different primary tumor sites, i.e. oesophagus, pancreas, stomach, liver, colon and rectum. The putative clinico-pathological relevance of LGR5-expression in terms of patient survival was studied in tissue micro arrays obtained from a cohort of 488 gastric carcinomas. Results: We succeeded to establish and characterize a highly specific antibody that recognizes the C-terminal tail of LGR5. LGR5 was differentially expressed on transcriptional and translational level in adenocarcinomas of the oesophagus, pancreas, stomach, colon, rectum, hepatocellular and cholangiocellular carcinoma of the liver compared with the adjacent non-neoplastic tissue. However, in gastric cancer tissue microarrays the expression of LGR5 had no impact on patient survival. Nevertheless, expression of LGR5 by endothelial cells of tumor vessels correlated significantly with the tumor type and tumor grade. Conclusion: Our results substantiate the significance of LGR5 on the biology of hepato-gastrointestinal carcinomas and provide evidence for its function as potential stem cell marker and candidate therapeutic target in the stomach. With our anti-LGR5 antibody we now have a useful tool for detection and further analyzes of LGR5 biological function. 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 LB-56. doi:10.1158/1538-7445.AM2011-LB-56

Bone Marrow-Derived Endothelial Progenitors Expressing Delta-Like 4 (Dll4) Regulate Tumor Angiogenesis

Neo-blood vessel growth (angiogenesis), which may involve the activation of pre-existing endothelial cells (EC) and/or the recruitment of bone marrow-derived vascular precursor cells (BM-VPC), is essential for tumor growth. Molecularly, besides the well established roles for Vascular endothelial growth factor (VEGF), recent findings show the Notch signalling pathway, in particular the ligand Delta-like 4 (Dll4), is also essential for adequate tumor angiogenesis; Dll4 inhibition results in impaired, non-functional, angiogenesis and reduced tumor growth. However, the role of BM-VPC in the setting of Notch pathway modulation was not addressed and is the subject of the present report. Here we show that SDF-1 and VEGF, which are produced by tumors, increase Dll4 expression on recruited BM-VPC. Mechanistically, BM-VPC activated, in a Dll4-dependent manner, a transcriptional program on mature EC suggestive of EC activation and stabilization. BM-VPC induced ICAM-2 and Fibronectin expression on EC, an effect that was blocked by a Dll4-specific neutralizing antibody. In vivo, transplantation of BM-VPC with decreased Dll4 into tumor-bearing mice resulted in the formation of microvessels with decreased pericyte coverage and reduced fibronectin expression. Consequently, transplantation of BM-VPC with decreased Dll4 resulted in impaired tumor angiogenesis, increased tumor hypoxia and apoptosis, and decreased tumor growth. Taken together, our data suggests that Dll4 expression by BM-VPC affects their communication with tumor vessel endothelial cells, thereby modulating tumor angiogenesis by affecting vascular stability.

Vascular infarction by subcutaneous application of tissue factor targeted to tumor vessels with NGR-peptides: Activity and toxicity profile

tTF-NGR consists of the extracellular domain of the (truncated) tissue factor (tTF), a central molecule for coagulation in vivo, and the peptide GNGRAHA (NGR), a ligand of the surface protein aminopeptidase N (CD13). After deamidation of the NGR-peptide moiety, the fusion protein is also a ligand for integrin αvβ3 (CD51/CD61). Both surface proteins are upregulated on endothelial cells of tumor vessels. tTF-NGR showed binding to specific binding sites on endothelial cells in vitro as shown by flow cytometry. Subcutaneous injection of tTF-NGR into athymic mice bearing human HT1080 fibrosarcoma tumors induced tumor growth retardation and delay. Contrast enhanced ultrasound detected a decrease in tumor blood flow in vivo after application of tTF-NGR. Histological analysis of the tumors revealed vascular disruption due to blood pooling and thrombotic occlusion of tumor vessels. Furthermore, a lack of resistance was shown by re-exposure of tumor-bearing mice to tTF-NGR after regrowth following a first cycle of treatment. However, after subcutaneous (s.c.) push injection with therapeutic doses (1-5 mg/kg bw) side effects have been observed, such as skin bleeding and reduced performance. Since lethality started within the therapeutic dose range (LD10 approximately 2 mg/kg bw) no safe therapeutic window could be found. Limiting toxicity was represented by thrombo-embolic events in major organ systems as demonstrated by histology. Thus, subcutaneous injection of tTF-NGR represents an active, but toxic application procedure and compares unfavourably to intravenous infusion.

Abstract 391: Both NGR and TNF portions of NGR-TNF contribute to tumor vessel specific homing and apoptosis in vivo

Abstract NGR-TNF is a novel peptide-targeted agent coupling CNGRCG peptide (NGR), homing to angiogenic blood vessels, and tumor necrosis factor alpha (TNF), presently in clinical development (phase II clinical studies in MPM, HCC and CRC). To further elucidate its mechanism of action, we investigated the NGR-TNF homing and downstream effects obtained with doses comparable to those used in clinical trials. Targeting studies carried-out using NGR-quantum dots (-Qd) administered in vivo demonstrate that the binding to CD13 occurs only in neo-angiogenic tissues, including tumor vessels, sparing CD13-expressing normal tissues. The specificity of this binding is due to the expression of an NGR-binding complex containing CD13. Indeed, in vitro RNA-interfering studies with CD13-specific shRNA demonstrate that the expression of CD13 on the target cells is essential but not sufficient for NGR-binding capability. Herein, for the first time, we formally demonstrated the physical interaction between NGR and CD13 on target cell surface by pool-down experiments using NGR-biotinylinated peptides. Although the molecular characterization of determinants responsible for NGR-binding capability is still ongoing, microvesicles released by NGR-binding cells are able to transfer the NGR-binding capability to NGR-non-binder cells, suggesting that the binding complex associates on the target cell before NGR engages CD13. In addition, the in vivo activity of NGR-TNF may be due to the NGR-mediated homing to target cells determining an increase in the affinity for TNF receptor (TNFR), and/or to an NGR-mediated cell signalling via CD13. Our data support the contribution of both mechanisms. First, using a recombinant tagged-human TNF (hTNF-Fc) as a probe along with NGR-TNF and TNF as competitors, NGR-TNF has an NGR-mediated higher affinity for TNFR than TNF. Second, in TNF-activated endothelial cells, NGR-TNF impairs survival signals via the significant inhibition of Mek, Erk1/2, and Akt, and activates apoptotic signals (caspases 3, 8 and 9) that induce apoptosis of tumor vessel endothelial cells in vivo. Of note, these pathways are similarly activated also using NGR and TNF as separate molecules engaging CD13 and TNFR, respectively, indicating that both NGR and TNF portions contribute to the activation of signaling pathways by NGR-TNF. In conclusion, the binding of NGR to CD13 determines not only the “mere homing” of NGR-TNF but also a) the stabilization of the NGR-CD13/TNF-TNFR interaction and b) a direct effect of CD13, leading to a highly selective homing on tumor blood vessels and apoptosis of angiogenic endothelial cells in vivo, thus significantly contributing to the control of tumor growth. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 391.

Autocrine effects of VEGF-D on endothelial cells after transduction with AD-VEGF-D ΔNΔC

Endothelial cells in tumor vessels display unusual characteristics in terms of survival and angiogenic properties which result from the increased expression of VEGF-D and its autocrine effect. To evaluate mechanisms by which VEGF-D leads to such abnormal phenotype, we searched for proteins with modified expression in HUVECs enriched in the recombinant mature VEGF-D (VEGFD ΔNΔC) delivered by adenovirus. Expression of membrane proteins in endothelial cells was characterized by FACS using anti-human IT-Box-135 antibodies. HUVECs transduced with Ad-VEGF-D ΔNΔC revealed markedly increased expression of proteins involved in adhesion and migration such as (a) integrins (αVβ5, α2β1, α5β1, αMβ2, αLβ2), (b) matrix metalloproteinases (MMP-2, MMP-9, and MMP-14), (c) components of fibrinolytic system (PAI-1, u-PAR), and (d) CD45, CD98, CD147. Interestingly, there also were numerous proteins with significantly reduced expression, particularly among surface exposed membrane proteins. Thus, it can be concluded that to induce proangiogenic phenotype and facilitate migration of HUVECs, VEGF-D ΔNΔC not only upregulates expression of proteins known to participate in the cell-matrix interactions but also silences some membrane proteins which could interfere with this process.

IL-12 Suppresses Vascular Endothelial Growth Factor Receptor 3 Expression on Tumor Vessels by Two Distinct IFN-γ–Dependent Mechanisms

IL-12 has been shown to be effective in enhancing antitumor responses. However, how IL-12 exerts its antiangiogenic effect is largely unknown. In this study, we elucidate this mechanism using B16 transfected to express IL-12 (B16/IL-12), a system that provides constant, local production of IL-12 within the tumor microenvironment. Intratumoral IL-12 resulted in a significant delay in tumor growth and phenotypic changes in the vasculature. Vessels found within B16 tumors are chaotic and poorly formed and express vascular endothelial growth factor receptor 3 (VEGFR3), a growth factor receptor not expressed on normal adult vessels. However, the vessels within B16/IL-12 tumors have a more normal morphology and do not express VEGFR3. We have shown that IFN-gamma is required for IL-12 to suppress the aberrant expression of VEGFR3. Indeed, the presence of intratumoral IL-12 stimulates the immune system resulting in more IFN-gamma-producing tumor-infiltrating lymphocytes per tumor when compared with parental B16 tumors, which may have a marked effect on control of tumor growth. Interestingly, within B16/IL-12 tumors, T cells are necessary to suppress VEGFR3 expression on tumor vessels. Finally, using IFN-gamma receptor knockout mice in a bone marrow chimera system, we show that the IFN-gamma produced within the tumor suppresses VEGFR3 expression in two ways: 1) acting directly on tumor vessel endothelial cells, and 2) acting on the tumor-infiltrating lymphocytes to indirectly alter endothelial cells' VEGFR3 expression. Our data indicate a mechanism in which tumor-infiltrating immune cells regulate tumor vessel phenotype.

Inhibiting Tumor Growth by Targeting Tumor Vasculature with Galectin-1 Antagonist Anginex Conjugated to the Cytotoxic Acylfulvene, 6-Hydroxylpropylacylfulvene

Targeted delivery of therapeutic drugs promises to become the norm to treat cancer. Here, we conjugated the cytotoxic agent 6-hydroxypropylacylfulvene (HPAF) to anginex, a peptide that targets galectin-1, which is highly expressed in endothelial cells of tumor vessels. In a human ovarian cancer model in mice, the conjugate inhibited tumor growth better than equivalent doses of either compound alone. Immunofluorescence on tumor tissue demonstrated that the conjugate, like parent anginex, selectively targeted tumor vasculature and inhibited tumor angiogenesis. Increased activity from the conjugate further suggests that HPAF retains at least some of its normal cytotoxic activity when linked to anginex. More importantly perhaps is the observation that the conjugate abrogates apparent systemic toxicity from treatment with HPAF. This work contributes to the development of tumor vascular targeting agents against cancer in the clinic.

Potential role of vascular targeted therapy to combat against tumor

Tumors, like other tissues, have a fundamental requirement for access to the nutrients, oxygen and waste removal functions of the circulatory system. Vascular targeted therapy exploits this basic need, along with molecular heterogeneity observed between normal and tumor blood vessels, to develop efficient and selective chemotherapies that essentially starve tumors by destroying their vasculature. As the basic principle on which this therapy is based differs from agents that directly target cancerous cells, combining it with traditional therapies such as radiation, surgery and existing chemotherapies has the potential to create powerful new anticancer strategies. As the requirement for vascularization is universal to solid tumors, vascular targeted therapies have the potential for broad applicability. Vascular targeted therapies include both angiogenesis inhibitors, which inhibit neovascularization, and vascular disrupting agents, which destroy existing vasculature. Applications of this model include finding peptides that bind specifically to cell surface markers on tumor vessel endothelial cells and might deliver chemotherapeutic agents. Expression profiling with microarrays, serial analysis of gene expression, and in vitro and in vivo screening of phage display libraries have identified candidate peptides for targeted delivery to the tumor endothelium.

αB-crystallin promotes tumor angiogenesis by increasing vascular survival during tube morphogenesis

Selective targeting of endothelial cells in tumor vessels requires delineation of key molecular events in formation and survival of blood vessels within the tumor microenvironment. To this end, proteins transiently up-regulated during vessel morphogenesis were screened for their potential as targets in antiangiogenic tumor therapy. The molecular chaperone alphaB-crystallin was identified as specifically induced with regard to expression level, modification by serine phosphorylation, and subcellular localization during tubular morphogenesis of endothelial cells. Small interfering RNA-mediated knockdown of alphaB-crystallin expression did not affect endothelial proliferation but led to attenuated tubular morphogenesis, early activation of proapoptotic caspase-3, and increased apoptosis. alphaB-crystallin was expressed in a subset of human tumor vessels but not in normal capillaries. Tumors grown in alphaB-crystallin(-/-) mice were significantly less vascularized than wild-type tumors and displayed increased areas of apoptosis/necrosis. Importantly, tumor vessels in alphaB-crystallin(-/-) mice were leaky and showed signs of caspase-3 activation and extensive apoptosis. Ultrastructural analyses showed defective vessels partially devoid of endothelial lining. These data strongly implicate alphaB-crystallin as an important regulator of tubular morphogenesis and survival of endothelial cell during tumor angiogenesis. Hereby we identify the small heat shock protein family as a novel class of angiogenic modulators.

Alterations in vascular architecture and permeability following OXi4503 treatment

OXi4503 retards tumor growth in a dose-dependent manner and improves survival in a murine model of colorectal liver metastases. This agent causes extensive vascular shutdown by selectively altering the tubulin cytoskeleton within the endothelial cells of tumor vessels. The destruction of tumor vessels is incomplete, however, and tumor revascularization occurs after the treatment. This study evaluates the pattern of microcirculatory changes and alterations to the ultrastructural properties of the tumor vasculature that result from OXi4503 treatment. Male CBA mice were induced with liver metastases via an intrasplenic injection of a murine-derived colorectal cell line. After administering a single intraperitoneal dose of OXi4503, changes in tumor perfusion, microvascular architecture and permeability were assessed at various time points. One hour after a 100-mg/kg dose of OXi4503, a significant decrease in the percentage of tumor perfusion (63.96+/-1.98 in controls versus 43.77+/-2.71 in treated mice, P<0.001) was observed, which was still evident 5 days after the treatment. Substantial tumor microvascular damage and minimal normal liver injury were observed. Tumor vascular permeability was significantly elevated 45 min after the OXi4503 treatment (67.5+/-3.60 in controls versus 80.5+/-2.24 microg/g, P<0.05). The findings suggest that OXi4503 selectively targets tumor vessels and causes immediate microvascular destruction. Even at the maximum tolerated dose, however, residual patent tumor vessels were still present after treatment, implying incomplete tumor destruction. A combination of OXi4503 with other chemotherapeutic modalities might achieve complete tumor eradication and improve long-term survival.

Cellular actions of angiogenesis inhibitors on blood vessels

Cellular actions of angiogenesis inhibitors on blood vessels

Robo1/Robo4: Differential expression of angiogenic markers in colorectal cancer

The family of roundabout (Robo) proteins is related to the transmembrane receptors and plays a major role in the process of axonal guidance in neurogenesis. It has recently been shown that Robo proteins are also associated with tumor angiogenesis with Slit2 acting as the corresponding ligand. The aim of this study was to validate the differential expression by means of microarray analysis and real-time PCR and to analyze the in situ expression of Robo1 and Robo4 in colorectal cancer. Quantitative analyses of Robo1, Robo4 and Slit2 mRNA expression measured by large scale gene expression studies (Affymetrix U133A) showed a significant up-regulation of Robo1 in tumor vs. normal tissue, whereas Robo4 and Slit2 showed no significant deregulation. For subsequent real-time PCR experiments, paired colorectal tissue samples from cancerous and corresponding non-cancerous tissues were obtained from 50 colorectal cancer patients who underwent surgical resection. Robo1 mRNA overexpression in cancerous tissue compared with normal counterparts was observed in 80% of the patients with a 4-fold expression in 45% and a 12-fold expression in 15%. For Robo4, an up-regulation was detected in >70% (36/50). For Slit2, no differential expression was observed. The overexpression of Robo1 and Robo4 in tumor vs. normal tissue was verified using real-time PCR. The histological analysis revealed an expression of Robo1 mainly in tumor cells, whereas Robo4 is located primarily in endothelial cells of tumor vessels. Therefore, the Robo proteins provide potential target structures for the anti-tumorigenic and anti-angiogenic therapy of colorectal carcinoma.