In vivo longitudinal and multimodal imaging of hypoxia-inducible factor 1a and angiogenesis in breast cancer

Background:Angiogenesis and hypoxia-inducible factor 1a(HIF-1a)play major roles in solid tumors.This study aimed to establish a longitudinal and multimodal imaging model for in vivo evaluation of HIF1a and angiogenesis in breast cancer.Methods:By transfection of a 5 hypoxia-responsive element(HRE)/green fluorescent protein(GFP)plasmid,the cell line Ca761-hregfp was established,which emitted green fluorescence triggered by HIF-1a under hypoxia.The cells were subjected to CoCl2-simulated hypoxia to confirm the imaging strategy.We grew Ca761-hre-gfp cells in the left rear flanks of twelve 615 mice.Experiments were conducted on days 4,9,15,and 19.For in vivo analysis,Ca761-hre-gfp subcutaneous allografted tumors were imaged in vivo using contrast-enhanced ultrasound(CEUS)and fluorescence imaging(FLI)during tumor development.The tumor size,CEUS peak intensity,and FLI photons were measured to evaluate tumor growth,angiogenesis,and HIF-1a activity,respectively.After each experiment,three mice were randomly sacrificed and tumor specimens were collected to examine HIF-1a activity and the microvessel density(MVD).Results:In vitro,both green fluorescence and HIF-1a expression were detected in Ca761-hre-gfp cells treated with CoCl2,indicating the suitability of the cells to detect HIF-1a activity.In vivo,HIF-1a activity first increased and then decreased,which was significantly correlated with angiogenic changes(r=0.803,P=0.005).These changes were confirmed by immunohistochemical staining of HIF-1a and MVD.Conclusions:The findings validated the Ca761-hre-gfp murine allograft model for reliable evaluation of HIF-1a activity and angiogenesis longitudinally using both molecular and pre-clinical non-invasive imaging modalities.The cell line may be useful for studies of anti-HIF pathway therapies.