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, 9 (11), e112667
eCollection

Photoacoustic Tomography of Human Hepatic Malignancies Using Intraoperative Indocyanine Green Fluorescence Imaging

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Photoacoustic Tomography of Human Hepatic Malignancies Using Intraoperative Indocyanine Green Fluorescence Imaging

Akinori Miyata et al. PLoS One.

Abstract

Recently, fluorescence imaging following the preoperative intravenous injection of indocyanine green has been used in clinical settings to identify hepatic malignancies during surgery. The aim of this study was to evaluate the ability of photoacoustic tomography using indocyanine green as a contrast agent to produce representative fluorescence images of hepatic tumors by visualizing the spatial distribution of indocyanine green on ultrasonographic images. Indocyanine green (0.5 mg/kg, intravenous) was preoperatively administered to 9 patients undergoing hepatectomy. Intraoperatively, photoacoustic tomography was performed on the surface of the resected hepatic specimens (n = 10) under excitation with an 800 nm pulse laser. In 4 hepatocellular carcinoma nodules, photoacoustic imaging identified indocyanine green accumulation in the cancerous tissue. In contrast, in one hepatocellular carcinoma nodule and five adenocarcinoma foci (one intrahepatic cholangiocarcinoma and 4 colorectal liver metastases), photoacoustic imaging delineated indocyanine green accumulation not in the cancerous tissue but rather in the peri-cancerous hepatic parenchyma. Although photoacoustic tomography enabled to visualize spatial distribution of ICG on ultrasonographic images, which was consistent with fluorescence images on cut surfaces of the resected specimens, photoacoustic signals of ICG-containing tissues decreased approximately by 40% even at 4 mm depth from liver surfaces. Photoacoustic tomography using indocyanine green also failed to identify any hepatocellular carcinoma nodules from the body surface of model mice with non-alcoholic steatohepatitis. In conclusion, photoacoustic tomography has a potential to enhance cancer detectability and differential diagnosis by ultrasonographic examinations and intraoperative fluorescence imaging through visualization of stasis of bile-excreting imaging agents in and/or around hepatic tumors. However, further technical advances are needed to improve the visibility of photoacoustic signals emitted from deeply-located lesions.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Establishment of PA tomography's ability to visualize ICG-containing tissue.
(A) Using a human liver tissue-mimicking phantom (top), human plasma containing ICG at concentrations of 0.001, 0.01, 0.1, and 1.0 mg/mL was encapsulated into holes that were 5 mm in diameter and located at a depth of 5 mm from the surface; PA amplitudes were measured using the Vevo LAZR imaging system (middle). Fluorescence images of each ICG-containing plasma sample were also obtained (bottom). (B) Fluorescence imaging in a mouse model with subcutaneously implanted well-differentiated human hepatoma cells (HuH-7) identified ICG accumulation in the subcutaneous tumor (left). PA tomography enabled differentiation of tumor-specific PA signals from those of surrounding organs under the conditions of 800-nm excitation light and 54-dB PA gain (right).
Figure 2
Figure 2. Photoacoustic tomography of surgically-resected hepatic tissue in humans.
(A) Fluorescence imaging identified uniform fluorescence of ICG on the cut surface of well-differentiated HCC tissue (left). PA tomography from the cut surface of the specimen visualized accumulation of ICG in cancerous tissues on US images (right, please see video S1). (B) Fluorescence imaging identified rim-type ICG fluorescence around CRLM lesions on the cut surface of the resected specimen (left). Photoacoustic tomography from the cut surface visualized accumulation of ICG in the peri-cancerous hepatic tissue on US images (right, please see video S2). Yellow squares and arrows in A and B indicate the site where a probe of the imaging system was attached and the boundaries between the tumors and non-cancerous liver parenchyma, respectively. (C) Photoacoustic amplitude of each ROI from the resected specimen (red bar indicates cancerous region; green bar, peri-cancerous region; and blue bar, non-cancerous hepatic parenchyma 2 mm from the tumor) according to the depth of the ROI from the sample's surface (depths of 1 to 5 mm). Indocyanine green accumulation was observed in the cancerous tissue in HCC specimens 1–4 (cancerous-type accumulation), while rim-type ICG accumulation was observed in HCC specimen 5, the ICC specimen, and CRLM specimens 1–4.
Figure 3
Figure 3. Fluorescence microscopy.
Left, hematoxylin-eosin staining; middle, fluorescence images; and right, fusion images of ICG fluorescence, indicated in green, and hematoxylin-eosin staining. (A) In well-differentiated HC lesions, ICG fluorescence was identified mainly in the cancerous tissue, as demonstrated in Figure 2A. (B) Magnified view of (A). Indocyanine green had accumulated in the pseudoglands (arrowheads) and the cytoplasm of cancer cells (arrow). (C) Indocyanine green fluorescence was identified in the peri-cancerous hepatic parenchyma surrounding a CRLM lesion, as demonstrated in Figure 2B. (D) Magnified view of (C). Indocyanine green had accumulated in the cytoplasm of relatively small hepatocytes rather than in the intracellular spaces.
Figure 4
Figure 4. Photoacoustic tomography from the body surface in NASH-HCC model mice.
(A) Spectrum computed tomography of a living NASH-HCC model mouse identified hepatic tumors with accumulation of ICG that had been intravenously injected 48 hours prior (IVIS Spectrum CT, PerkinElmer, Hopkinton, US; excitation 745 nm, emission 800 nm). (B) Photoacoustic tomography failed to visualize any cancer-specific PA signals on US images. (C, D) Three liver nodules were macroscopically identified in this mouse (C); one was visualized on fluorescence imaging (D). (E) Fluorescence microscopy identified ICG fluorescence (demonstrated in green in this fusion image with hematoxylin-eosin staining) in the cytoplasm of some of the cancerous cells in this NASH-HCC model mouse.

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Grant support

This work was supported by grants from the Takeda Science Foundation, the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Ministry of Health, Labour and Welfare of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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