Intraoperative Near-Infrared Fluorescence-Guided Peripheral Lung Tumor Localization in Rabbit Models Hironobu Wada, MD, PhD, Jinzi Zheng, PhD, Alexander.

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Intraoperative Near-Infrared Fluorescence-Guided Peripheral Lung Tumor Localization in Rabbit Models Hironobu Wada, MD, PhD, Jinzi Zheng, PhD, Alexander Gregor, MD, Kentaro Hirohashi, MD, PhD, Hsin-Pei Hu, BASc, Priya Patel, MD, Hideki Ujiie, MD, PhD, Tatsuya Kato, MD, PhD, Takashi Anayama, MD, PhD, David A. Jaffray, PhD, Kazuhiro Yasufuku, MD, PhD The Annals of Thoracic Surgery Volume 107, Issue 1, Pages 248-256 (January 2019) DOI: 10.1016/j.athoracsur.2018.08.020 Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 (A) Schematic diagram (left) of a PEGylated liposome co-encapsulating indocyanine green (ICG) and iohexol (IOX) (CF800). Modified from [12, 13]. (B) Photograph of CF800 that is being injected into an ear vein of a rabbit. The Annals of Thoracic Surgery 2019 107, 248-256DOI: (10.1016/j.athoracsur.2018.08.020) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 (A) Mean iodine concentrations of a tumor, inferior vena cava (IVC), and a back muscle were calculated per tissue volumes. Significant difference in the concentration of IVC was found between 48 and 72 hours after systemic administration (p = 0.019). (B) Percentage of injection dose per cubed millimeter of tumor (%ID/cm3) is presented. (C) Representative computed tomography images from a single rabbit are shown from pre-injection, a few minutes post-injection, and 72 hours post-injection. A scale bar represents 1 cm. The Annals of Thoracic Surgery 2019 107, 248-256DOI: (10.1016/j.athoracsur.2018.08.020) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 (A) Near-infrared (NIR) images in vivo and ex vivo from representative cases on days 2, 4, and 6 are shown. Values in the adjacent boxes express the mean fluorescence intensity and SD. Scale bars in the ex vivo images represent 5 mm. (B) Eleven rabbits were separated into three groups based on their day of thoracotomy, including day 2, days 4 and 5, and days 6 and 7. The fluorescence intensity was measured in three different way. Solid black, gray, and white present data obtained by in vivo NIR thoracoscope, ex vivo NIR thoracoscope, and ex vivo Maestro, respectively. A tumor was not visible on the NIR thoracoscope in 2 cases (†) and 1 case (‡), respectively. The lung was not inflated in 1 case (¶). (C) The fluorescence intensity of each organ was measured in three different ways. Each symbol is assigned to a single rabbit. The fluorescence intensity of the tumor was significantly greater than that of the lung parenchyma (*p = 0.013, n = 5, **p = 0.016, n = 8, ***p = 0.047, n = 8, paired t test). NIR fluorescence was not detected in 3 of 8 rabbits in vivo. (IVC = inferior vena cava; L = right lung; Lt = left; Med = mediastinal; Rt = right; T = tumor; TBR = tumor-to-background ratio; WL = white light.) The Annals of Thoracic Surgery 2019 107, 248-256DOI: (10.1016/j.athoracsur.2018.08.020) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 A rabbit sacrificed on day 4 had small pulmonary metastases (arrows) in the left lung which were incidentally identified on ex vivo analysis. A vertical band on the scale bar in the macroscopic figure represents 1 mm. A black and white scale bar in microscopic figures shows 1 mm and 50 μm, respectively. The Annals of Thoracic Surgery 2019 107, 248-256DOI: (10.1016/j.athoracsur.2018.08.020) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Pathologic assessment of a rabbit euthanized on day 4 (rabbit 3 in Fig 3). (A) A cross section of the tumor, with a vertical band on the scale bar representing 1 mm. A (B) near-infrared (NIR) and a (C) merged image of the tumor obtained by the SPY scope. (D) Frozen sections were obtained from the tumor cross section and evaluated with (E) hematoxylin and eosin staining, (F) 4ʹ6-diamidino-2-phenylindole staining, and (G) NIR fluorescence. The Annals of Thoracic Surgery 2019 107, 248-256DOI: (10.1016/j.athoracsur.2018.08.020) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions

Fig 6 Iodine concentrations of the (A) tumor and the (B) inferior vena cava (IVC) correlated with intraoperative near-infrared (NIR) fluorescence intensities of the tumor and the lung, respectively (tumor: R2 = 0.878, p = 0.0059, lung: R2 = 0.734, p = 0.024). The symbols used correspond with the legend included in Figure 6E. Preoperative iodine concentrations in the (C) tumor and (D) inferior vena cava did not differ between detectable and undetectable cases. (E) The tumor fluorescence intensity obtained by Maestro did not vary by depth from the lung surface measured on computed tomography. However, it did correlate with intraoperative tumor detection. The Annals of Thoracic Surgery 2019 107, 248-256DOI: (10.1016/j.athoracsur.2018.08.020) Copyright © 2019 The Society of Thoracic Surgeons Terms and Conditions