Differential Mechanisms Associated with Vascular Disrupting Action of Electrochemotherapy: Intravital Microscopy on the Level of Single Normal and Tumor Blood Vessels

Electropermeabilization/electroporation (EP) provides a tool for the introduction of molecules into cells and tissues. In electrochemotherapy (ECT), cytotoxic drugs are introduced into cells in tumors, and nucleic acids are introduced into cells in gene electrotransfer. The normal and tumor tissue blood flow modifying effects of EP and the vascular disrupting effect of ECT in tumors have already been determined. However, differential effects between normal vs. tumor vessels, to ensure safety in the clinical application of ECT, have not been determined yet. Therefore, the aim of our study was to determine the effects of EP and ECT with bleomycin on the HT-29 human colon carcinoma tumor model and its surrounding blood vessels. The response of blood vessels to EP and ECT was monitored in real time, directly at the single blood vessel level, by in vivo optical imaging in a dorsal window chamber in SCID mice with 70 kDa fluorescently labeled dextrans. The response of tumor blood vessels to EP and ECT started to differ within the first hour. Both therapies induced a vascular lock, decreased functional vascular density (FVD) and increased the diameter of functional blood vessels within the tumor. The effects were more pronounced for ECT, which destroyed the tumor blood vessels within 24 h. Although the vasculature surrounding the tumor was affected by EP and ECT, it remained functional. The study confirms the current model of tumor blood flow modifying effects of EP and provides conclusive evidence that ECT is a vascular disrupting therapy with a specific effect on the tumor blood vessels.     

Effects of the calcium channel blocker flunarizine on the hemodynamics and oxygenation of tumor microvasculature.

Flunarizine is a diphenylpiperazine calcium entry blocker that has been shown previously to increase tumor blood flow and sensitivity to radiotherapy via reduction in the radiobiologically significant hypoxic fraction. Two mechanisms of action have been proposed previously (vasodilation, altered blood viscosity), but no studies have been performed to examine its mechanisms of action in vivo. Such information would be invaluable in determining the role of flunarizine in multimodality approaches to reduce tumor hypoxia. Fisher-344 rats bearing R3230Ac tumors transplanted into dorsal flap window chambers were used to examine microcirculatory changes after administration of flunarizine (1.0 mg/kg, iv). The drug increased the diameters of the microvasculature and red cell velocities specifically in central tumor regions (producing an average increase in vessel flow by a factor of 1.96), which was accompanied by an increase in perivascular pO2 of 12 mm Hg, on the average. The drug did not change the diameters of tumor "feeding" vessels, nor did it change vascular length densities. Thus the improvement in central tumor blood flow and oxygenation could not be attributed to dilation of feeding vessels. The oxygen-carrying capacity of the blood was not altered either since hemoglobin saturation (measured in vitro) and the hematocrits of the microvasculature were unchanged after drug administration. Therefore, by a process of elimination, the most likely explanation for the effect of the drug is modification of blood viscosity. Additional studies are under way in this laboratory to examine whether changes in viscosity occur after flunarizine administration.     

A comparison of the effects of photodynamic therapy on normal and tumor blood vessels in the rat microcirculation

The effects of light activation of the tumor photosensitizer dihematoporphyrin ether (DHE) were studied in the microcirculation of the rat cremaster muscle. Arterioles and venules in an implanted chondrosarcoma were studied by in vivo television microscopy and were compared to normal vessels of the same size elsewhere in the preparation and in control preparations. Activation with green light (530-560 nm, 200 mW/cm2, 120 J/cm2) 48 h after intraperitoneal injection of DHE (10 mg/kg body wt) resulted in significant narrowing of diameters of red blood cell columns in tumor arterioles and venules. The response in normal and control arterioles and venules was not significantly different from that seen in the tumor vessels except that the control arterioles did not remain significantly constricted during the treatment period. Treatment resulted in stasis of blood flow in 90% of tumor and normal arterioles at the completion of light activation. In venules, stasis of blood flow was observed in 75% of tumor and 70% of normal vessels. Vasoconstriction was the primary response in arterioles, while thrombosis predominated in venules. Morphologic assessment of light-activated vessels in the cremaster preparation by transmission electron microscopy revealed platelet aggregation with damage to endothelial cells and smooth muscle cells. Perivascular effects observed included interstitial edema and damage to skeletal muscle cells. In the tumor-bearing preparation, no direct cytotoxic effect on the tumor cells was shown. The surrounding vessels exhibited similar vascular stasis, but the lining cells appeared minimally affected. Photoactivation of DHE results in significant changes in the microcirculation which lead to stasis of blood flow. In this model, the response was similar for the normal microvasculature and for the microcirculation of an implanted chondrosarcoma. These effects may account, in part, for the mechanism of action of photodynamic therapy.     

Study of tumor blood perfusion and its variation due to vascular normalization by anti-angiogenic therapy based on 3D angiogenic microvasculature

The coupling of intravascular and interstitial flow is a distinct feature of tumor microcirculation, due to high vessel permeability, low osmotic pressure gradient and absence of functional lymphatic system inside tumors. We have previously studied the tumor microcirculation by using a 2D coupled model. In this paper, we extend it to a 3D case with some new considerations, to investigate tumor blood perfusion on a more realist microvasculature, and the effects of vascular normalization by anti-angiogenic therapy on tumor microenvironment.The model predict the abnormal tumor microcirculation and the resultant hostile microenvironment: (1) in the intra-tumoral vessels, blood flows slowly with almost constant pressure values, haematocrit is much lower which contributes to hypoxia and necrosis formation of the tumor centre; (2) the total transvascular flux is at the same order of magnitude as intravascular flux, the intravasation appears inside of the tumor, the ratio of the total amount of intravasation flux to extravasation flux is about 16% for the present model; (3) the interstitial pressure is uniformly high throughout the tumor and drops precipitously at the periphery, which leads to an extremely slow interstitial flow inside the tumor, and a rapidly rising convective flow oozing out from the tumor margin into the surrounding normal tissue. The investigation of the sensitivity of flows to changes in transport properties of vessels and interstitium as well as the vascular density of the vasculature, gains an insight into how normalization of tumor microenvironment by anti-angiogenic therapies influences the blood perfusion.     

Monitoring sunitinib-induced vascular effects to optimize radiotherapy combined with soy isoflavones in murine xenograft tumor

Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) to monitor vascular changes induced by sunitinib within a murine xenograft kidney tumor, we previously determined a dose that caused only partial destruction of blood vessels leading to "normalization" of tumor vasculature and improved blood flow. In the current study, kidney tumors were treated with this dose of sunitinib to modify the tumor microenvironment and enhance the effect of kidney tumor irradiation. The addition of soy isoflavones to this combined antiangiogenic and radiotherapy approach was investigated based on our studies demonstrating that soy isoflavones can potentiate the radiation effect on the tumors and act as antioxidants to protect normal tissues from treatment-induced toxicity. DCE-MRI was used to monitor vascular changes induced by sunitinib and schedule radiation when the uptake and washout of the contrast agent indicated regularization of blood flow. The combination of sunitinib with tumor irradiation and soy isoflavones significantly inhibited the growth and invasion of established kidney tumors and caused marked aberrations in the morphology of residual tumor cells. DCE-MRI studies demonstrated that the three modalities, sunitinib, radiation, and soy isoflavones, also exerted antiangiogenic effects resulting in increased uptake and clearance of the contrast agent. Interestingly, DCE-MRI and histologic observations of the normal contralateral kidneys suggest that soy could protect the vasculature of normal tissue from the adverse effects of sunitinib. An antiangiogenic approach that only partially destroys inefficient vessels could potentially increase the efficacy and delivery of cytotoxic therapies and radiotherapy for unresectable primary renal cell carcinoma tumors and metastatic disease.     

Effect of isoproterenol or exercise on pulmonary lymph flow and hemodynamics

Experiments were performed to determine whether different methods of increasing cardiac output would have similar effects on lung lymph flow, and to assess the contribution of the microvasculature (fluid-exchanging vessels) to the total calculated pulmonary vascular resistance. Yearling unanesthetized sheep with chronic vascular catheters and lung lymph fistulas underwent intravenous infusions of isoproterenol at 0.2 micrograms X kg-1. min-1 (n = 8) or were exercised on a treadmill (n = 16). Both isoproterenol and exercise increased cardiac output, lowered calculated total pulmonary and systemic vascular resistances, and had no effect on the calculated pulmonary microvascular pressure. Isoproterenol infusions did not affect lung lymph flow, whereas exercise increased lung lymph flow in proportion to the increase in cardiac output. We conclude that 1) the sheep has a different pulmonary hemodynamic response to exercise than dogs and man, 2) the microvasculature is recruited during exercise-induced but not isoproterenol-induced increases in cardiac output, and 3) the microvasculature represents only a small proportion of the total calculated pulmonary vascular resistance.     

Arterial supply and venous drainage of the brain of the black bear (Ursus americanus). II. Intracranial microvasculature

A morphological study of the intracranial microvasculature of the Black bear (Ursus americanus) using vascular casts was undertaken. The object was to provide basic information regarding structural modifications of the microvasculature that might provide insight into the ability to cope with low blood flow states that occur during winter sleep. Vascular casts were prepared from 6 animals. The microvasculature of the brain disclosed characteristic features of the small vessels in mammals, including vascular sphincters of two types and numerous arterial and arteriolar anastomoses.     

IAP antagonization promotes inflammatory destruction of vascular endothelium

In this study, we show for the first time that the therapeutic antagonization of inhibitor of apoptosis proteins (IAPs) inhibits B16 melanoma growth by disrupting tumor vasculature. Specifically, the treatment of mice bearing B16 melanoma with an IAP antagonist compound A (Comp A) inhibits tumor growth not by inducing direct cytotoxicity against B16 cells but rather by a hitherto unrecognized antiangiogenic activity against tumor vessels. Our detailed analysis showed that Comp A treatment induces NF-κB activity in B16 tumor cells and facilitates the production of TNF. In the presence of Comp A, endothelial cells (ECs) become highly susceptible to TNF and undergo apoptotic cell death. Accordingly, the antiangiogenic and growth-attenuating effects of Comp A treatment were completely abolished in TNF-R knockout mice. This novel targeting approach could be of clinical value in controlling pathological neoangiogenesis under inflammatory condition while sparing blood vessels under normal condition.     

Effects of bradykinin on the hemodynamics of tumor and granulating normal tissue microvasculature.

Bradykinin (BK) is an important endogenous mediator of microvascular flow modulation. Since the structure of the microcirculation is very different in tumor tissues than in normal tissues, bradykinin may elicit different responses in tumors. This study was designed to test the hypothesis that local administration of bradykinin increases blood flow preferentially in normal tissue relative to adjacent tumor tissue, resulting in a "vascular steal" phenomenon. Microvessel diameters (D), velocities (Vc), length densities, shear rates, and intermittent flow frequencies were measured every 10 min before, during, and after 40 min exposure to BK in rats with dorsal flap window chambers 9 days after tumor implantation. Separate studies were made of normal vessels outside the tumor margin, the hypervascular tumor periphery, and the tumor center. Bradykinin was administered with a suffusion medium flowing over the tissue at 1-2 ml/min with a BK concentration of 1.6 x 10(7) M. Administration of BK created five distinct changes in normal and tumor vessel function that varied over time, but coincidentally reached a maximum effect after 20 min exposure to BK. In normal vessels, increased Vc and D led to increased flow, which reached a peak 20 min after onset of suffusion with BK. In contrast, in centrally located tumor vessels, decreased D and Vc were observed in most vessels during the initial 10-20 min of suffusion. In addition, there was a significant increase in intermittent flow frequency in tumor central vessels, which peaked after 20 min of suffusion with BK. These five separate observations that coincided at 20 min of suffusion are consistent with a "vascular steal" phenomenon. The increase in normal microvessel D and Vc at 20 min suggests that BK causes vasodilation in arterioles. The coincident decrease in tumor microvessel D and Vc suggests that tumor feeding vessels are less able to respond to BK by vasodilating. The concomitant increase in intermittent flow frequency in tumor vessels suggests that a reduction in pressure drop occurred after 20 min exposure to BK, which is also consistent with "vascular steal." Since BK is also known to increase vascular permeability, it is possible that increases in interstitial fluid pressure brought on by exposure to BK contributed to the observed reduction in tumor blood flow. In normal vessels, reduced D and Vc, relative to peak values, were noted after 40 min suffusion with BK. Adherence of leukocytes to the vessel walls was prominent and microthrombi were also observed during this period. No evidence of such adhesion was seen in tumor vessels, although microthrombi were observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin regulates its own delivery to skeletal muscle by feed-forward actions on the vasculature

Insulin, at physiological concentrations, regulates the volume of microvasculature perfused within skeletal and cardiac muscle. It can also, by relaxing the larger resistance vessels, increase total muscle blood flow. Both of these effects require endothelial cell nitric oxide generation and smooth muscle cell relaxation, and each could increase delivery of insulin and nutrients to muscle. The capillary microvasculature possesses the greatest endothelial surface area of the body. Yet, whether insulin acts on the capillary endothelial cell is not known. Here, we review insulin's actions at each of three levels of the arterial vasculature as well as recent data suggesting that insulin can regulate a vesicular transport system within the endothelial cell. This latter action, if it occurs at the capillary level, could enhance insulin delivery to muscle interstitium and thereby complement insulin's actions on arteriolar endothelium to increase insulin delivery. We also review work that suggests that this action of insulin on vesicle transport depends on endothelial cell nitric oxide generation and that insulin's ability to regulate this vesicular transport system is impaired by inflammatory cytokines that provoke insulin resistance.     

Coupled modeling of blood perfusion in intravascular, interstitial spaces in tumor microvasculature

The coupling of intravascular and interstitial flow is a distinct feature of tumor microcirculation, due to the high vessel permeability, the low osmotic pressure gradient as well as the absence of functional lymphatic system inside tumors. In this paper, a coupled mathematical model of tumor microcirculation is developed, which provides the link between microvasculature and interstitial space perfusion through the matrices determining a neighbor point belonging to either connected vessel (matrix B) or interstitial space (matrix A), and combines the intravascular and interstitial flow by vascular leaky terms. In addition, the compliance of tumor vessels, blood rheology with hematocritic distribution at branches is also considered. The microvascular network, on which the microcirculation calculation is carried out, is generated from our two-dimensional 9-point (2D9P) model of tumor angiogenesis, improved from the previous 2D5P one. A specific coupling procedure is developed in the study to couple the intravascular and interstitial flow. It is based on the iteratively numerical simulation techniques, including local iterations at individual parameter level and one global loop to provide coupling and simulation convergence. The simulation results not only present the basic features and characteristics of tumor microcirculation, which agree with the corresponding experimental observations reported, but also predict an intimate relationship between the tumor intravascular and interstitial flow quantitatively. Among the parameters, the vascular leakiness is a key to govern the systemic flowing pattern, influence the tumor internal environment and contribute to the metastasis of tumor cells, which could not be presented by the previous uncoupled models.     

Quantitative Ex-Vivo Micro-Computed Tomographic Imaging of Blood Vessels and Necrotic Regions within Tumors

Techniques for visualizing and quantifying the microvasculature of tumors are essential not only for studying angiogenic processes but also for monitoring the effects of anti-angiogenic treatments. Given the relatively limited information that can be gleaned from conventional 2-D histological analyses, there has been considerable interest in methods that enable the 3-D assessment of the vasculature. To this end, we employed a polymerizing intravascular contrast medium (Microfil) and micro-computed tomography (micro-CT) in combination with a maximal spheres direct 3-D analysis method to visualize and quantify ex-vivo vessel structural features, and to define regions of hypoperfusion within tumors that would be indicative of necrosis. Employing these techniques we quantified the effects of a vascular disrupting agent on the tumor vasculature. The methods described herein for quantifying whole tumor vascularity represent a significant advance in the 3-D study of tumor angiogenesis and evaluation of novel therapeutics, and will also find potential application in other fields where quantification of blood vessel structure and necrosis are important outcome parameters.     

Microvasculature of the bear heart demonstrated by scanning electron microscopy

Morphological features of the coronary microvasculature were examined in ten Black bears (Ursus americanus). Tissue samples were collected for scanning electron microscopy from five animals, and vascular casts of the coronary vessels were prepared from five. Each portion of the microvasculature examined had specific morphological characteristics which may contribute to the control of blood flow. All indications from the present study suggest that the structure of the coronary microvasculature in the Black bear is similar to that of mammalian species previously examined.     

低剂量化疗联合中药抗血管生成作用的研究进展

自从八十年代初Folkman提出肿瘤生长依赖于新血管生成的理论,抗血管生成治疗已逐渐成为肿瘤研究的热点和肿瘤治疗的新策略。对抗血管生成治疗的深入研究也使人们对许多细胞毒化疗药物的功能活性有了新的审视,近期一些国外研究表明降低化疗药物的剂量可特异地杀伤新生肿瘤血管内皮细胞,利用化疗药物的这一新靶点,采取合理的给药方式和计划,可能帮助解决常规高剂量化疗引起的毒副作用和耐药性的难题。我国传统的中医药在肿瘤诊治上已经积累了许多宝贵的经验,对这些初步筛选出采的抗肿瘤中药借助现代技术进行精细分析、模拟修饰和药理机制研究,发现我国传统中药提取物中许多有效成分显示出了抗血管生成作用,将其与低剂量化疗联合应用也显示出良好抑瘤效果,同时毒副作用小,患者生存质量提高,因此这种联合疗法为晚期肿瘤患者提供了一种新的安全有效的治疗途径,本文对目前低剂量化疗与中药联合应用的抗肿瘤血管生成机制及应用前景作一综述。     

Tubulin-Destabilizing Agent BPR0L075 Induces Vascular-Disruption in Human Breast Cancer Mammary Fat Pad Xenografts

BPR0L075, 6-methoxy-3-(3',4',5'-trimethoxy-benzoyl)-1H-indole, is a tubulin-binding agent that inhibits tubulin polymerization by binding to the colchicine-binding site. BPR0L075 has shown antimitotic and antiangiogenic activity in vitro. The current study evaluated the vascular-disrupting activity of BPR0L075 in human breast cancer mammary fat pad xenografts using dynamic bioluminescence imaging. A single dose of BPR0L075 (50 mg/kg, intraperitoneally (i.p.)) induced rapid, temporary tumor vascular shutdown (at 2, 4, and 6 hours); evidenced by rapid and reproducible decrease of light emission from luciferase-expressing orthotopic MCF7 and MDA-MB-231 breast tumors after administration of luciferin substrate. A time-dependent reduction of tumor perfusion after BPR0L075 treatment was confirmed by immunohistological staining of the perfusion marker Hoechst 33342 and tumor vasculature marker CD31. The vasculature showed distinct recovery within 24 hours post therapy. A single i.p. injection of 50 mg/kg of BPR0L075 initially produced plasma concentrations in the micromolar range within 6 hours, but subsequent drug distribution and elimination caused BPR0L075 plasma levels to drop rapidly into the nanomolar range within 24 h. Tests with human umbilical vein endothelial (HUVEC) cells and tumor cells in culture showed that BPR0L075 was cytotoxic to both tumor cells and proliferating endothelial cells, and disrupted pre-established vessels in vitro and ex vivo. In conclusion, BPR0L075 caused rapid, albeit, temporary tumor vascular shutdown and led to reduction of tumor perfusion in orthotopic human breast cancer xenografts, suggesting that this antimitotic agent may be useful as a vascular-disrupting cancer therapy.     

Differential arrest and adhesion of tumor cells and microbeads in the microvasculature

To investigate the mechanical mechanisms behind tumor cell arrest in the microvasculature, we injected fluorescently labeled human breast carcinoma cells or similarly sized rigid beads into the systemic circulation of a rat. Their arrest patterns in the microvasculature of mesentery were recorded and quantified. We found that 93 % of rigid beads were arrested either at arteriole–capillary intersections or in capillaries. Only 3 % were at the capillary–postcapillary venule intersections and in postcapillary venules. In contrast, most of the flexible tumor cells were either entrapped in capillaries or arrested at capillary or postcapillary venule–postcapillary venule intersections and in postcapillary venules. Only 12 % of tumor cells were arrested at the arteriole–capillary intersections. The differential arrest and adhesion of tumor cells and microbeads in the microvasculature was confirmed by a $\chi ^{2}$ test ( $p<0.001$ ). These results demonstrate that mechanical trapping was responsible for almost all the arrest of beads and half the arrest of tumor cells. Based on the measured geometry and blood flow velocities at the intersections, we also performed a numerical simulation using commercial software (ANSYS CFX 12.01) to depict the detailed distribution profiles of the velocity, shear rate, and vorticity at the intersections where tumor cells preferred to arrest and adhere. Simulation results reveal the presence of localized vorticity and shear rate regions at the turning points of the microvessel intersections, implying that hemodynamic factors play an important role in tumor cell arrest in the microcirculation. Our study helps elucidate long-debated issues related to the dominant factors in early-stage tumor hematogenous metastasis.     

Blood flow regulation in the cerebral microvasculature with an arcadal network: a numerical simulation

Blood flow regulation in the cerebral microvasculature with an arcadal network was investigated using a numerical simulation. A mathematical model for blood flow in the arcadal network, based on in vivo data of cat cerebral microvasculature and flow velocity was developed. The network model consists of 45 vessel segments and 25 branching points. To simulate microvascular response to blood flow, non-reactive (solid), cerebral arteriole-like, or skeletal muscle arteriole-like responses to wall shear stress were taken into account. Numerical calculation was carried out in the flow condition where the inlet (arterial) pressure was changed from 60 to 120 mmHg. Flow-rate in each efferent vessel and the mean flow-rate over all efferent vessels were evaluated for assessment of blood supply to the local area of cerebral tissue. The simulation demonstrated the wall shear stress-induced vasodilation in the arcadal network worked to maintain the blood flow at a constant level with pressure variable in a wide range. It is suggested that an individual microvessel (segment) should join in the regulatory process of flow, interacting with other microvessels (cooperative regulation).     

Lingering Dynamics in Microvascular Blood Flow

The microvascular networks in the body of vertebrates consist of the smallest vessels such as arterioles, capillaries, and venules. The flow of red blood cells (RBCs) through these networks ensures the gas exchange in as well as the transport of nutrients to the tissues. Any alterations in this blood flow may have severe implications on the health state. Because the vessels in these networks obey dimensions similar to the diameter of RBCs, dynamic effects on the cellular scale play a key role. The steady progression in the numerical modeling of RBCs, even in complex networks, has led to novel findings in the field of hemodynamics, especially concerning the impact and the dynamics of lingering events when a cell meets a branch of the network. However, these results are yet to be matched by a detailed analysis of the lingering experiments in vivo. To quantify this lingering effect in in vivo experiments, this study analyzes branching vessels in the microvasculature of Syrian golden hamsters via intravital microscopy and the use of an implanted dorsal skinfold chamber. It also presents a detailed analysis of these lingering effects of cells at the apex of bifurcating vessels, affecting the temporal distribution of plasmatic zones of blood flow in the branches and even causing a partial blockage in severe cases.     

Dynamic contrast enhanced MRI of mouse fibrosarcoma using small-molecular and novel macromolecular contrast agents

The aim of the study was a comparison of 2 novel macromolecular contrast agents, Gadomer-17 and Polylysine-Gd-DTPA, with commercially available Gd-DTPA in determining the quality of tumor microvasculature by dynamic contrast enhanced MRI. Three groups of 5 mice with SA-1 tumors were studied. To each group of animals one contrast agent was administered; i.e. the first group got Gd-DTPA, the second group Gadomer-17 and the third group Polylysine-Gd-DTPA. To perform dynamic contrast enhanced MRI a standard keyhole approach was used by which consecutive signal intensity change due to contrast agent accumulation in the tumor was measured. From the obtained data, tissue permeability surface area product PS and fractional blood volume BV were calculated on a pixel-by-pixel basis. PS and BV values were calculated for each contrast agent. Based on the values, contrast agents were classified according to their performance in characterizing tumor microvasculature. Results of our study suggest that Gadomer-17 and Polylysine-Gd-DTPA are significantly superior to Gd-DTPA in characterizing tumor microvasculature.     

Vascular disrupting agents

It has been well established that a functioning vascular supply is essential for solid tumor growth and metastases. In the absence of a viable vascular network, tumors are unable to grow beyond a few millimeters and therefore remain dormant. Initiation of angiogenesis allows for continued tumor growth and progression. Targeting tumor vasculature, either by inhibiting growth of new tumor blood vessels (antiangiogenic agents) or by destroying the already present tumor vessels (vascular disrupting agents; VDA), is an area of extensive research in the development of new antitumor agents. These two groups differ in their direct physiological target, the type or extent of disease that is likely to be susceptible, and the treatment schedule. VDAs target the established tumor blood vessels, resulting in tumor ischemia and necrosis. These agents show more immediate effects compared to antiangiogenic agents and may have more efficacy against advanced bulky disease. VDAs can be divided into two groups--ligand-bound and small molecule agents. Both VDA groups have demonstrated antitumor effects and tumor core necrosis, but consistently leave a thin rim of viable tumor cells at the periphery of the tumor. More evidence suggests VDAs will have their greatest effect in combination with conventional chemotherapy or other modes of treatment that attack this outer rim of cells.