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.

We have reviewed the studies on radiation-induced vascular changes in human and experimental tumors reported in the last several decades. Although the reported results are inconsistent, they can be generalized as follows. In the human tumors treated with conventional fractionated radiotherapy, the morphological and functional status of the vasculature is preserved, if not improved, during the early part of a treatment course and then decreases toward the end of treatment. Irradiation of human tumor xenografts or rodent tumors with 5–10 Gy in a single dose causes relatively mild vascular damages, but increasing the radiation dose to higher than 10 Gy/fraction induces severe vascular damage resulting in reduced blood perfusion. Little is known about the vascular changes in human tumors treated with high-dose hypofractionated radiation such as stereotactic body radiotherapy (SBRT) or stereotactic radiosurgery (SRS). However, the results for experimental tumors strongly indicate that SBRT or SRS of human tumors with doses higher than about 10 Gy/fraction is likely to induce considerable vascular damages and thereby damages the intratumor microenvironment, leading to indirect tumor cell death. Vascular damage may play an important role in the response of human tumors to high-dose hypofractionated SBRT or SRS.

Purpose: To test whether a direct antiangiogenic peptide (anginex) and a vascular endothelial growth factor antibody (bevacizumab, Avastin) can transiently normalize vasculature within tumors to improve oxygen delivery, alleviate hypoxia, and increase the effect of radiation therapy. Experimental Design: Tumor oxygenation levels, microvessel density and pericyte coverage were monitored in three different solid tumor models (xenograft human ovarian carcinoma MA148, murine melanoma B16F10, and murine breast carcinoma SCK) in mice. Multiple treatment schedules were tested in these models to assess the influence on the effect of radiation therapy. Results: In all three tumor models, we found that tumor oxygenation levels, monitored daily in real time, were increased during the first 4 days of treatment with both anginex and bevacizumab. From treatment day 5 onward, tumor oxygenation in treated mice decreased significantly to below that in control mice. This “tumor oxygenation window” occurred in all three tumor models varying in origin and growth rate. Moreover, during the treatment period, tumor microvessel density decreased and pericyte coverage of vessels increased, supporting the idea of vessel normalization. We also found that the transient modulation of tumor physiology caused by either antiangiogenic therapy improved the effect of radiation treatment. Tumor growth delay was enhanced when single dose or fractionated radiotherapy was initiated within the tumor oxygenation window as compared with other treatment schedules. Conclusions: The results are of immediate translational importance because the clinical benefits of bevacizumab therapy might be increased by more precise treatment scheduling to ensure radiation is given during periods of peak radiosensitivity. The oxygen elevation in tumors by non–growth factor–mediated peptide anginex suggests that vessel normalization might be a general phenomenon of agents directed at disrupting the tumor vasculature by a variety of mechanisms.

In many past clinical studies in which hyperthermia enhanced the efficacy of radiotherapy, the tumor temperatures could be raised only to 40-42 degrees C range in most cases. The heat-induced cell death, cellular radiosensitization, and vascular damage induced by such mild temperature hyperthermia (MTH) are likely to be insignificant despite the increased response of tumors to radiotherapy. Heating rodent tumors at 40-42 degrees C was found to cause an enduring increase in blood flow and oxygenation in the tumors. Recent studies with canine soft tissue sarcoma and human tumor clinical studies also demonstrated that MTH improves tumor oxygenation, and enhances response of the tumors to radiotherapy or chemoradiotherapy. The increased blood flow and vascular permeability caused by MTH may also improve the delivery of various therapeutic agents such as chemotherapy drugs, immunotherapeutic agents and genetic constructs for gene therapy to tumor cells. MTH as a means to potentiate the efficacy of radiotherapy and others warrants further investigation.

Tumor necrosis factor-α (TNF-α) is a potent cytokine with anticancer efficacy that can significantly enhance hyperthermic injury. However, TNF-α is systemically toxic, thereby creating a need for its selective tumor delivery. We used a newly developed nanoparticle delivery system consisting of 33-nm polyethylene glycol–coated colloidal gold nanoparticles (PT-cAu-TNF-α) with incorporated TNF-α payload (several hundred TNF-α molecules per nanoparticle) to maximize tumor damage and minimize systemic exposure to TNF-α. SCK mammary carcinomas grown in A/J mice were treated with 125 or 250 μg/kg PT-cAu-TNF-α alone or followed by local heating at 42.5°C using a water bath for 60 minutes, 4 hours after nanoparticle injection. Increases in tumor growth delay were observed for both PT-cAu-TNF-α alone and heat alone, although the most dramatic effect was found in the combination treatment. Tumor blood flow was significantly suppressed 4 hours after an i.v. injection of free TNF-α or PT-cAu-TNF-α. Tumor perfusion, imaged by contrast enhanced ultrasonography, on days 1 and 5 after treatment revealed perfusion defects after the injection of PT-cAu-TNF-α alone and, in many regions, complete flow inhibition in tumors treated with combination treatment. The combination treatment of SCK tumors in vivo reduced the in vivo/in vitro tumor cell survival to 0.05% immediately following heating and to 0.005% at 18 hours after heating, suggesting vascular damage–mediated tumor cell killing. Thermally induced tumor growth delay was enhanced by pretreatment with TNF-α-coated gold nanoparticles when given i.v. at the proper dosage and timing. [Mol Cancer Ther 2006;5(4):1014–20]