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© 2018, The Author(s). The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Conventional drug delivery systems for solid tumors are composed of a nano-carrier that releases its therapeutic load. These two-stage nanoparticles utilize the enhanced permeability and retention (EPR) effect to enable preferential delivery to tumor tissue. However, the size-dependency of the EPR, the limited penetration of nanoparticles into the tumor as well as the rapid binding of the particles or the released cytotoxic agents to cancer cells and stromal components inhibit the uniform distribution of the drug and the efficacy of the treatment. Here, we employ mathematical modeling to study the effect of particle size, drug release rate and binding affinity on the distribution and efficacy of nanoparticles to derive optimal design rules. Furthermore, we introduce a new multi-stage delivery system. The system consists of a 20-nm primary nanoparticle, which releases 5-nm secondary particles, which in turn release the chemotherapeutic drug. We found that tuning the drug release kinetics and binding affinities leads to improved delivery of the drug. Our results also indicate that multi-stage nanoparticles are superior over two-stage nano-carriers provided they have a faster drug release rate and for high binding affinity drugs. Furthermore, our results suggest that smaller nanoparticles achieve better treatment outcome. © 2015, Biomedical Engineering Society. 43 9 2291 2300 Cited By :25

Delivery of blood-borne molecules and nanoparticles from the vasculature to cells in the tissue differs dramatically between tumor and normal tissues due to differences in their vascular architectures. Here we show that two simple measures of vascular geometry— δ max and λ—readily obtained from vascular images, capture these differences and link vascular structure to delivery in both tissue types. The longest time needed to bring materials to their destination scales with the square of δ max , the maximum distance in the tissue from the nearest blood vessel, whereas λ, a measure of the shape of the spaces between vessels, determines the rate of delivery for shorter times. Our results are useful for evaluating how new therapeutic agents that inhibit or stimulate vascular growth alter the functional efficiency of the vasculature and more broadly for analysis of diffusion in irregularly shaped domains.

In solid tumors, angiogenesis occurs in the setting of a defective vasculature and impaired lymphatic drainage that is associated with increased vascular permeability and enhanced tumor permeability. These universal aspects of the tumor microenvironment can have a marked influence on intratumoral drug delivery that may often be underappreciated. In this study, we investigated the effect of blood vessel normalization in tumors by the antiangiogenic drug bevacizumab on antibody uptake by tumors. In mouse xenograft models of human ovarian and esophageal cancer (SKOV-3 and OE19), we evaluated antibody uptake in tumors by positron emission tomographic imaging 24 and 144 hours after injection of (89)Zr-trastuzumab (SKOV-3 and OE19), (89)Zr-bevacizumab (SKOV-3), or (89)Zr-IgG (SKOV-3) before or after treatment with bevacizumab. Intratumor distribution was assessed by fluorescence microscopy along with mean vessel density (MVD) and vessel normalization. Notably, bevacizumab treatment decreased tumor uptake and intratumoral accumulation compared with baseline in the tumor models relative to controls. Bevacizumab treatment also reduced MVD in tumors and increased vessel pericyte coverage. These findings are clinically important, suggesting caution in designing combinatorial trials with therapeutic antibodies due to a possible reduction in tumoral accumulation that may be caused by bevacizumab cotreatment.

Despite their great promise, only a few nanoparticle formulations have been approved for clinical use in oncology. The failure of nano-scale drugs to enhance cancer therapy is in large part due to inefficient delivery. To overcome this outstanding problem, a better understanding of how the physical properties (i.e., size, surface chemistry, and shape) of nanoparticles affect their transvascular transport in tumors is required. In this study, we developed a mathematical model for nanoparticle delivery to solid tumors taking into account electrostatic interactions between the particles and the negatively-charged pores of the vessel wall. The model predictions suggest that electrostatic repulsion has a minor effect on the transvascular transport of nanoparticles. On the contrary, electrostatic attraction, caused even by small cationic charges (surface charge density less than 3 × 10-3 C/m2) can lead to a twofold or more increase in the transvascular flux of nanoparticles into the tumor interstitial space. Importantly, for every nanoparticle size, there is a value of charge density above which a steep increase in transvascular transport is predicted. Our model provides important guidelines for the optimal design of nanoparticle formulation for delivery to solid tumors. © 2012 Biomedical Engineering Society. 41 68 77 68-77

The enhanced permeability and retention (EPR) effect has been a key rationale for the development of nanoscale carriers to solid tumors. As a consequence of EPR, nanotherapeutics are expected to improve drug and detection probe delivery, have less adverse effects than conventional chemotherapy, and thus result in improved detection and treatment of tumors. Physiological barriers posed by the abnormal tumor microenvironment, however, can hinder the homogeneous delivery of nanomedicine in amounts sufficient to eradicate cancer. To effectively enhance the therapeutic outcome of cancer patients by nanotherapeutics, we have to find ways to overcome these barriers. One possibility is to exploit the abnormal tumor microenvironment for selective and improved delivery of therapeutic agents to tumors. Recently, we proposed a multistage nanoparticle delivery system as a potential means to enable uniform delivery throughout the tumor and improve the efficacy of anticancer therapy. Here, we describe the synthesis of a novel multistage nanoparticle formulation that shrinks in size once it enters the tumor interstitial space to optimize the delivery to tumors as well as within tumors. Finally, we provide detailed experimental methods for the characterization of such nanoparticles. © 2012 Elsevier Inc. All rights reserved. 508 109 130 109-130

The use of nanotechnology has offered new hope for cancer detection, prevention and treatment. Nanoparticle formulations are advantageous over conventional chemotherapy because they can incorporate multiple diagnostic and therapeutic agents and are associated with significantly less adverse effects due to selective accumulation to tumor tissue. Despite their great promise, however, only a few nanoparticle formulations have been approved for clinical use in oncology. The failure of nano-scale drugs to enhance cancer therapy is in large part due to inefficient delivery. Indeed, physiological barriers posed by the tumor micro-environment inhibit homogeneous distribution of drugs to the interstitial space of tumors and compromise the efficacy of the treatment. To overcome this outstanding problem, a better understanding of how the physical properties (i.e., size, and surface charge) of nanoparticles affect their transport in tumors is required. Here we use a mathematical model to provide basic design guidelines for the optimal delivery of nanoparticle formulations. © 2012 IEEE. 529 534 529-534

The blood vessels of cancerous tumours are leaky and poorly organized. This can increase the interstitial fluid pressure inside tumours and reduce blood supply to them, which impairs drug delivery. Anti-angiogenic therapiesĝwhich ĝ̃ normalizeĝ™ the abnormal blood vessels in tumours by making them less leakyĝhave been shown to improve the delivery and effectiveness of chemotherapeutics with low molecular weights, but it remains unclear whether normalizing tumour vessels can improve the delivery of nanomedicines. Here, we show that repairing the abnormal vessels in mammary tumours, by blocking vascular endothelial growth factor receptor-2, improves the delivery of smaller nanoparticles (diameter, 12 nm) while hindering the delivery of larger nanoparticles (diameter, 125 nm). Using a mathematical model, we show that reducing the sizes of pores in the walls of vessels through normalization decreases the interstitial fluid pressure in tumours, thus allowing small nanoparticles to enter them more rapidly. However, increased steric and hydrodynamic hindrances, also associated with smaller pores, make it more difficult for large nanoparticles to enter tumours. Our results further suggest that smaller (1/412 nm) nanomedicines are ideal for cancer therapy due to their superior tumour penetration. © 2012 Macmillan Publishers Limited. 7 383 388 383-388

Current Food and Drug Administration-approved cancer nanotherapeutics, which passively accumulate around leaky regions of the tumor vasculature because of an enhanced permeation and retention (EPR) effect, have provided only modest survival benefits. This suboptimal outcome is likely due to physiological barriers that hinder delivery of the nanotherapeutics throughout the tumor. Many of these nanotherapeutics are ≈100 nm in diameter and exhibit enhanced accumulation around the leaky regions of the tumor vasculature, but their large size hinders penetration into the dense collagen matrix. Therefore, we propose a multistage system in which 100-nm nanoparticles "shrink" to 10-nm nanoparticles after they extravasate from leaky regions of the tumor vasculature and are exposed to the tumor microenvironment. The shrunken nanoparticles can more readily diffuse throughout the tumor's interstitial space. This size change is triggered by proteases that are highly expressed in the tumor microenvironment such as MMP-2, which degrade the cores of 100-nm gelatin nano-particles, releasing smaller 10-nm nanoparticles from their surface. We used quantum dots (QD) as a model system for the 10-nm particles because their fluorescence can be used to demonstrate the validity of our approach. In vitro MMP-2 activation of the multistage nanoparticles revealed that the size change was efficient and effective in the enhancement of diffusive transport. In vivo circulation half-life and intratumoral diffusion measurements indicate that our multistage nanoparticles exhibited both the long circulation half-life necessary for the EPR effect and the deep tumor penetration required for delivery into the tumor's dense collagen matrix. 108 2426 2431 2426-2431

In vivo imaging technologies drive the development of improved cancer therapies by revealing critical aspects of the complex pathophysiology of solid tumors in small animal models[1]. The abnormal vascular function, which predicts tumor malignant potential and presents broad barriers to effective treatment, has been studied at the subcellular size scale using multiphoton (MP) microscopy [2], and at significantly larger size scales using ultrasound, μCT and μMRI[3-5]. However, limited in vivo imaging approaches exist to study the vascular function at the network level, i.e., with sufficient resolution to discern smaller vessels while maintaining a field of view and penetration depth large enough to reveal interconnectivity and inhomogeneities across the tumor and surrounding tissue. One promising technology operating at this size scale is optical frequency domain imaging (OFDI) using Doppler-methods to detect blood flow. We have recently designed and constructed a Doppler OFDI system specifically for the application of vascular imaging in tumor models[6]. The technical aspects of this system that enable the required levels of flow sensitivity and imaging speed are described. Beam scanning patterns used for this system will be reviewed and analyzed. The construction of the imaging system including high-rate data acquisition with the ability to continuously stream data at rates of 400 MB/sec will be described. Finally, the algorithms used to process, filter, and display the acquired volumetric datasets as vascular projections will be described. To validate the developed Doppler OFDI instrument for this application, its capabilities and limitations were explored relative to those of multiphoton microscopy, the standard optical imaging approach applied to the study of tumor biology. We investigated both the resolution and penetration depth, as well as differences in vascular visibility resulting from the differing mechanisms of contrast (endogenous flow in Doppler OFDI, exogenous fluorescent agent in multiphoton microscopy). Figure 1 illustrates a comparison between Doppler and MP in vivo imaging of a region of normal brain in a mouse cranial window model. Semi-automated vessel tracing algorithms were applied to each dataset, allowing quantitative comparison of visualized vessel sizes. As expected, multiphoton microscopy provides higher resolution, but, as indicated in Fig. 1(e), each modality provides consistent sizing of vessels exceeding 10 microns in diameter. To compare the ability of each modality to image the abnormal vessels within and surrounding tumors, we performed imaging with each modality in a series of tumors in a dorsal skin chamber models. ©2010 IEEE. 59 60 59-60

Intravital multiphoton microscopy has provided powerful mechanistic insights into health and disease and has become a common instrument in the modern biological laboratory. The requisite high numerical aperture and exogenous contrast agents that enable multiphoton microscopy, however, limit the ability to investigate substantial tissue volumes or to probe dynamic changes repeatedly over prolonged periods. Here we introduce optical frequency domain imaging (OFDI) as an intravital microscopy that circumvents the technical limitations of multiphoton microscopy and, as a result, provides unprecedented access to previously unexplored, crucial aspects of tissue biology. Using unique OFDI-based approaches and entirely intrinsic mechanisms of contrast, we present rapid and repeated measurements of tumor angiogenesis, lymphangiogenesis, tissue viability and both vascular and cellular responses to therapy, thereby demonstrating the potential of OFDI to facilitate the exploration of physiological and pathological processes and the evaluation of treatment strategies. © 2009 Nature America, Inc. All rights reserved. 15 1219 1223 1219-1223

Unlike vascular endothelial growth factor (VEGF)-A, the effect of VEGF-C on tumor angiogenesis, vascular permeability, and leukocyte recruitment is not known. To this end, we quantified in vivo growth and vascular function in tumors derived from two VEGF-C-overexpressing (VC+) and mock-transfected cell lines (T241 fibrosarcoma and VEGF-A-/- embryonic stem cells) grown in murine dorsal skinfold chambers. VC+ tumors grew more rapidly than mock-transfected tumors and exhibited parallel increases in tumor angiogenesis. Furthermore, VEGF-C overexpression elevated vascular permeability in T241 tumors, but not in VEGF-A-/- tumors. Surprisingly, unlike VEGF-A, VEGF-C did not increase leukocyte rolling or adhesion in tumor vessels. Administration of VEGF receptor (VEGFR)-2 neutralizing antibody DC101 reduced vascular density and permeability of both VC+ and mock-transduced T241 tumors. These data suggest that VEGFR-2 signaling is critical for tumor angiogenesis and vascular permeability and that VEGFR-3 signaling does not compensate for VEGFR-2 blockade. An alternate VEGFR, VEGFR-1 or neuropilin-1, may modulate adhesion of leukocytes to tumor vessels.

Tumor oxygenation is critical for tumor survival as well as for response to therapy, e.g., radiation therapy. Hormone ablation therapy in certain hormone-dependent tumors and antiangiogenic therapy lead to vessel regression and have also shown beneficial effects when combined with radiation therapy. These findings are counterintuitive because vessel regression should reduce oxygen tension (pO2) in tumors, decreasing the effectiveness of radiotherapy. Here we report on the dynamics of pO2 and oxygen consumption in a hormone-dependent tumor following hormone ablation and during treatment with an anti-VEGFR-2 monoclonal antibody (mAb) or a combination of doxorubicin and cyclophosphamide; the latter combination is not known to cause vessel regression at doses used clinically. Androgen-dependent male mouse mammary carcinoma (Shionogi) was implanted into transparent dorsal skin-fold chambers in male severe combined immunodeficient mice. Thirteen days after the tumors were implanted, mice were treated with antiangiogenic therapy (anti-VEGFR-2 mAb, 1.4 mg/30 g body weight), hormone ablation by castration, or doxorubicin (6.5 mg/kg every 7 days) and cyclophosphamide (100 mg/kg every 7 days). A non-invasive in vivo method was used to measure pO2 profiles and to calculate oxygen consumption rates (Q(O2)) in tumors. Tumors treated with anti-VEGFR-2 mAb exhibited vessel regression and became hypoxic. Initial vessel regression was followed by a "second wave" of angiogenesis and increases in both pO2 and Q(O2). Hormone ablation led to tumor regression followed by an increase in pO2 coincident with regrowth. Chemotherapy led to tumor growth arrest characterized by constant Q(O2) and elevated pO2. The increased pO2 during anti-VEGFR-2 mAb and hormone ablation therapy may explain the observed beneficial effects of combining antiangiogenic or hormone therapies with radiation treatment. Thus, understanding the microenvironmental dynamics is critical for optimal scheduling of these treatment modalities.

As a result of deprivation of oxygen (hypoxia) and nutrients, the growth and viability of cells is reduced. Hypoxia-inducible factor (HIF)-1alpha helps to restore oxygen homeostasis by inducing glycolysis, erythropoiesis and angiogenesis. Here we show that hypoxia and hypoglycaemia reduce proliferation and increase apoptosis in wild-type (HIF-1alpha+/+) embryonic stem (ES) cells, but not in ES cells with inactivated HIF-1alpha genes (HIF-1alpha-/-); however, a deficiency of HIF-1alpha does not affect apoptosis induced by cytokines. We find that hypoxia/hypoglycaemia-regulated genes involved in controlling the cell cycle are either HIF-1alpha-dependent (those encoding the proteins p53, p21, Bcl-2) or HIF-1alpha-independent (p27, GADD153), suggesting that there are at least two different adaptive responses to being deprived of oxygen and nutrients. Loss of HIF-1alpha reduces hypoxia-induced expression of vascular endothelial growth factor, prevents formation of large vessels in ES-derived tumours, and impairs vascular function, resulting in hypoxic microenvironments within the tumour mass. However, growth of HIF-1alpha tumours was not retarded but was accelerated, owing to decreased hypoxia-induced apoptosis and increased stress-induced proliferation. As hypoxic stress contributes to many (patho)biological disorders, this new role for HIF-1alpha in hypoxic control of cell growth and death may be of general pathophysiological importance.

It is generally accepted that the host microenvironment influences tumor biology. There are discrepancies in growth rate, metastatic potential, and efficacy of systemic treatment between ectopic and orthotopic tumors. Liver is the most common and critical site of distant metastasis of colorectal carcinoma. Tumorigenicity and efficacy of chemotherapeutic agents in colorectal tumors are different in liver and subcutaneous sites. Thus, we hypothesize that the liver (orthotopic) versus subcutaneous (ectopic) microenvironment would have different effects on the angiogenesis and maintenance of the microcirculation of colorectal tumor. To this end, we developed a new method to monitor and to quantify microcirculatory parameters in the tumor grown in the liver. Using this approach, we compared the microcirculation of LS174T, a human colon adenocarcinoma, metastasized to the liver with that of the host liver vessels and that of the same tumor grown in the subcutaneous space. In the liver metastasis model, 5 x 10(6) LS174T cells were injected into the spleen of nude mice. Four to eight weeks later, the liver with metastatic tumors was exteriorized and placed on a special stage and observed under an intravital fluorescence microscope. The dorsal skinfold chamber model was used to study the subcutaneous tumors. Red blood cell velocity, vessel diameter, density, permeability, and leukocyte-endothelial interactions were measured using fluorescence microscopy and image analysis. Vascular endothelial growth factor/ vascular permeability factor (VEGF/VPF) mRNA expression was determined by the Northern blot analysis. LS174T tumor foci in the liver had tortuous vascular architecture, heterogeneous blood flow, significantly lower vascular density, and significantly higher vascular permeability than normal liver tissue. Tumors grown in the liver had significantly lower vessel density, especially in the center coincident with central necrosis, than the subcutaneous tumors. The frequency distribution of vessel diameters of liver tumor was slightly shifted to smaller size compared with that of subcutaneous tumor. Leukocyte rolling in liver tumor was twofold lower than that in subcutaneous tumor. These physiological findings were consistent with the measurement of VEGF/VPF in that the VEGF/VPF mRNA level was lower in the liver tumor than that in the subcutaneous tumor. However, macromolecular vascular permeability in the liver tumor was significantly higher than in the subcutaneous tumor. Liver sinusoidal endothelial cells, the origin of liver tumor vessel endothelium, are known to be fenestrated and not to have a basement membrane, suggesting that the difference in endothelial cell origin may explain the difference in tumor vascular permeability in two sites. These findings demonstrate that liver microenvironment has different effects on some aspects of the tumor angiogenesis and microcirculation compared with the subcutaneous tissues. The new model/method described in this paper has significant implications in two research areas: 1) the liver microenvironment and its effect on tumor pathophysiology in conjunction with cytokine/ growth factor regulation and 2) the delivery of drugs, cells, and genes to liver tumors.

The present study was designed to define the role of nitric oxide (NO) in tumor microcirculation, through the direct intravital microcirculatory observations after administration of NO synthase (NOS) inhibitor and NO donor both regionally and systemically. More specifically, we tested the following hypotheses: 1) endogenous NO derived from tumor vascular endothelium and/or tumor cells increases and/or maintains tumor blood flow, decreases leukocyte-endothelial interactions, and increases vascular permeability, 2) exogenous NO can increase tumor blood flow via vessel dilatation and decrease leukocyte-endothelial interactions, and 3) NO production and tissue responses to NO are tumor dependent. To this end, a murine mammary adenocarcinoma (MCaIV) and a human colon adenocarcinoma (LS174T) were implanted in the dorsal skinfold chamber in C3H and severe combined immunodeficient mice, respectively, and observed by means of intravital fluorescence microscopy. Both regional and systemic inhibition of endogenous NO by N omega-nitro-L-arginine methyl ester (L-NAME; 100 mumol/L superfusion or 10 mg/kg intravenously) significantly decreased vessel diameter and local blood flow rate. The diameter change was dominant on the arteriolar side. Superfusion of NO donor (spermine NO, 100 mumol/L) increased tumor vessel diameter and flow rate, whereas systemic injection of spermine NO (2.62 mg/kg) had no significant effect on these parameters. Rolling and stable adhesion of leukocytes were significantly increased by intravenous injection of L-NAME. In untreated animals, both MCaIV and LS174T tumor vessels were leaky to albumin. Systemic NO inhibition significantly attenuated tumor vascular permeability of MCaIV but not of LS174T tumor. Immunohistochemical studies, using polyclonal antibodies to endothelial NOS and inducible NOS, revealed a diffuse pattern of positive labeling in both MCaIV and LS174T tumors. Nitrite and nitrate levels in tumor interstitial fluid of MCaIV but not of LS174T were significantly higher than that in normal subcutaneous interstitial fluid. These results support our hypotheses regarding the microcirculatory response to NO in tumors. Modulation of NO level in tumors is a potential strategy for altering tumor hemodynamics and thus improving oxygen, drug, gene vector, and effector cell delivery to solid tumors.