Imaging of tumor-induced sentinel lymph node lymphangiogenesis with LyP-1 peptide

Objectives Lymphangiogenesis in tumor-draining lymph nodes (LNs) starts before the onset of metastasis and is associated with metastasis to distant LNs and organs. In this study, we aimed to visualize tumor induced lymphangiogensis with a tumor-lymphatics-specific peptide Lyp-1.

Methods The LyP-1 peptide was labeled with a near-infrared fluorophore (Cy5.5) for optical imaging. A tumor model was developed by subcutaneous injection of 4T1 murine breast cancer cells. At day 3, 7, 14 and 21 after tumor inoculation, Cy5.5-LyP-1 was administered through the middle phalanges of the upper extremities to the tumor bearing mice. At 45 min and 24 hr postinjection, brachial LN fluorescence imaging was performed with a Maestro II small-animal imaging system. Ex vivo fluorescence images were acquired for quantitative analysis of the fluorescence intensity. PET imaging also was performed with ⁶⁴Cu-DOTA-LyP-1. MRI was acquired to provide anatomical positions of the tumor and LNs.

Results Tumor induced lymphangiogenesis was confirmed by increased size of tumor side brachial LNs and LYVE-1 immunostaining. Cy5.5-LyP-1 staining in LNs co-localized with LYVE-1, indicating the lymphatics-specific binding of LyP-1 peptide. The brachial LNs were clearly visualized by optical imaging at both time points. The tumor side LNs showed significantly higher fluorescence intensities than the contralateral brachial LNs at day 7, 14, and 21 after tumor inoculation. Tumor-draining brachial LNs showed extensive growth of lymphatic sinuses throughout the cortex and medulla. Static small animal PET images also revealed higher uptake of ⁶⁴Cu-DOTA-LyP-1 in the tumor-draining brachial LNs at day 21 after tumor inoculation.

Conclusions Optical and PET imaging using LyP-1 based imaging probes offer a powerful tool to study tumor induced lymphangiogenesis. LyP-1 may serve as a marker of lymphangiogenesis for detecting “high risk” lymph nodes before tumor metastasis and after micro-metastasis, as well as for screening of potential anti-lymphatic therapies