OBJECTIVE. Tumor growth and progression require the formation of new blood vessels from preexisting vasculature, a process called angiogenesis. The ability to noninvasively visualize angiogenesis may provide new opportunities to more appropriately select patients for antiangiogenesis treatment and to monitor treatment efficacy.
CONCLUSION. The superior molecular sensitivity of PET and the lack of radiation from MRI and contrast-enhanced ultrasound put these techniques at the forefront of clinical translation.
The formation of new blood vessels from preexisting vasculature (angiogenesis) is a critical part of tumor growth and progression. Solid tumors cannot grow beyond the size of a few millimeters without inducing the proliferation of endothelium and the formation of new blood vessels. In the absence of angiogenesis, cells reach equilibrium rapidly with their rate of death and stop expanding. Imaging the various components of angiogenesis may provide new insights into cancer biology pathways.
Tumor growth is affected by inhibition of angiogenesis, and this may even result in tumor regression, as shown in various experimental models. Antiangiogenic drugs have proven to be mainly cytostatic, slowing or stopping tumor growth and preventing metastasis. Numerous molecular pathways regulate angiogenesis, including vascular endothelial growth factor (VEGF), matrix metalloproteinases (MMPs), endoglin (CD105), α v β 3 integrin, and E-selectin. Each of these pathways can be a target for diagnostic and therapeutic interventions. The ability to noninvasively evaluate angiogenesis may lead to opportunities to more appropriately select patients for antiangiogenesis treatment or to monitor the efficacy of targeted treatments. VEGF, α v β 3 integrin, and MMPs have been pursued as potential targets for imaging angiogenesis.
Note—MMP = matrix metalloproteinases, VEGF = vascular endothelial growth factor.
In this article, we review preclinical and clinical studies evaluating angiogenesis imaging and summarize current advances in this field for a spectrum of imaging techniques.
Conventional imaging with contrast-enhanced CT relies on changes in size to evaluate response to therapy. Response Evaluation Criteria in Solid Tumors (RECIST) were published in 2000. Key features of the original RECIST include definitions of minimum size of measurable lesions; instructions on how many lesions to follow (up to 10, a maximum five per organ site); and the use of unidimensional, rather than bidimensional, measures for overall evaluation of tumor burden. The recently revised RECIST concluded that, at present, there is not sufficient standardization or evidence to abandon anatomic assessment of tumor burden. However, targeted therapies, especially early on, may not cause a significant change in the size of the lesions, despite efficacy at the molecular level. Thus, early assessment of response to such treatments may not be accurate using conventional imaging. In contrast, molecular imaging is the visualization, characterization, and measurement of biologic processes at the molecular and cellular levels in humans and other living systems. The techniques used in molecular imaging include PET, SPECT, molecular MRI, optical fluorescence, optical bioluminescence, and targeted contrast-enhanced ultrasound.
Functional imaging is used for detection or measurement of changes in metabolism, blood flow, regional chemical composition, or absorption. Perfusion MRI and CT perfusion are examples of functional imaging. In SPECT and PET, the patients are imaged after they have been injected with a radiotracer that circulates within them and is incorporated into various cellular processes, providing functional information. The introduction of the combined SPECT and CT (SPECT/CT), as well as the combination of PET and CT (PET/CT), greatly enhanced the utility of these techniques. The expanding use of PET/CT for oncology brought molecular and functional imaging to the clinical front line. The PET radiopharmaceutical 18 F-FDG is useful for functional imaging of a wide variety of malignancies and contributes to the clinical success of PET. However, FDG PET is not directly evaluating the vascular changes occurring in a malignant tumor. Both CT perfusion and molecular MRI have been investigated as possible methods for imaging angiogenesis. Molecular MRI can be used to noninvasively measure the efficacy of angiogenesis inhibitors during the course of therapy, as shown by Mulder et al., who used α v β 3 integrin–targeted bimodal liposomes to quantitate angiogenesis and to evaluate the therapeutic efficacy of angiogenesis inhibitors in a tumor mouse model. Others used molecular MRI with gadolinium 3+-complexed nanoparticle T1 sequences and iron oxide nanoparticle T2 sequences for angiogenesis imaging.
Targeted contrast-enhanced ultrasound imaging is increasingly being recognized as a powerful imaging tool for the detection and quantification of tumor angiogenesis at the molecular level. In an era of legitimate public concern and scrutiny related to radiation exposure resulting from medical imaging, the use of a nonionizing radiation technique such as ultrasound is very attractive to investigators. Contrast-enhanced ultrasound uses IV-administered gas-filled microbubbles as the contrast medium. The microbubbles are too large to leave the vasculature and, therefore, can be used to target vascular endothelial proteins of interest by coating the surface of the bubbles with specific ligands. Contrast-enhanced ultrasound with microbubbles targeted to α v integrin expressed on the neovascular endothelium has been used to image tumor vascular integrin status. Willmann et al. have shown that integrin-binding knottin peptides (a small disulfide-rich protein characterized by a special “disulfide through disulfide knot”) can be conjugated to the surface of microbubbles and used for in vivo targeted contrast-enhanced ultrasound imaging of tumor angiogenesis. Investigators in our group also showed that dual-targeted contrast-enhanced ultrasound directed not only at α v β 3 integrin but also at VEGF receptor 2 improves the in vivo visualization of tumor angiogenesis in a human ovarian cancer xenograft in mice.
Fig. 1 —Mice with human colon tumor xenografts. Molecular ultrasound imaging with microbubbles targeted to human kinase domain insert receptor cross-reacting with mouse vascular endothelial growth factor receptor 2 can be used to quantify human kinase domain insert receptor and vascular endothelial growth factor receptor 2 expression levels. Brightness-mode ultrasound scanning of human colon tumor xenografts in nude mice was performed using dedicated small-animal ultrasound imaging system and 40-MHz transducer (Vevo770, VisualSonics). Molecular ultrasound signal was calculated and displayed (green overlay) by postprocessing of destruction replenishment imaging sequence. (Courtesy of Pysz MA and Willmann JK, Stanford University, Stanford, CA)
Because Arg-Gly-Asp (RGD) peptides strongly bind to α v β 3 integrin, a significant number of research projects focused on multimodality imaging of integrin expression are based on this RGD peptide sequence. RGD peptides exhibit high α v β 3 integrin affinity in vitro and receptor-specific tumor uptake in vivo. Near-infrared fluorescent dyes conjugated with the cyclic RGD peptide were used to visualize subcutaneously inoculated integrin-positive tumors. Chen et al. reported that the combination of the specificity of the RGD peptide-integrin interaction with the near-infrared fluorescence detection could be applied to noninvasive imaging of integrin expression and monitoring antiintegrin treatment efficacy and provided near real-time measurements in U87MG glioblastoma xenografts.
Although methods such as optical fluorescence and optical bioluminescence are attractive to preclinical investigators and can be used for proof-of-concept studies, the future clinical translation of angiogenesis imaging must rely on techniques that can be widely used in patient cohorts. Dynamic contrast-enhanced (DCE) MRI, which is particularly attractive for its noninvasiveness, lack of ionizing radiation, and high spatial resolution, has been used for measuring properties of tumor microvasculature. DCE-MRI provides the tracking of low-molecular-weight contrast agents through blood vessels, in particular the vasculature of tumors. By analyzing the pharmacokinetics of the contrast agent into a specific tumor, it is possible to measure alterations in vascular permeability, blood flow, and extracellular volumes. Several measurable hemodynamic parameters, such as the bidirectional transfer coefficient K trans, are already established and have been used in evaluation of antiangiogenic therapies. DCE-MRI may be useful for establishing whether a specific cancer therapy has been successful and has been tested in preclinical studies. In an animal model, Sipkins et al. found that it is feasible to image α v β 3 integrin expression using MRI and paramagnetic liposomes coated with a monoclonal antibody. Such work laid the foundation for clinical translation of m
