Abstract P4-02-14: PD-1-IRDye800CW as a novel probe for fluorescence molecular imaging and image guided intraoperative surgery in breast tumor mouse model
Authors: Du Y , Chi C , Yang X , Tian J 
Published: March 2017

Abstract
Background:Breast cancer, especially triple-negative breast cancer (TNBC), characterized by lack of expression of estrogen receptor (ER), progesterone receptor (PR), and HER2/neu (HER2), are generally high-grade, aggressive tumors with poorer disease-specific survival than other breast cancer subtypes. Therefore, novel imaging and therapeutic strategies are needed to improve the management of TNBC. Programmed cell death (PD-1) is an immune inhibitory receptor expressed by activated T cells that play a pivotal role in tumor immune evasion. Previous studies showed that intratumoral expression of T-cell inhibitory molecule PD-L1 was associated with high histologic grade and TNBC. Therefore, PD-1 was suggested to be an attractive target for antibody-guided fluorescent tumor imaging of breast tumor. Aim: The aim of this study is to create a novel near infrared fluorescence imaging probe PD-1-IRDye800 CW, which may facilitate a targeted in vivo fluorescence imaging and guide the intraoperative surgery of breast tumors. Methods: The 4T1 breast tumor bearing Balb/c mice were used in this study. PD-1-IRDye800CW probes were injected intravenously, and the ratIgG-IRDye800CW was used as control. The fluorescence imaging was performed at different time points including 0, 1 h, 4 h, 8 h, 12 h and 24 h after probe injection. And the surgery was performed 8 h post-injection with the aid of fluorescence imaging guided surgical navigation system. Results: It showed that PD-1-IRDye800CW reached the tumor site 30 min after tail vein injection. The fluorescence signal gradually accumulated at the tumor region 1 h to 8 h post-injection and then the tumor signal decreased 12 h thereafter. While the IgG-IRDye800CW control mice, the fluorescence signal can also be detected in the tumor region 30 min after injection, and then the signal was restricted to the tumor and kidney regions 1 to 4 h post-injection. Thereafter, most of probe was urinated, and the signal in the tumor regions decreased after 8 h. The fluorescence signal intensities of PD-1-IRDye800CW and IgG-IRDye 800CW probes at the tumor regions were compared under the same fluorescence scale bar, and we found that the PD-1-IRDye800CW probe showed a specific targeting and stable signal at the tumor region. To confirm the in vivo observation, the tumors and major organs were dissected out 8 h post-injection, and we found that the fluorescence signal of the PD-1-IRDye800CW probe in the tumors was stronger than the control probe. Conclusion: Our data suggested that through specific binding to the PD-1 expressing tumor infiltrating lymphocytes (TIL), the PD-1-IRDye800CW probe achieved a targeted imaging of breast tumors in vivo. Furthermore, the PD-1-IRDye800CW can work as an intraoperative imaging probe for the guidance of breast tumor surgery. These findings reinforce the potential of PD-1-IRDye800CW for the theranostic applications in clinics in the future.Background:Breast cancer, especially triple-negative breast cancer (TNBC), characterized by lack of expression of estrogen receptor (ER), progesterone receptor (PR), and HER2/neu (HER2), are generally high-grade, aggressive tumors with poorer disease-specific survival than other breast cancer subtypes. Therefore, novel imaging and therapeutic strategies are needed to improve the management of TNBC. Programmed cell death (PD-1) is an immune inhibitory receptor expressed by activated T cells that play a pivotal role in tumor immune evasion. Previous studies showed that intratumoral expression of T-cell inhibitory molecule PD-L1 was associated with high histologic grade and TNBC. Therefore, PD-1 was suggested to be an attractive target for antibody-guided fluorescent tumor imaging of breast tumor. Aim: The aim of this study is to create a novel near infrared fluorescence imaging probe PD-1-IRDye800 CW, which may facilitate a targeted in vivo fluorescence imaging and guide the intraoperative surgery of breast tumors. Methods: The 4T1 breast tumor bearing Balb/c mice were used in this study. PD-1-IRDye800CW probes were injected intravenously, and the ratIgG-IRDye800CW was used as control. The fluorescence imaging was performed at different time points including 0, 1 h, 4 h, 8 h, 12 h and 24 h after probe injection. And the surgery was performed 8 h post-injection with the aid of fluorescence imaging guided surgical navigation system. Results: It showed that PD-1-IRDye800CW reached the tumor site 30 min after tail vein injection. The fluorescence signal gradually accumulated at the tumor region 1 h to 8 h post-injection and then the tumor signal decreased 12 h thereafter. While the IgG-IRDye800CW control mice, the fluorescence signal can also be detected in the tumor region 30 min after injection, and then the signal was restricted to the tumor and kidney regions 1 to 4 h post-injection. Thereafter, most of probe was urinated, and the signal in the tumor regions decreased after 8 h. The fluorescence signal intensities of PD-1-IRDye800CW and IgG-IRDye 800CW probes at the tumor regions were compared under the same fluorescence scale bar, and we found that the PD-1-IRDye800CW probe showed a specific targeting and stable signal at the tumor region. To confirm the in vivo observation, the tumors and major organs were dissected out 8 h post-injection, and we found that the fluorescence signal of the PD-1-IRDye800CW probe in the tumors was stronger than the control probe. Conclusion: Our data suggested that through specific binding to the PD-1 expressing tumor infiltrating lymphocytes (TIL), the PD-1-IRDye800CW probe achieved a targeted imaging of breast tumors in vivo. Furthermore, the PD-1-IRDye800CW can work as an intraoperative imaging probe for the guidance of breast tumor surgery. These findings reinforce the potential of PD-1-IRDye800CW for the theranostic applications in clinics in the future.
Citation Format: Du Y, Chi C, Yang X, Tian J. PD-1-IRDye800CW as a novel probe for fluorescence molecular imaging and image guided intraoperative surgery in breast tumor mouse model [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-02-14.