Ultrasound molecular imaging as a non-invasive companion diagnostic for netrin-1 interference therapy in breast cancer

Abstract

In ultrasound molecular imaging (USMI), ligand-functionalized microbubbles (MBs) are used to visualize vascular endothelial targets. Netrin-1 is upregulated in 60% of metastatic breast cancers and promotes tumor progression. A novel netrin-1 interference therapy requires the assessment of netrin-1 expression prior to treatment. In this study, we studied netrin-1 as a target for USMI and its potential as a companion diagnostic in breast cancer models.Methods: To verify netrin-1 expression and localization, an in vivo immuno-localization approach was applied, in which anti-netrin-1 antibody was injected into living mice 24 h before tumor collection, and revealed with secondary fluorescent antibody for immunofluorescence analysis. Netrin-1 interactions with the cell surface were studied by flow cytometry. Netrin-1-targeted MBs were prepared using MicroMarker Target-Ready (VisualSonics), and validated in in vitro binding assays in static conditions or in a flow chamber using purified netrin-1 protein or netrin-1-expressing cancer cells. In vivo USMI of netrin-1 was validated in nude mice bearing human netrin-1-positive SKBR7 tumors or weakly netrin-1-expressing MDA-MB-231 tumors using the Vevo 2100 small animal imaging device (VisualSonics). USMI feasibility was further tested in transgenic murine FVB/N Tg(MMTV/PyMT634Mul) (MMTV-PyMT) mammary tumors.Results: Netrin-1 co-localized with endothelial CD31 in netrin-1-positive breast tumors. Netrin-1 binding to the surface of endothelial HUVEC and cancer cells was partially mediated by heparan sulfate proteoglycans. MBs targeted with humanized monoclonal anti-netrin-1 antibody bound to netrin-1-expressing cancer cells in static and dynamic conditions. USMI signal was significantly increased with anti-netrin-1 MBs in human SKBR7 breast tumors and transgenic murine MMTV-PyMT mammary tumors compared to signals recorded with either isotype control MBs or after blocking of netrin-1 with humanized monoclonal anti-netrin-1 antibody. In weakly netrin-1-expressing human tumors and normal mammary glands, no difference in imaging signal was observed with anti-netrin-1- and isotype control MBs. Ex vivo analysis confirmed netrin-1 expression in MMTV-PyMT tumors.Conclusions: These results show that USMI allowed reliable detection of netrin-1 on the endothelium of netrin-1-positive human and murine tumors. Significant differences in USMI signal for netrin-1 reflected the significant differences in netrin-1 mRNA & protein expression observed between different breast tumor models. The imaging approach was non-invasive and safe, and provided the netrin-1 expression status in near real-time. Thus, USMI of netrin-1 has the potential to become a companion diagnostic for the stratification of patients for netrin-1 interference therapy in future clinical trials.

Ultrasound Molecular Imaging as a Potential Non-invasive Diagnosis to Detect the Margin of Hepatocarcinoma via CSF-1R Targeting

“…Nanobubble CSF-1R had an average size of about 428 nm and were ultrasound-visible even at 20 MHz both in vitro and in vivo ; imaging was still viable even when diluted 2000 times. Notably, NBs were administered at a low concentration compared with our previous research and other studies ( Wischhusen et al, 2018 ), a technique which can be employed to reduce the level of background signal and modulate facilitate the comparison of heterogeneous tumor models ( Wang et al, 2010 , 2016 ).…”

Applications in Molecular Ultrasound Imaging: Present and Future

“…Xu explored the dual-targeting capability of this novel contrast agent further and found that dual-targeted agent had more intense signal than single-targeted agent [16]. Wischhusen created netrin-1 microbubbles as a potential diagnostic and therapeutic marker for interference therapy in breast cancer [17].…”

Molecular ultrasound imaging of neutrophil membrane-derived biomimetic microbubbles for quantitative evaluation of hepatic ischemia-reperfusion injury

“…Contrast-enhanced ultrasound (CEUS) combining ultrasound contrast agents (UCAs) and contrast-specific imaging techniques has already become a valuable clinical technique for assessing hepatic perfusion after OLT due to its advantages including no ionizing radiation, real-time detection, and reproducibility 8 , 9 . As an important branch of CEUS, molecular ultrasound (US) imaging has emerged as a new method for visualizing molecular targets using ligand-functionalized microbubbles (MBs), thus providing anatomical and functional information simultaneously with high sensitivity and specificity 10 , 11 . Currently, molecular US imaging is widely used in preclinical research and has shown good accuracy 12 , 13 .…”