Although clinical applications of intraoperative fluorescence imaging of liver cancer using indocyanine green (ICG) have begun, the mechanistic background of ICG accumulation in the cancerous tissues remains unclear.
In 170 patients with hepatocellular carcinoma cells (HCC), the liver surfaces and resected specimens were intraoperatively examined by using a near-infrared fluorescence imaging system after preoperative administration of ICG (0.5 mg/kg i.v.). Microscopic examinations, gene expression profile analysis, and immunohistochemical staining were performed for HCCs, which showed ICG fluorescence in the cancerous tissues (cancerous-type fluorescence), and HCCs showed fluorescence only in the surrounding non-cancerous liver parenchyma (rim-type fluorescence).
ICG fluorescence imaging enabled identification of 273 of 276 (99 %) HCCs in the resected specimens. HCCs showed that cancerous-type fluorescence was associated with higher cancer cell differentiation as compared with rim-type HCCs (P < 0.001). Fluorescence microscopy identified the presence of ICG in the canalicular side of the cancer cell cytoplasm, and pseudoglands of the HCCs showed a cancerous-type fluorescence pattern. The ratio of the gene and protein expression levels in the cancerous to non-cancerous tissues for Na+/taurocholate cotransporting polypeptide (NTCP) and organic anion-transporting polypeptide 8 (OATP8), which are associated with portal uptake of ICG by hepatocytes that tended to be higher in the HCCs that showed cancerous-type fluorescence than in those that showed rim-type fluorescence.
Preserved portal uptake of ICG in differentiated HCC cells by NTCP and OATP8 with concomitant biliary excretion disorders causes accumulation of ICG in the cancerous tissues after preoperative intravenous administration. This enables highly sensitive identification of HCC by intraoperative ICG fluorescence imaging.
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This work was supported by grants from the Takeda Science Foundation, the Kanae Foundation for the Promotion of Medical Science, and the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 23689060 and No. 23249067). The authors acknowledge the significant contribution made by Drs. N. Harada, S. Tamura, T. Aoki, Y. Sakamoto, and Y. Sugawara, the members of this study group.
Takeaki Ishizawa and Koichi Masuda equally contributed as first authors.
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Supplementary Fig. 1 Fluorescence patterns of hepatocellular carcinoma cell (HCC). (a) Total-type fluorescence (well-differentiated HCC, 7 mm in diameter), (b) partial-type fluorescence (moderately differentiated HCC, 35 mm in diameter), (c) rim-type fluorescence (poorly differentiated HCC, 25 mm in diameter). (TIFF 1698 kb)
Supplementary Fig. 2. Representative gene sets associated with the fluorescence pattern by GSEA. (a,b) A GSEA plot (a) and list of genes (b) showing enrichment, in association with the fluorescence pattern, originally identified in CAIRO_LIVER_DEVELOPMENT_DN (genes downregulated in the early fetal liver stage [embryonic days E11.5 - E12.5] as compared to the late fetal liver stage [embryonic days E14.5 - E16.5]). (c,d) A GSEA plot (c) and list of genes (d) showing enrichment, in association with the fluorescence pattern, originally identified in CAIRO_HEPATOBLASTOMA_DN (genes downregulated in hepatoblastoma samples as compared to normal liver tissues). (e,f) A GSEA plot (e) and list of genes (f) showing enrichment, in association with the fluorescence pattern, originally identified in CAIRO_HEPATOBLASTOMA_CLASSES_DN (genes downregulated in robust Cluster 2 [rC2] of hepatoblastoma samples as compared to those in robust Cluster 1 [rC1]). (TIFF 2751 kb)
Supplementary Fig. 3 Suggested background of indocyanine green (ICG) fluorescence in hepatocellular carcinoma cell (HCC) tissues. (a) In non-cancerous liver tissues, ICG is taken up into hepatocytes by NTCP and OATP8, and excreted into the bile canaliculi by MRP2. (b, c) In differentiated HCC cells, portal uptake of ICG is mediated mainly by Na+/taurocholate cotransporting polypeptide (NTCP), but its biliary excretion is deteriorated, possibly because of a functional disorder of MRP2, (b) and/or morphological changes in the biliary system (c), leading to retention of ICG in the cytoplasm and/or pseudoglands, which is the basis for the cancerous ICG fluorescence from HCC tissues detected on fluorescence imaging. (d) In poorly differentiated HCC tissues, ICG is not taken up by the cancer cells at all because of downregulation of NTCP and organic anion-transporting polypeptide 8 (OATP8). On the other hand, it is retained in the non-cancerous liver tissues around the tumor, probably as a result of the biliary congestion caused by tumor compression and/or hepatic microenvironmental changes associated with cancer progression, making this type of HCC appear as a rim-fluorescence-type tumor on ICG fluorescence imaging. (TIFF 385 kb)
Supplementary Video 1 (a) Detection of a new lesion during liver resection for hepatocellular carcinoma cell (HCC) by using intraoperative indocyanine green (ICG) fluorescence imaging. (b) Intraoperative identification of the HCC that could not be identified by visual inspection, manual palpation, or contrast-enhanced intraoperative ultrasonography (IOUS). (MPG 39586 kb)
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Ishizawa, T., Masuda, K., Urano, Y. et al. Mechanistic Background and Clinical Applications of Indocyanine Green Fluorescence Imaging of Hepatocellular Carcinoma. Ann Surg Oncol 21, 440–448 (2014). https://doi.org/10.1245/s10434-013-3360-4
- Hepatocellular Carcinoma Cell
- Fluorescence Pattern
- Portal Uptake
- Fluorescence Imaging System
- Small HCCs