Gene Silencing in Human Tumor Xenograft Models: The Effect of VEGF and ECM Downregulation on Ultrasound Backscatter

Abstract

Purpose: : Using an experimental tumor system with upregulated VEGF and laminin expression (important factors in tumor growth and metastasis) an increase US backscatter can be shown to relate to increased vascular and extracellular matrix density. In this study using a more biologically relevant model, where the constitutive expression of genes is reduced using systemic application of short interfering RNA, we evaluated the effect of these critical growth factors on tumor backscatter.

Methods: : M619 human tumor xenografts were grown in nu/nu athymic nude mice. Selected tumors were treated with SQ siRNA shown to reduce tumor growth in–vivo. Control tumors were untreated. Mice were scanned with a 3–D VHF ultrasound system to obtain parameter images and calibrated ensemble–averaged power spectrum measurements, as well as tumor volume. We examined the relationship between US backscatters and angiogenesis semi–quantitatively using immunohistochemistry with antibodies to CD–31 and laminin and quantitatively using FACS to determine percentage of cells expressing the given protein.

Results: : Both VEGF–siRNA and laminin–siRNA tumors show global reduction in backscatter (MBF) compared with control tumors. A correlation with reduction in vascular and ECM expression as measured by FACS was also seen.

Conclusions: : The downregulation of genes that control angiogenesis and the deposition of extracellular matrix patterns both influence tumor backscatter in xenograft models. This is consistent with clinical studies that correlate vascular density and ECM features with US backscatter. To further improve clinical evaluation of tumor backscatter the interactions of these and other genes active in tumor growth and metastasis need to be evaluated in multiple downregulated systems.

Keywords: melanoma • imaging/image analysis: non-clinical • gene modifiers