Theranostics 2018; 8(18):5126-5142. doi:10.7150/thno.27221 This issue

Research Paper

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

Jennifer Wischhusen1,2,3, Katheryne E. Wilson2, Jean-Guy Delcros3, Rodolfo Molina-Peña1, Benjamin Gibert3, Shan Jiang3, Jacqueline Ngo1, David Goldschneider4, Patrick Mehlen3,4*, Juergen K. Willmann2*†, Frederic Padilla1✉*

1. Univ Lyon, Université Lyon 1, Centre Léon Bérard, INSERM, LabTAU, F-69003, LYON, France.
2. Department of Radiology, School of Medicine, Stanford University, 94305 Stanford, USA.
3. Apoptosis, Cancer and Development Laboratory - Equipe labellisée 'La Ligue', LabEx DEVweCAN, Centre de Cancérologie de Lyon, INSERM U1052-CNRS UMR5286, Centre Léon Bérard, 69008 Lyon, France ;
4. Netris Pharma, 69008 Lyon, France.
*P. Mehlen, J. K. Willmann and F. Padilla contributed equally as senior authors of this article.
†deceased on January 8, 2018

This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (https://creativecommons.org/licenses/by-nc/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Citation:
Wischhusen J, Wilson KE, Delcros JG, Molina-Peña R, Gibert B, Jiang S, Ngo J, Goldschneider D, Mehlen P, Willmann JK, Padilla F. Ultrasound molecular imaging as a non-invasive companion diagnostic for netrin-1 interference therapy in breast cancer. Theranostics 2018; 8(18):5126-5142. doi:10.7150/thno.27221. Available from https://www.thno.org/v08p5126.htm

File import instruction

Abstract

Graphic 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.

Keywords: ultrasound molecular imaging, netrin-1, companion diagnostic, breast cancer, targeted microbubbles