• He-Wen Tang, Hai-Liang Feng, Ming Wang, Qing-Li Zhu, Yu-Qin Liu, and Yu-Xin Jiang.
• Department of Ultrasound, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, China.
• Chin. Med. J. 2020 Jan 20; 133 (2): 205-211.

BackgroundAngiogenesis and hypoxia-inducible factor 1α (HIF-1α) play major roles in solid tumors. This study aimed to establish a longitudinal and multimodal imaging model for in vivo evaluation of HIF1α and angiogenesis in breast cancer.MethodsBy transfection of a 5 hypoxia-responsive element (HRE)/green fluorescent protein (GFP) plasmid, the cell line Ca761-hre-gfp was established, which emitted green fluorescence triggered by HIF-1α 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-1α activity, respectively. After each experiment, three mice were randomly sacrificed and tumor specimens were collected to examine HIF-1α activity and the microvessel density (MVD).ResultsIn vitro, both green fluorescence and HIF-1α expression were detected in Ca761-hre-gfp cells treated with CoCl2, indicating the suitability of the cells to detect HIF-1α activity. In vivo, HIF-1α 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-1α and MVD.ConclusionsThe findings validated the Ca761-hre-gfp murine allograft model for reliable evaluation of HIF-1α 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.

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