Experimental Study On Intraoperative Imaging And Postoperative Prevention Of Recurrence And Metastasis By Targeting PD-1 In Triple Negative Breast Cancer Mice

Background and Aim:Breast cancer is the leading cause of cancer in Chinese women,among which triple negative breast cancer(TNBC)accounts for about 20%of all breast cancer types.TNBC is characterized by rapid progress,easy recurrence,and high metastasis and lack specific therapeutic targets.Surgical removal is considered as the standard treatment for it.The aim of this study was to develop a near-infrared fluorescence imaging probe PD-1-IRDye800CW and explore its application to guide immunocompetent 4T1 mouse tumor resection.Further,the effects of the combination immunotherapy with anti-PD-1 and anti-CTLA-4 were investigated on postoperative recurrence and lung metastasis and its potential mechanism.Methods:Immunohistochemistry and flow cytometry were used to analyze the expression position of PD-1 in 4T1 tumor and the expression ratio of PD-1 in lymphocyte population in tumor microenvironment.PD-1 mAbs and near-infrared dye IRDye800CW was conjugated with chemical coupling.The fluorescent probe PD-1-IRDye800CW physicochemical property was analyzed by ultraviolet-visible spectrometer and fluorescence spectrometer.PD-1-IRDye800CW immunoreactivity was examined in mouse splenic cells and analyzed by flow cytometry.Fluorescence molecular imaging was performed to investigate the probe distribution and its tumor targeting effects in vivo with the IVIS Imaging Spectrum System.HE staining further was used to verified the probe toxicity in vivo.For real-time intraoperative fluorescence image-guided surgery,surgery was performed 24h after probe injection under a fluorescence stereomicroscope(ex 745 nm,em 800 nm).The mice were imaged by bioluminescence imaging before and after surgery to detect the tumor residuals.Postoperative survival was observed between PD-1-IRDye800CW and traditional surgery groups over 30-day observation.In order to inhibit tumor postoperative metastasis,PD-1 inhibitor was used after near-infrared fluorescence surgery.Serum IFN-y and IL-18 were used to monitor the treatment effect of anti-PD-1.To further enhance the inhibition of tumor recurrence and metastasis after surgery,the combination immunotherapy with anti-PD-1 and anti-CTLA-4 were used in postoperative mice.Bioluminescence imaging was used to dynamically monitor the postoperative recurrence and lung metastasis after each treatment.And the survival time of postoperative mice was used as the main prognostic indicator.To explore the mechanism of combined therapy in TNBC,multispectral immunofluorescence staining was used to analyze the changes of CD8+ and CD4+T cells at different groups before and after combination immunotherapy.And the accumulation of CD8+T cells at the tumor margin with different treatments was also analyzed in 4T1 tumor-bearing mice.Results:PD-1 was mainly expressed on the immune cell populations in the TNBC tumor microenvironment.The expression rates of CD4+ and CD8+ T cells are 27.6%and 25.6%respectively,suggesting that PD-1 has the potential to be used as an imaging and therapeutic target for TNBC.Successful synthesis of PD-1-IRDye800CW near-infrared fluorescence molecular probe,the probe has good fluorescence properties.The stability of the probe was further investigated.Moreover,the probe had good live-cell-binding affinity with mouse splenic cells.PD-1-IRDye800CW had more specific tumor targeting effects after 48h injection.The quantification of the tumor-to-background revealed that the signals produced by PD-1-IRDye800CW were more specific than the control probe,and the intensity of the targeted probe at 8h was about 1.7 times higher.For fluorescence image-guided surgery,the targeted probe can determine the tumor location,distinguish between the tumor and surrounding tissues,reduce the postoperative residuals,and extend the postoperative survival time,suggesting that the targeted probe can effectively guide 4T1-fLuc mice tumor resection.Postoperative combined with anti-PD-1 can reduce metastasis by upregulation of serum IFN-y and IL-18 and further prolong the postoperative mice survival.Moreover,the combination of anti-PD-1 and anti-CTLA-4 could further prevent the traditional postoperative recurrence and lung metastasis and prolong mice survival time.The potential mechanism of combination immunotherapy was by upregulation of CD8+and CD4+T cells and increasing the CD8+T infiltration of the tumor margin.Conclusion:PD-1-IRDye800CW could be used for TNBC image-guided tumor resection and the combination immunotherapy with anti-PD-1 and anti-CTLA-4 could inhibit postoperative recurrence and lung metastasis.