Fig. 2. Assessment of efficacy of NanoLuc-miniSOG system expressed by BT-474 cells in vivo.

a Schematic presentation of the experiment. BT-474 cells stably expressing NanoLuc-miniSOG are inoculated subcutaneously into an animal. Gene expression of NanoLuc-miniSOG leads to production of fusion protein NanoLuc-miniSOG, which leads to BRET-activation of miniSOG after the injection of furimazine, followed by the ROS production and cancer cell kill. b Tumor-growth curves for various treatment conditions: mice bearing BT-474 tumor xenograft, treated with PBS (red curve); BT-474/NanoLuc-expressing tumors treated with furimazine (blue curve); mice bearing NanoLuc-miniSOG-expressing tumors treated with furimazine and riboflavin (green curve); mice bearing NanoLuc-miniSOG-expressing tumors treated with riboflavin (yellow curve). Data are presented as the mean ± SD (n = 6). c Evidence of BRET effect in vivo. An image of a living animal imaging after i.v. furimazine administration with (top row) and without (bottom row) Rf pre-injection. The difference in the distribution of average luminescence signals is a sign of BRET in NanoLuc-miniSOG system. The tumor region is indicated as a red-circled area. d Luminescence intensity signal from the tumor region measured in photons per second per cm² per steradian with and without RF pre-injection. e Hematoxylin-eosin-stained histological sections of different organs and tumors at the end of furimazine treatment (I-VI) and after completion of the experiment (VII, VIII). I—Liver. In the center of the lobules weakly expressed plethora and hydropic degeneration of hepatocytes are circled, necrosis of individual hepatocytes with small focal leukocyte infiltration are pointed with arrows, magnification ×200. II—Kidney with focal hydropic dystrophy of the epithelium of the proximal convoluted tubules (arrows) and small focal hemorrhages (circled), magnification ×200. III—Hydropic dystrophy of cardiomyocytes (arrows), magnification ×400. IV—Heart with a fibrin thrombus (circled) in the lumen of the left ventricle, magnification ×200. V—Lungs with plethora, confluent hemorrhages, fibrin thrombus in the lumen of the vessel (circled), magnification ×200. VI—Tumor with a focus of necrosis (circled), magnification ×200. VII—Tumor. Poorly differentiated adenocarcinoma, magnification ×200. VIII—Tumor with necrosis and decay cavity (decay cavity with necrotic masses in the lumen is circled), magnification ×100