F4/80-targeted MR Molecular Imaging In Mice With Colon Cancer:In Vivo MR Detection Of Tumor-associated Macrophages

Objective:To detect tumor-associated macrophages(TAMs)and monitor their dynamic migration in mice with colon cancer using a targeted USPIO contrast agent F4/80-USPIO.Methods:(1)The physicochemical properties of F4/80-USPIO and USPIO were determined,and T2 relaxation rates of them were compared.(2)Cell cytotoxicity was examined by CCK-8 assay to assess whether F4/80-USPIO had an effect on cell viability.(3)Specific molecular probes developed by conj ugating F4/80 antibodies with USPIO,were co-incubated with macrophages,and the uptake of probes by macrophages was assessed by using Prussian blue staining.(4)The expression rate of F4/80 in colon cancer tissues was detected by incubating F4/80 antibodies with colon cancer tissue sections and DAB staining.(5)The phenotypes of macrophages were identified by flow cytometry.The fluorescent-labeled F4/80 antibodies were co-incubated with macrophages and then macrophage F4/80 receptor cell surface expression rate and its binding rate with F4/80 antibodies were assessed by flow cytometry.(6)A subcutaneous transplantation tumor model of human colon cancer was established using mice.(7)MR axial T2WI plain scan and enhanced scan were performed respectively in the tumor bearing mice.Measurement of T2 signal intensity changes were performed respectively before and after tail vein injection of F4/80-USPIO.Results:The experimental results show that the USPIO conjugated with F4/80 has a proper particle size,maintaining a relatively constant concentration in blood,with a high relaxation rate(R2)and without obvious cytotoxicity to normal tissues.It was confirmed that F4/80-USPIO can be used as T2 contrast agent in colon cancer based on physicochemical and biological properties.Prussian blue staining,immunohistochemistry and flow cytometry experiments confirmed binding characteristics of F4/80 antibodies to macrophages,in other words,F4/80-USPIO can be specifically uptaken by macrophages,which lay a solid foundation for achieving the target imaging in this study.F4/80-USPIO has a high relaxation rate,and accumulated nano-iron particles showed.low signal intensity on T2WI.But in vivo serial MR imaging of subcutaneous transplantation tumor model of human colon cancer,signal intensity decrease on T2WI caused by F4/80-USPIO was not significant.Conclusion:It was confirmed that F4/80-USPIO can be used as T2 contrast agent in colon cancer based on physicochemical and biological properties and F4/80-USPIO can be specifically uptaken by macrophages.But in vivo serial MR imaging of subcutaneous transplantation tumor model of human colon cancer,signal intensity decrease on T2WI caused by F4/80-USPIO was not significant.Listed below are some possible reasons.(1)The amount of macrophages in mice model of colon cancer was too little to be detected;(2)This can be attributed to permeability and retention enhancement effect of nanoparticles,causing a low level of F4/80-USPIO in mice models of colon cancer;(3)Mild signal change caused by nano-iron particles was beyond the detection limit of clinical magnetic resonance scanner with relatively low field strength.