In Vivo, Longitudinal, Multimodal Imaging Study Of Hypoxia-inducible Factor-1? Regulating Breast Cancer Angiogenesis

Objective:To establish a hypoxia-dependent breast cancer cell model with green fluorescent protein expressed as a HIF-la imaging system.Methods:Plasmid 5HRE/GFP was transfected into the murine breast cancer cell Ca761.G418 was used to screen stable transfected cells and monoclonal cells were separated by limiting dilution method.The 100 ?M CoCl2 was treated for 24h to simulate a hypoxia condition.The fluorescent microscope was used to select the cell lines with the strongest green fluorescence,and the cell line was cultured for amplification and storage.Subcellular cells with mimic hypoxia pretreatment were observed under fluorescence microscope and was detected for HIF-la expression with Western Blot.2×106 cells(0.1ml)were subcutaneously inoculated into the left flank of the 615 mouse,and the tumor growth was observed.Fluorescence imaging platform was used to detect the fluorescence signal of allograft tumor.Results:Cells with stable transfected plasmid were named Ca761-gre-gfp.With mimic hypoxia pretreatment,green fluorescence emitted by these cells could be seen under the fluorescence microscope,and the expression of HIF-la protein could be detected by Western Blot,whereas cells without mimic hypoxia pretreatment did not express the fluorescent signal and HIF-laprotein.An enlarged tumor was seen after Ca761-gre-gfp cells inoculation,and fluorescence signal was detected in the tumor with fluorescence imaging.Conclusion:A hypoxia-dependent breast cancer cell model with green fluorescent protein expressed as a HIF-la imaging system was successfully established.This cell model can be utilized for researches in both cellular and animal level.Objective:To validate a multimodal imaging in a longitudinal study of HIF-la regulation in breast cancer angiogenesis.Methods:hypoxia-dependent with green fluorescence protein expressed breast cancer cells Ca761-hre-gfp were subcutaneously injected into the left flank of 615 mouse to build an allograft breast cancer model.Allograft tumors were imaged in vivo using ultrasound,contrast enhanced ultrasound(CEUS)and fluorescence imaging(FLI)on day 4,9,15,19 after inoculation.Tumor growth,angiogenesis and HIF-la activity level was measured through tumor size,CEUS quantitative parameters and FLI photons,respectively.After each experiment,3 mice were sacrificed randomly to harvest tumor specimens for immunohistochemistry study of CD34 and HIF-1?.The relation of CEUS peak intensity,FLI photons,CD34-MVD,HIF-1? were analyzed.Results:12 allograft breast cancer models have been successfully built.The average tumor volume was 0.06±0.02 cm3?0.23±0.11 cm3?1.33±0.72 cm3 and 2,23±0.18cm3,average CEUS peak intensity was 55.60±2.43?68.80±7.20,70.57±5.51 and 47.9(p=0.018),average FLI photons was 1.02±0.82ph/s,2.13±1.09 ph/s,4.01±0.57 ph/s and 1.1 ph/s(p = 0.04),and average CD34-MVD was 49.3±7.5/200HPF,37.1±2.9/200HPF,36.8±1.7/200HPF and 44.1±5.5/200 HPF(p = 0.040)on Day 4,9,15 and 19.There were three grade 1 on Day 4,three grade 2 on Day 9,two grade 3 and one grade 2 on Day 15,and three grade 3 on Day 19(p=0.021).There is a significant correlation between photons and peak intensity(r = 0.803,p=0.005),and between expression of HIF-la and FLI photons(r=0.688,p=0.019).There is no correlation between CD34-MVD and CEUS peak intensity(r=-0.430,p=0.215).Conclusion:The findings of this study validate the multimodal imaging in evaluating HIF-1? activity level and angiogenesis longitudinally in Ca761-hre-gfp murine allograft tumor;and HIF-la activity and angiogenesis had the same changing rule with HIF-la during tumor growth.