Vascular targeting tumor therapy: non-invasive contrast enhanced ultrasound for quantitative assessment of tumor microcirculation.

The aim of the study was to quantitatively assess tumor microcirculation upon vascular targeting tumor therapy by non-destructive contrast enhanced ultrasonography (CEUS) and to validate this technology by correlation with high-resolution intravital fluorescence microscopy (IVM). Subcutaneous Lewis Lung carcinomas (LLC-1) carcinomas were established in mice. A-MEL-3 melanomas were grown in dorsal skinfold chambers of hamsters to permit bimodal imaging of tumor microcirculation by CEUS and IVM. Animals were treated by i.p. injection of ZD6126 and CEUS imaging after bolus injection of microbubbles was performed. Red blood cell velocity (VRBC), segmental blood flow (Q) and microcirculatory perfusion (PI) of tumors was quantified by IVM. Change in signal intensity (SI) from baseline (ΔSI), rate of SI increase (RSI) and area below intensity time curves (AUC) were calculated in tumors by analysis of CEUS data. Microvessel density was measured by quantitative analysis of CD31 immunohistochemistry. The Mann-Whitney test was used to evaluate differences between groups. Spearman correlation test was used to investigate the relation between CEUS and IVM parameters or histologic CD31 count. ΔSI, RSI and AUC values in ZD6126 treated tumors were lower compared to untreated controls. Comparing central and peripheral tumor regions a vascularized viable rim in the tumor periphery could be detected by CEUS imaging. For the entire cohort ΔSI, RSI and AUC values positively correlated with VRBC, Q and PI quantified by IVM. In LLC-1 carcinomas a positive correlation between ΔSI, RSI and AUC and histological assessment of tumor vascularity was found. In conclusion tumor vascular response to vascular targeting therapy can be quantified non-invasively by CEUS. Bimodal tumor imaging by intravital microscopy and CEUS represents an experimental tool to further develop molecular imaging of tumor microcirculation by CEUS.