Ultrasound assessment of angiogenesis in a matrigel model in rats

Abstract

Matrigel, a basement membrane extract, has been extensively used in in vivo angiogenesis. Contrast ultrasound imaging (CUI) of implanted Matrigel plugs with (+bFGF) and without basic fibroblast growth factor (−bFGF) was performed 7 and 14 d after implantation, followed by histologic analysis. Statistically significant differences between +bFGF and −bFGF plugs were apparent at d 7 in both plug size and contrast enhancement (both p < 0.05). Histopathology revealed differences in microvessel density (MVD) between +bFGF and −bFGF at d 7 and d 14. A significant correlation between MVD and both power Doppler contrast-enhanced area (r = 0.65, p < 0.05) and fraction of plug enhanced (r = 0.59, p < 0.05) was present. CUI of Matrigel plugs was shown to be a robust method for distinguishing between two different angiogenic states. Ultrasound measurements of blood flow in the plugs correlated with MVD, a histologic technique used to quantify tumor angiogenesis. (E-mail: [email protected])

Introduction

Angiogenesis is a fundamental process by which new blood capillaries develop from a preexisting microvascular network stimulated by different growth factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and platelet derived growth factor. Solid tumors must continuously stimulate the growth of new blood vessels to enlarge, and furthermore, these blood vessels provide a gateway for tumor cell metastasis (Folkman and Shing 1992, Nehls et al 1994, Wiggins et al 1995, Carmeliet and Jain 2000, Liekens et al 2001, Bergers and Benjamin 2003). Researchers have shown that angiogenesis is involved in many disease processes and is, in particular, an important prognostic factor in cancer development, progression and recurrence (Horak et al 1992, Weidner et al 1992, Wiggins et al 1995).

Because of the fundamental role of angiogenesis in tumor development, several angiogenesis assays have been developed over the past few decades to investigate angiogenesis and antiangiogenic therapies. Some examples of commonly used assays include the chicken embryo chorioallantoic membrane (CAM) assay (Auerbach et al. 1974), the dorsal skin fold chamber (Algire 1943), the corneal micropocket assay (Gimbrone et al 1974, Ausprunk and Folkman 1977) and Matrigel plug or sponge assays (Malinda2001, Akhtar et al 2002, Kragh et al 2003). Although in vitro angiogenesis assays have the advantages of simplicity and repeatability, they lack the biologic complexity of in vivo angiogenesis assays. Therefore, in vivo angiogenesis assays are necessary to more thoroughly understand this complex process. The Matrigel angiogenesis assay is easy to perform and reproducible, and the progression of angiogenesis can be observed within a few days. Matrigel is a soluble basement membrane extract of the Engelbreth-Holm-Swarm tumor. It has been demonstrated that when Matrigel is injected subcutaneously, endothelial cells migrate into the Matrigel plug and form vessels. This angiogenic process can be accelerated by adding known angiogenic growth factors, such as bFGF, to the Matrigel. The newly formed vessels can contain erythrocytes, indicating that they are functional capillaries (Passaniti et al 1992, Malinda2001, Auerbach et al 2003).

Angiogenic activity is an important feature of solid neoplasms that may be predictive of biologic behavior and response to therapy but few techniques exist for noninvasively quantifying angiogenesis in a clinical setting. The only existing validated method for angiogenesis measurement in vivo is the histologic measurement of microvessel density (MVD). The MVD measurement in isolated regions of high vessel density (hotspots) from biopsy specimens or excised tumors appears to be useful in predicting metastasis risk, tumor growth and recurrence. However, the degree of MVD has not been proven to be a prognostic factor for angiogenic activity in a tumor or useful in planning anti-angiogenesis treatment (Hlatky et al 2002, McDonald and Choyke2003). In addition, MVD measurement is an invasive technique and representative tumor sampling is difficult due to tumor heterogeneity. Therefore, there is an acute need for developing imaging techniques that can quantitatively estimate angiogenesis in whole tumors, and in using these techniques to direct cancer therapy (McDonald and Choyke2003, Miller et al 2005).

Developing imaging strategies to evaluate angiogenic and anti-angiogenic responses and comparing the accuracy, specificity and sensitivity of different imaging techniques requires a standardized, reproducible model. Many angiogenesis imaging studies have been performed in tumor models (Fleming and Brekken 2003, Kiessling et al 2003, Krix et al 2003), but comparison across different studies is difficult because of differences in tumor cell lines, animal subjects, techniques and equipment. The Matrigel implant model allows the evaluation of pro- and antiangiogenic compounds. In addition, this model mimics the hypoxic microenvironment of a tumor, providing a good model to study tumor angiogenesis (Hasan et al. 2004). However, published studies using imaging methods for the evaluation of angiogenesis in Matrigel models are sparse. Phongkitkarun et al. (2004) used contrast-enhanced functional computed tomography (fCT) in a rat Matrigel model, but reported that fCT-derived measurements did not correlate well with MVD or other histologic measures of angiogenesis. Leong-Poi et al. (2003) successfully used contrast-specific ultrasound imaging of a mouse Matrigel model to study both blood flow and targeted contrast agent retention, but did not compare ultrasound-derived measurements to MVD. Lucidarme et al. (2004) used contrast ultrasound to study Matrigel plugs in both mice and rats. This study reported a near-significant correlation between ultrasound degree of enhancement and macroscopic angiogenic response in mice. However, these researchers found that the thicker rat skin interfered with ultrasound assessment of Matrigel plugs injected on either side of the spine.

Here, we report the results of a preliminary study using Matrigel plugs implanted in the caudo-ventral region of a rat. We were interested both in evaluating this model for angiogenesis imaging, and in comparing our contrast ultrasound-derived measurements to MVD, a standard measurement of angiogenesis.