Ultrasound Molecular Imaging of Netrin-1

Project Description

In ultrasound molecular imaging (USMI), ligand-functionalized microbubbles (MBs) are used to detect specific vascular endothelial targets. Netrin-1 is a laminin-like, secreted protein of 75 kDa and  upregulated in 60% of metastatic breast cancers1. Overexpressed Netrin-1 promotes cell survival through dependence receptor binding and tumor angiogenesis2. A newly developed targeted therapy which interferes with Netrin-1 binding requires the identification of eligible patients3. Here, we explore Netrin-1 as a new target for USMI and its potential as a companion diagnostic in different murine breast cancer models.

Flow chamber assays showed specific binding of anti-Netrin-1-MBs to human and murine breast cancer cell lines overexpressing Netrin-1, confirming binding specificity and affinity of the MBs for Netrin-1. In tumor models positively expressing Netrin-1 (MMTV, SKBR7, 4T1), USMI showed a statistically significant increased differential targeted enhancement (dTE) with anti-Netrin-1 MBs compared to dTE values of isotype control MBs or dTE values after blocking of Netrin-1. In tumor models negative for Netrin-1 (MDA-MB-231, 67NR) and in normal MMTV glands, USMI showed statistically significant lower dTE values than in positive tumors. Immunohistochemistry confirmed the expression of Netrin-1 and co-localization of Netrin-1 with the vascular marker CD31 in MMTV, 4T1, and SKBR7 tumors. In conclusion, USMI allowed detection of Netrin-1 on tumor vessels and differentiation between Netrin-1-positive and Netrin-1-negative breast cancers. USMI of Netrin-1 may become a companion diagnostic for breast cancer patient stratification and therapy monitoring.

Figure 1: USMI in MMTV breast cancer. 
B-mode imaging served for anatomical guidance. For USMI, Netrin-1-targeted MBs (MBsNetrin-1) and isotype control MBs (MBsIsotype) were used. USMI signal is presented as differential targeted enhancement (dTE). A significant difference in imaging signal between MBsNetrin-1 and MBsIsotype confirmed binding specificity. In the blocking experiment, mice were injected with anti-Netrin-1 antibody 24 h prior to imaging. The significant decrease in signal intensity after blocking indicated specificity of the novel anti-Netrin-1 contrast agent.

Staff

LabTAU: Jennifer Wischhusen (PhD student), Rodolfo Molina-Peña (internship student), Jacqueline Ngo (Engineer), Dr. Frederic Padilla (CNRS researcher)

Cancer Research Center Lyon: Dr. Jean-Guy Delcros (INSERM researcher), Dr. Benjamin Gibert (INSERM researcher), Dr. Patrick Mehlen (INSERM Researcher)

University of Stanford, School of Medicine, Department of Radiology: Dr. Katheryne E. Wilson (Instructor), Dr. Juergen K. Willmann (Professor)

 

Publications

1.     Fitamant et al. Proc Natl Acad Sci 105, 4850–4855 (2008)

2.     Castets & Mehlen. Cell Cycle 9, 1466-1471 (2010)

3.     Mehlen & Guenebeaud. Curr Opin Oncol 22, 46–54 (2010)