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

在超声分子成像 (USMI) 中，配体功能化微泡 (MB) 用于可视化血管内皮靶标。Netrin-1 在 60% 的转移性乳腺癌中上调并促进肿瘤进展。一种新型的 netrin-1 干扰疗法需要在治疗前评估 netrin-1 的表达。在这项研究中，我们研究了 netrin-1 作为 USMI 的靶标及其作为乳腺癌模型伴随诊断的潜力。

方法：为了验证 netrin-1 的表达和定位，采用体内免疫定位方法，即在肿瘤采集前 24 小时将抗 netrin-1 抗体注射到活体小鼠体内，并用荧光二抗显影以进行免疫荧光分析。通过流式细胞术研究了 Netrin-1 与细胞表面的相互作用。使用 MicroMarker Target-Ready (VisualSonics) 制备靶向 Netrin-1 的 MB，并在静态条件下或在流动室中使用纯化的 netrin-1 蛋白或表达 netrin-1 的癌细胞在体外结合测定中进行验证。体内使用 Vevo 2100 小动物成像设备 (VisualSonics) 在携带人 netrin-1 阳性 SKBR7 肿瘤或弱表达 netrin-1 的 MDA-MB-231 肿瘤的裸鼠中验证 netrin-1 的 USMI。在转基因小鼠 FVB/N Tg(MMTV/PyMT634Mul) (MMTV-PyMT) 乳腺肿瘤中进一步测试了 USMI 的可行性。

结果： Netrin-1 与 netrin-1 阳性乳腺肿瘤中的内皮 CD31 共定位。Netrin-1 与内皮 HUVEC 和癌细胞表面的结合部分由硫酸乙酰肝素蛋白多糖介导。以人源化单克隆抗 netrin-1 抗体为目标的 MB 在静态和动态条件下与表达 netrin-1 的癌细胞结合。与用同种型对照 MB 记录的信号或用人源化单克隆抗 netrin-1 抗体阻断 netrin-1 后记录的信号相比，人 SKBR7 乳腺肿瘤和转基因鼠 MMTV-PyMT 乳腺肿瘤中抗 netrin-1 MB 的 USMI 信号显着增加. 在弱表达 netrin-1 的人类肿瘤和正常乳腺中，用抗 netrin-1 和同种型对照 MB 观察到成像信号没有差异。离体分析证实了 netrin-1 在 MMTV-PyMT 肿瘤中的表达。

结论：这些结果表明，USMI 可以可靠地检测 netrin-1 对 netrin-1 阳性人和小鼠肿瘤的内皮细胞。netrin-1 的 USMI 信号的显着差异反映了在不同乳腺肿瘤模型之间观察到的 netrin-1 mRNA 和蛋白质表达的显着差异。成像方法是无创且安全的，并且近乎实时地提供了 netrin-1 表达状态。因此，netrin-1 的 USMI 有可能成为未来临床试验中 netrin-1 干扰治疗患者分层的伴随诊断。

关键词：超声分子成像，netrin-1，伴随诊断，乳腺癌，靶向微泡