Vascular Targeting Approaches Research Articles

Targeting the tumour vasculature: from vessel destruction to promotion.

As angiogenesis was recognized as a core hallmark of cancer growth and survival, several strategies have been implemented to target the tumour vasculature. Yet to date, attempts have rarely been so diverse, ranging from vessel growth inhibition and destruction to vessel normalization, reprogramming and vessel growth promotion. Some of these strategies, combined with standard of care, have translated into improved cancer therapies, but their successes are constrained to certain cancer types. This Review provides an overview of these vascular targeting approaches and puts them into context based on our subsequent improved understanding of the tumour vasculature as an integral part of the tumour microenvironment with which it is functionally interlinked. This new knowledge has already led to dual targeting of the vascular and immune cell compartments and sets the scene for future investigations of possible alternative approaches that consider the vascular link with other tumour microenvironment components for improved cancer therapy.

Recombinant Annexin A2 Administration Improves Neurological Outcomes After Traumatic Brain Injury in Mice.

Microvascular failure is one of the key pathogenic factors in the dynamic pathological evolution after traumatic brain injury (TBI). Our laboratory and others previously reported that Annexin A2 functions in blood-brain barrier (BBB) development and cerebral angiogenesis, and recombinant human Annexin A2 (rA2) protected against hypoxia plus IL-1β-induced cerebral trans-endothelial permeability in vitro, and cerebral angiogenesis impairment of AXNA2 knock-out mice in vivo. We thereby hypothesized that ANXA2 might be a cerebrovascular therapy candidate that targets early BBB integrity disruption, and subacute/delayed cerebrovascular remodeling after TBI, ultimately improve neurological outcomes. In a controlled cortex impact (CCI) mice model, we found rA2 treatment (1 mg/kg) significantly reduced early BBB disruption at 24 h after TBI; and rA2 daily treatment for 7 days augmented TBI-induced mRNA levels of pro-angiogenic and endothelial-derived trophic factors in cerebral microvessels. In cultured human brain microvascular endothelial cells (HBMEC), through MAPKs array, we identified that rA2 significantly activated Akt, ERK, and CREB, and the activated CREB might be responsible for the rA2-induced VEGF and BDNF expression. Moreover, rA2 administration significantly increased cerebral angiogenesis examined at 14 days and vessel density at 28 days after TBI in mice. Consistently, our results validated that rA2 significantly induced angiogenesis in vitro, evidenced by tube formation and scratched migration assays in HBMEC. Lastly, we demonstrated that rA2 improved long-term sensorimotor and cognitive function, and reduced brain tissue loss at 28 days after TBI. Our findings suggest that rA2 might be a novel vascular targeting approach for treating TBI.

The Evolution of Safe and Effective Coaguligands for Vascular Targeting and Precision Thrombosis of Solid Tumors and Vascular Malformations.

In cardiovascular and cerebrovascular biology, control of thrombosis and the coagulation cascade in ischemic stroke, myocardial infarction, and other coagulopathies is the focus of significant research around the world. Ischemic stroke remains one of the largest causes of death and disability in developed countries. Preventing thrombosis and protecting vessel patency is the primary goal. However, utilization of the body’s natural coagulation cascades as an approach for targeted destruction of abnormal, disease-associated vessels and tissues has been increasing over the last 30 years. This vascular targeting approach, often termed “vascular infarction”, describes the deliberate, targeted delivery of a thrombogenic effector to diseased blood vessels with the aim to induce localized activation of the coagulation cascade and stable thrombus formation, leading to vessel occlusion and ablation. As systemic delivery of pro-thrombotic agents may cause consternation amongst traditional stroke researchers, proponents of the approach must suitably establish both efficacy and safety to take this field forward. In this review, we describe the evolution of this field and, with a focus on thrombogenic effectors, summarize the current literature with respect to emerging trends in “coaguligand” development, in targeted tumor vessel destruction, and in expansion of the approach to the treatment of brain vascular malformations.

Vascular-immuno-phenotypic (VIP) model for locally advanced and oligo-metastatic cancer: A hypothesis

Primary Hypothesis: In cancer therapy, normalization of the vasculature, and not disruption, to facilitate the reversal of the immuno-phenotypic changes, is the sine-qua-non for cancer elimination. The triad of normalization of the vasculature, leading to the improved immunological tumour microenvironment and increased susceptibility of resistant phenotypic cancer cells (VIP model), forms the basis of this hypothesis. This article hypothesizes the absolute need for vascular normalization for the eradication of cancer. Locally advanced and oligometastatic cancers have the potential to be cured with aggressive therapy. The focus on vascular normalization its clinical relevance in this situation is essential. Most traditional approaches have focused on the elimination of cancer by targeting and disrupting vasculature. Initially, antiangiogenic drugs showed significant promise in animal experiments. However, this vascular disruption approach has not paid the expected long-term dividends in the clinical setup. However, antiangiogenics are playing a significant role when used concurrently with chemotherapy/immunotherapy. Antiangiogenics have dual temporal actions - an initial normalization effect with improved oxygenation followed by pruning of blood vessels, resulting in exaggerated hypoxia along with a rebound progression. The literature is replete with phenomena of initial vascular normalization with a paradigm shift in the immuno-phenotypic milieu of cancer as part of vascular targeting approaches. The hypothesis in this article stresses the need to have strategies to extend this normalization window or to have pre-clinical trials to optimize the dose scheduling of antiangiogenics cyclically along with chemo/targeted/immune therapy and other combination therapies. We can implement this hypothesis by a combinatorial harmonization of present-day cancer therapies in the setting of tumor vasculature integrity. In addition, based on the proposed hypothesis, the current normalization effect of antiangiogenics and newer therapy development should focus primarily on normalization of the vasculature as well as targeting hypoxia-Inducible-factor-1 alpha (HIF-1 α) in the presence of differential genetic modulation of vascular endothelial cell resistance enhancement along with cancer cell sensitization. Also, the article enumerates six supporting hypotheses supplementing the primary hypothesis.

Occlusion of Animal Model Arteriovenous Malformations Using Vascular Targeting.

Brain arteriovenous malformations (AVMs) are a significant cause of intracerebral hemorrhage in children and young adults. Currently, one third of patients have no viable treatment options. Vascular targeting agents (VTAs) are being designed to deliver pro-thrombotic molecules to the abnormal AVM vessels for rapid occlusion and cure. This study assessed the efficacy of a pro-thrombotic VTA targeting phosphatidylserine (PS) in a radiation-primed AVM animal model. The model AVM was surgically created in rats by anastomosis of the left external jugular vein to the adjacent common carotid artery. After 6weeks, the AVM was irradiated (20Gy) using gamma knife surgery (GKS). A PS-targeting VTA was created by conjugation of annexin V with human thrombin and administered intravenously 3weeks post-GKS or sham. Unconjugated thrombin was used as a non-targeting control. AVM thrombosis and occlusion was monitored 3weeks later by angiography and histology. Preliminary experiments established a safe dose of active thrombin for systemic administration. Subsequently, a single dose of annexin V-thrombin conjugate (0.77mg/kg) resulted in angiographic AVM occlusion in sham (75%) and irradiated (63%) animals, while non-targeted thrombin did not. Lowering the conjugate dose (0.38mg/kg) decreased angiographic AVM occlusion in sham (13%) relative to irradiated (80%) animals (p= 0.03) as did delivery of two consecutive doses of 0.38mg/kg, 2days apart (sham (0%); irradiated (78%); p= 0.003). These findings demonstrate efficacy of the PS-targeting VTA and the feasibility of a vascular targeting approach for occlusion of high-flow AVMs. Targeting specificity can be enhanced by radiation-sensitization and VTA dose modification.

Overcoming Biological Barriers in Neuroblastoma Therapy: The Vascular Targeting Approach with Liposomal Drug Nanocarriers.

Neuroblastoma is a rare pediatric cancer characterized by a wide clinical behavior and adverse outcome despite aggressive therapies. New approaches based on targeted drug delivery may improve efficacy and decrease toxicity of cancer therapy. Furthermore, nanotechnology offers additional potential developments for cancer imaging, diagnosis, and treatment. Following these lines, in the past years, innovative therapies based on the use of liposomes loaded with anticancer agents and functionalized with peptides capable of recognizing neuroblastoma cells and/or tumor-associated endothelial cells have been developed. Studies performed in experimental orthotopic models of human neuroblastoma have shown that targeted nanocarriers can be exploited for not only decreasing the systemic toxicity of the encapsulated anticancer drugs, but also increasing their tumor homing properties, enhancing tumor vascular permeability and perfusion (and, consequently, drug penetration), inducing tumor apoptosis, inhibiting angiogenesis, and reducing tumor glucose consumption. Furthermore, peptide-tagged liposomal formulations are proved to be more efficacious in inhibiting tumor growth and metastatic spreading of neuroblastoma than nontargeted liposomes. These findings, herein reviewed, pave the way for the design of novel targeted liposomal nanocarriers useful for multitargeting treatment of neuroblastoma.

Phosphatidylserine Translocation after Radiosurgery in an Animal Model of Arteriovenous Malformation.

Phosphatidylserine (PS) is asymmetrically distributed across the plasma membrane, located predominantly on the inner leaflet in healthy cells. Translocation of PS to the outer leaflet makes it available as a target for biological therapies. We examined PS translocation after radiosurgery in an animal model of brain arteriovenous malformation (AVM). An arteriovenous fistula was created by end-to-side anastomosis of the left external jugular vein to the common carotid artery in 6-week-old, male Sprague Dawley rats. Six weeks after AVM creation, 15 rats underwent Gamma Knife stereotactic radiosurgery receiving a single 15 Gy dose to the margin of the fistula; 15 rats received sham treatment. Externalization of PS was examined by intravenous injection of a PS-specific near-infrared probe, PSVue-794, and in vivo fluorescence optical imaging at 1, 7, 21, 42, 63 and 84 days postirradiation. Fluorescent signaling indicative of PS translocation to the luminal cell surface accumulated in the AVM region, in both irradiated and nonirradiated animals, at all time points. Fluorescence was localized specifically to the AVM region and was not present in any other anatomical sites. Translocated PS increased over time in irradiated rats (P < 0.001) but not in sham-irradiated rats and this difference reached statistical significance at day 84 (P < 0.05). In summary, vessels within the mature rat AVM demonstrate elevated PS externalization compared to normal vessels. A single dose of ionizing radiation can increase PS externalization in a time-dependent manner. Strict localization of PS externalization within the AVM region suggests that stereotactic radiosurgery can serve as an effective priming agent and PS may be a suitable candidate for vascular-targeting approaches to AVM treatment.

Tumor vascular infarction: prospects and challenges.

Induction of thrombosis in tumor vasculature represents an appealing strategy for combating cancer. Formation of fibrin clots may be sufficient to occlude the blood vessels that feed tumor cells, contributing to massive ischemia, vascular infarction, and the subsequent necrosis and apoptosis of neoplastic cells. This approach called as tumor vascular infarction was pioneered by Huang et al. (Science 275:547-550, 1997). Since then, different vascular targeting moieties were linked to a truncated form of human tissue factor (tTF), to generate coaguligands with selective thrombotic activities on tumor neovasculature. In contrast to the wide clinical application of angiogenesis inhibitors and tumor vascular disrupting agents, tTF-NGR is the only example of clinically tested coaguligands. Notably, among these three tumor vascular targeting approaches, tumor vascular infarction is the only modality manifesting long-term curative potential in mice. Translation of this worthy approach has been limited, as induction of thrombosis by TF fusion proteins is leaky. In this review, we describe the clinical significance of tumor vascular infarction, highlight its advantages and disadvantages, and propose a novel strategy for expediting its translation to clinical settings.

The pivotal role of reactivity in the design of novel biotinylation reagents for the chemical-proteomics-based identification of vascular accessible biomarkers

A promising approach for the development of novel therapeutics with fewer side effects in healthy tissues is the targeted delivery of bioactive molecules directly to the site of disease. Thus, one prerequisite is the identification of a robust, disease-specific, vascular accessible biomarker localized on the surface of diseased cells, in the surrounding extracellular matrix or on newly formed blood vessels. One avenue towards the identification of such biomarkers consists in the enrichment of the vascular accessible surface proteome fraction prior to analysis. This can be achieved by covalent modification of the target proteins with membrane-impermeable ester derivatives of biotin, followed by streptavidin-based affinity capturing. The properties of the respective reagents are determined by the linker between the biotin moiety and the reactive group for protein coupling. In the frame of this study, novel, reactivity-improved peptide-based biotinylation reagents as well as reagents based on highly hydrophilic heparin linkers were synthesized and validated. The comprehensive evaluation of different biotinylation reagent classes with aliphatic, PEGylated, peptide-based and heparin-based linkers on single model protein BSA, HeLa cells as well as perfused kidney tissue revealed that the linker-dependent chemical reactivity is the crucial factor for the design of novel biotinylation reagents for vascular targeting approaches. SignificanceTo obtain a reliable identification and stable quantification of vascular accessible protein targets by means of mass spectrometry, covalent modification with a membrane-impermeable ester derivative of biotin, followed by streptavidin-based affinity capturing, is frequently applied for in vivo or ex vivo biomarker identification studies. Nevertheless, no comprehensive evaluation of different biotinylation reagent classes has been performed so far. Within this study, we systematically evaluated novel peptide- and heparin-based biotinylation reagents as well as established compounds based on aliphatic and PEGylated linkers. We identified the linker-dependant chemical reactivity of biotinylation reagents to be the critical factor for the design of novel reagents with high efficiency. The novel, site-specifically activated peptide-based reagents were found to be efficient compounds for application in mass spectrometry-based discovery of novel vascular-accessible biomarkers.

The Champalimaud Vision Award: honouring basic and translational research

The Champalimaud Vision Award: honouring basic and translational research

Photoactivation of lysosomally sequestered sunitinib after angiostatic treatment causes vascular occlusion and enhances tumor growth inhibition.

The angiogenesis inhibitor sunitinib is a tyrosine kinase inhibitor that acts mainly on the VEGF and PDGF pathways. We have previously shown that sunitinib is sequestered in the lysosomes of exposed tumor and endothelial cells. This phenomenon is part of the drug-induced resistance observed in the clinic. Here, we demonstrate that when exposed to light, sequestered sunitinib causes immediate destruction of the lysosomes, resulting in the release of sunitinib and cell death. We hypothesized that this photoactivation of sunitinib could be used as a vaso-occlusive vascular-targeting approach to treating cancer. Spectral properties of sunitinib and its lysosomal accumulation were measured in vitro. The human A2780 ovarian carcinoma transplanted onto the chicken chorioallantoic membrane (CAM) and the Colo-26 colorectal carcinoma model in Balb/c mice were used to test the effects of administrating sunitinib and subsequently exposing tumor tissue to light. Tumors were subsequently resected and subject to immunohistochemical analysis. In A2780 ovarian carcinoma tumors, treatment with sunitinib+light resulted in immediate specific angio-occlusion, leading to a necrotic tumor mass 24 h after treatment. Tumor growth was inhibited by 70% as compared with the control group (**P<0.0001). Similar observations were made in the Colo-26 colorectal carcinoma, where light exposure of the sunitinib-treated mice inhibited tumor growth by 50% as compared with the control and by 25% as compared with sunitinib-only-treated tumors (N≥4; P=0.0002). Histology revealed that photoactivation of sunitinib resulted in a change in tumor vessel architecture. The current results suggest that the spectral properties of sunitinib can be exploited for application against certain cancer indications.

A34 Session 10: Adjunctive Therapeutic Approaches: Vascular targeting approaches to enhance thermal therapy applications

The physiological reaction of tumor tissue and the progression of normal vascular and cellular repair processes in the body after thermal ablation suggest specific ways that adjuvant therapies may improve tumor control. This is a topic of growing importance as thermal ablation techniques are being used more than ever yet continue to have significant and unpredictable recurrence rates in clinical settings. We hypothesized that incomplete ablation of tumor tissue near the margin and around major blood vessels allows re-oxygenation and repopulation of the tumor. Microvessel functionality after partial thermal ablation of experimental murine tumors has been investigated after both high and low temperature thermal ablations. Interestingly, the oxygenation in the tumor margin increased from 1.2 ± 0.98 mmHg to 10.4 ± 6.3 mmHg during and for the first 20–30 min after a typical hot or cold ablation session, suggesting that the vascular response is a common feature to all of ablative medicine. Vessels with active blood flow in the peripheral regions of the tumor were seen to persist at varying levels from 24–72 h post-ablation. The level of hypoxia detected by pimonidazole binding was inversely related to the number of functional vessels detected. Our results suggest that simultaneous or tightly sequenced ablation and ionizing radiation therapy may have merit as an efficient means to completely sterilize tumor volume since radiation is most cytotoxic in areas of improved oxygenation and ablation most effectively debulks volumes with low or impaired perfusion. Furthermore, nanomedicine or other vascular-targeted drugs that can selectively impair the vasculature of the tumor may significantly increase the volume of complete ablation obtained and improve reproducibility of the ablation zone from tumor to tumor. A vascular sensitizing/damaging agent may also increase the overall effect of radiotherapy and thus improve tumor control. We have also documented a contribution of progenitor cells in repairing sublethally damaged tissue after ablation and promoting establishment of a vascular niche capable of regenerating the tumor mass which suggests agents that block the mobilization of bone marrow derived cells could also serve to improve the results of thermal ablative therapy for a variety of tumor types. These aspects of combined treatments will be discussed for both high temperature ablations and cryosurgery. Supported by NCI CA44114, Arkansas Breast Cancer Research Program and the Winthrop P. Rockefeller Cancer Institute at UAMS.

Murine endoglin-specific single-chain Fv fragments for the analysis of vascular targeting strategies in mice

Endoglin has been identified as a promising cell surface antigen for vascular targeting approaches in cancer therapy, e.g. employing antibody molecules as targeting moieties. However, in vivo analysis of such strategies in mouse models requires antibodies recognizing endoglin on mouse endothelial cells. Here we describe the isolation of single-chain Fv fragments (scFvs) from phage display libraries, which bind to the extracellular region of mouse endoglin. One of these clones, scFv mE12, showed strong ( K d = 11 nM) and selective binding to purified endoglin and also to the endoglin-expressing mouse endothelioma cell line eEnd.2. This antibody recognized a linear epitope located in the N-terminal region (aa 27–361) of endoglin. Cell binding was further increased by generating a bivalent scFv–Fc fusion protein composed of scFv mE12 and the human γ1 Fc part. Moreover, scFv mE12 was endowed with an additional cysteine residue in the linker region and applied for the generation of anti-endoglin scFv immunoliposomes capable of selectively binding to endoglin-expressing cells. Thus, anti-mouse endoglin scFv mE12 should be useful to analyze vascular targeting strategies in mice.

Cationic lipid complexed camptothecin (EndoTAG®-2) improves antitumoral efficacy by tumor vascular targeting

Neo-vascular targeting by cationic colloidal carriers enables to realize an innovative approach for tumor therapy. EndoTag®-2 is a novel vascular targeting agent, comprising the mammalian topoisomerase I inhibitor camptothecin in its carboxylate form complexed to cationic lipid (cationic lipid complexed camptothecin). Here we studied tumor vascular targeting properties, antitumoral effects and mode of action of EndoTag®-2. Tumor vascular targeting properties of fluorescently labelled EndoTag®-2 were investigated by in vivo microscopy using A-MEL-3 tumors grown in the dorsal skinfold chamber preparation and by fluorescence histology of s.c. LLC-1 carcinomas. Therapeutic effects have been investigated in the s.c. LLC-1 carcinoma model and the L3.6pl human pancreatic cancer model implanted orthotopically in athymic nude mice. Antivascular effects have been studied by histological investigation of tumor microvessel density and non invasive investigation of tumor blood flow by dynamic contrast enhanced MRI imaging (DCE-MRI). EndoTag®-2 selectively targeted tumor microvessels as confirmed by quantitative fluorescence microscopy. Compared to controls EndoTag®-2 revealed remarkable antitumoral efficiency in s.c. LLC-1 carcinomas implanted in C57/Bl6 mice. Growth and metastasis of orthotopic L3.6pl human pancreatic tumors was significantly inhibited by EndoTag®-2 treatment. Quantitative analysis of tumor microvessel density revealed significant reduction of microvessel density in lewis lung carcinomas up to 50%. DCE-MRI confirmed significant reduction of intratumoral vascular volume as well as tumor perfusion upon EndoTag®-2 treatment. In conclusion this study shows that cationic lipid complexed camptothecin (EndoTag®-2) is a markedly active antitumor agent based on an innovative vascular targeting approach.

Coupling Tumor Necrosis Factor-α with αV Integrin Ligands Improves Its Antineoplastic Activity

Despite the impressive results obtained in animal models, the clinical use of tumor necrosis factor-alpha (TNF) as an anticancer drug is limited by severe toxicity. We have shown previously that targeted delivery of TNF to aminopeptidase N (CD13), a marker of angiogenic vessels, improved the therapeutic index of this cytokine in tumor-bearing mice. To assess whether the vascular-targeting approach could be extended to other markers of tumor blood vessels, in this work, we have fused TNF with the ACDCRGDCFCG peptide, a ligand of alpha(V) integrins by recombinant DNA technology. We have found that subnanogram doses of this conjugate are sufficient to induce antitumor effects in tumor-bearing mice when combined with melphalan, a chemotherapeutic drug. Cell adhesion assays and competitive binding experiments with anti-integrin antibodies showed that the Arg-Gly-Asp moiety interacts with cell adhesion receptors, including alpha(V)beta(3) integrin, as originally postulated. In addition, ACGDRGDCFCG-mouse TNF conjugate induced cytotoxic effects in standard cytolytic assays, implying that ACGDRGDCFCG-mouse TNF conjugate can also bind TNF receptors and trigger death signals. These results indicate that coupling TNF with alpha(V) integrin ligands improves its antineoplastic activity and supports the concept that vascular targeting is a strategy potentially applicable to different endothelial markers, not limited to CD13.

Strategies for vascular targeting in tumors.

Strategies for vascular targeting in tumors.

Potent antitumor effects of an antitumor endothelial cell immunotoxin in a murine vascular targeting model.

Immunotoxins and other antibody-based therapeutic reagents have proved effective agonist lymphomas and leukemias, but results with carcinomas and other solid tumors have thus far been less impressive. A major reason for this difference is that macromolecules penetrate poorly and unevenly into solid tumors. A solution to this problem would be to attack the endothelial cells of the tumor vascular bed rather than the tumor cells themselves. We have developed a murine model of this vascular targeting approach where transfection of the tumor cells with a cytokine gene causes them to induce the expression of an experimental marker (MHC Class II) on tumor endothelium. In this report we show that an anti-Class II-deglycosylated Ricin A-chain immunotoxin kills IFN-gamma-activated endothelial cells in culture and, when injected into tumor-bearing Balb/c nude mice, causes complete thrombosis of the tumor vasculature, widespread infarction, and dramatic regressions of large solid tumors. These findings suggest that immunoconjugates prepared with recently described antibodies against human tumor endothelium could provide a broad-based therapy for variety of solid cancers in humans.