Abstract Body:

Introduction:

The standard treatment for early breast cancer is conservative surgery; however, positive surgical margins pose a significant risk factor for local recurrence post-surgery. Currently, there is a lack of accurate methods to assess surgical margins, resulting in a high proportion of positive margins and subsequent postoperative recurrence. Studies have shown that the rate of positive surgical margins in HER2-positive breast cancer patients is significantly higher than other subtypes. Near-infrared fluorescence imaging has the advantages of in vivo, real-time, and dynamic imaging, and the Near-infrared II (NIR-II) provides higher resolution and deeper tissue visualization compared to the Near-infrared I (NIR-I) . Indocyanine green (ICG) fluorescent dye has a long tail signal in the NIR-II , which has good application prospects in visualizing malignant tumors and intraoperative navigation, and can help doctors complete the resection of the tumor lesion.

Methods:

First, Trastuzumab was conjugated with ICG to obtain an NIR-II fluorescent molecular probe, ICG-Tra, which targets HER2, and its properties were characterized and optimized. Then, a stable transfectant cell line, MDA-MB-231-luc-HER2, with high expression of HER2 was constructed using the triple-negative breast cancer cell line MDA-MB-231-luc, and the safety and targeting of the probe were verified in cell and mouse models. In addition, the feasibility of the probe to visualize HER2 in vivo was validated in MMTV-PyVT mouse model.

Results:

1. The ICG-Tra has been successfully synthesized, exhibiting excellent NIR-II optical properties.
2. The ICG-Tra demonstrated negligible cytotoxicity towards cells and tissues.
3. When the ICG-Tra was incubated with cells expressing different levels of HER2，the fluorescence intensity of MDA-MB-231-luc-HER2 cells was significantly enhanced compared with MDA-MB-231-luc-NC, which was about 3 times that of the former. When the trastuzumb was added early for blocking，the fluorescence intensity decreased by about 70%.
4. Following injection of ICG-Tra into the tail vein of HER2-overexpressing tumor-bearing mice, the TBR(Tumor-to-Background Ratio) reached its peak after 36 hours（TBR=6.4）, which was significantly reduced （TBR=3.0）when Trastuzumab was early injected.
5. MMTV-PyVT mouse showed that the fluorescence intensity of different mammary gland tumors in different weeks and in the same mouse were different, but they were higher than that in normal mammary gland tissues without cancer, which was related to HER2 expression.

Conclusions:

The results of the present study demonstrate that the ICG-Tra exhibits excellent specificity for HER2 targeting on both cellular and animal levels, enabling real-time in vivo visualization of HER2-positive breast cancer lesions and margins. In future investigations, we will further explore its potential application in precision image guide surgery. 

Image/Figure:

Click to view full size

Image/Figure Caption:

A.The protein expressions of HER2 and GAPDH were determined by western blotting in MDA-MB-231-Luc-NC and MDA-MB-231-Luc-HER2 cells.

B.MDA-MB-231-Luc-NC and MDA-MB-231-Luc-HER2 cells incubated for 4 hours with ICG-Tra，with or without free trastuzumab blocking.

C.Quantified fluorescence intensities of different cells , unblock and block groups in B.

D.NIR-II fluorescence imaging of tumor-bearing mice with different expression of HER2 after 36h intravenous injection of ICG-Tra with or without the injection of trastuzumab.

E.Quantification analysis and the contrast of TBR between unblock and block groups in D.

F.Tumor targeting by ICG-Tra in MMTV-PyVT mouse in different weeks.

Author