Journal of Gastroenterology

, Volume 49, Issue 1, pp 110–116 | Cite as

Photodynamic therapy for human hepatoma-cell–line tumors utilizing biliary excretion properties of indocyanine green

  • Junichi Kaneko
  • Yoshinori Inagaki
  • Takeaki Ishizawa
  • Jianjun Gao
  • Wei Tang
  • Taku Aoki
  • Yoshihiro Sakamoto
  • Kiyoshi Hasegawa
  • Yasuhiko Sugawara
  • Norihiro Kokudo
Original Article—Liver, Pancreas, and Biliary Tract



Photodynamic therapy (PDT) has not been reported for human hepatoma, because cancer cells only weakly take up the photosensitizer. Indocyanine green (ICG) is a photosensitizer normally excreted into the bile, and bile excretion is impaired in human hepatomas. We examined whether human hepatoma cell lines preferentially take up the ICG and then assessed the effectiveness of PDT using ICG and near-infrared (NIR) laser.


HuH-7 and HepG2 human hepatoma cell lines were transplanted subcutaneously into mice. Developing HuH-7 and HepG2 tumors were confirmed that preferentially took up the ICG in 24 h after ICG was administered to mice via tail vein. The HuH-7 tumor showed a high tumor-to-background fluorescence intensity ratio, 255:1, whereas fluorescence intensity of HuH-7 is increased twofold compared to HepG2. HuH-7 cell transplanted mice were divided into three groups: ICG administration only (ICG+NIR−, n = 8), ICG and NIR laser exposure (ICG+NIR+, n = 12), and NIR laser exposure only (ICG−NIR+, n = 5).


Mean tumor volume in the ICG+NIR− and ICG−NIR+ groups increased steadily. In contrast, mean tumor volume in the ICG+NIR+ group did not change between days 0 and 3. Mean tumor volume did not differ significantly between the ICG−NIR+ and ICG−NIR− groups, but was significantly different between the ICG+NIR+ group and both the ICG−NIR+ and ICG+NIR− groups (p < 0.01).


ICG is preferentially taken up by HuH-7 and HepG2 human hepatoma cell line tumors. The tumor-to-background ratio of HuH-7 tumors, in particular, was extremely high. PDT with NIR laser irradiation suppressed HuH-7 human hepatoma cell line tumor growth.


Photodynamic therapy Human hepatoma Indocyanine green Near-infrared laser 



This work was supported by grants 21791271 (Kaneko) and 23249067 (Kokudo) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and 2011 Tokyo Igakukai Medical Research Grants (Kaneko). We thank Dr. Yutaka Takazawa for helpful comments and suggestions regarding the pathology findings; Harukuni Tsuda for excellent technical assistance with the microscopic fluorescence evaluation; Yasuyuki Morishita for excellent technical assistance with histological preparation.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

When the power density of the NIR light was increased from 0 to 4 mW/cm2, the entire mouse body (Immediately after ICG administration) and HuH-7 tumor (24 h after ICG administration) showed increased fluorescence intensity. When the NIR light was turned off, the fluorescence disappeared (MPG 10196 kb)


  1. 1.
    Christensen E, Mork C, Skogvoll E. High and sustained efficacy after two sessions of topical 5-aminolevulinic acid photodynamic therapy for basal cell carcinoma: a prospective, clinical and histological 10-year follow-up study. Br J Dermatol. 2012;166(6):1342–8. (Epub 2012/02/09).PubMedCrossRefGoogle Scholar
  2. 2.
    Peng Q, Juzeniene A, Chen J, Svaasand LO, Warloe T, Giercksky K-E, et al. Lasers in medicine. Rep Prog Phys. 2008;71(5):056701.CrossRefGoogle Scholar
  3. 3.
    Klein A, Szeimies RM, Baumler W, Zeman F, Schreml S, Hohenleutner U, et al. Indocyanine green-augmented diode laser treatment of port-wine stains: clinical and histological evidence for a new treatment option from a randomized controlled trial. Br J Dermatol. 2012;167(2):333–42. (Epub 2012/03/23).PubMedCrossRefGoogle Scholar
  4. 4.
    Urbanska K, Romanowska-Dixon B, Matuszak Z, Oszajca J, Nowak-Sliwinska P, Stochel G. Indocyanine green as a prospective sensitizer for photodynamic therapy of melanomas. Acta Biochimica Polonica. 2002;49(2):387–91. (Epub 2002/10/05).PubMedGoogle Scholar
  5. 5.
    Hirano T, Kohno E, Gohto Y, Obana A. Singlet oxygen generation by irradiation of indocyanine green and its effect to tissues. J Japan Soc Laser Surg Med. 2007;28:122–8.Google Scholar
  6. 6.
    Shafirstein G, Bäumler W, Hennings LJ, Siegel ER, Friedman R, Moreno MA, et al. Indocyanine green enhanced near-infrared laser treatment of murine mammary carcinoma. Int J Cancer. 2012;130(5):1208–15.PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Ishizawa T, Fukushima N, Shibahara J, Masuda K, Tamura S, Aoki T, et al. Real-time identification of liver cancers by using indocyanine green fluorescent imaging. Cancer. 2009;115(11):2491–504.PubMedCrossRefGoogle Scholar
  8. 8.
    Nakabayashi H, Taketa K, Miyano K, Yamane T, Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. Cancer Res. 1982;42(9):3858–63. (Epub 1982/09/01).PubMedGoogle Scholar
  9. 9.
    Aden DP, Fogel A, Plotkin S, Damjanov I, Knowles BB. Controlled synthesis of HBsAg in a differentiated human liver carcinoma-derived cell line. Nature. 1979;282(5739):615–6. (Epub 1979/12/06).PubMedCrossRefGoogle Scholar
  10. 10.
    Inagaki Y. Effect of c-Met inhibitor SU11274 on hepatocellular carcinoma cell growth. BioScience Trends. 2011;5(2):52–6.PubMedCrossRefGoogle Scholar
  11. 11.
    Diehl KH, Hull R, Morton D, Pfister R, Rabemampianina Y, Smith D, et al. A good practice guide to the administration of substances and removal of blood, including routes and volumes. J Appl Toxicol. 2001;21(1):15–23. (Epub 2001/02/17).PubMedCrossRefGoogle Scholar
  12. 12.
    Kwon S, Sevick-Muraca EM. Non-invasive, dynamic imaging of murine intestinal motility. Neurogastroenterol Motility. 2011;23(9):881–e344. (Epub 2011/06/01).Google Scholar
  13. 13.
    Reindl S, Penzkofer A, Gong SH, Landthaler M, Szeimies RM, Abels C, et al. Quantum yield of triplet formation for indocyanine green. J Photochem Photobiol A. 1997;105(1):65–8.CrossRefGoogle Scholar
  14. 14.
    Brown SB, Brown EA, Walker I. The present and future role of photodynamic therapy in cancer treatment. Lancet Oncol. 2004;5(8):497–508. (Epub 2004/08/04).PubMedCrossRefGoogle Scholar
  15. 15.
    Kessel D. Death pathways associated with photodynamic therapy. Med Laser Appl. 2006;21(4):219–24. (Epub 2006/11/15).PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Thayer D, Unlu MB, Lin Y, Yan K, Nalcioglu O, Gulsen G. Dual-contrast dynamic MRI-DOT for small animal imaging. Technol Cancer Res Treat. 2010;9(1):61–70. (Epub 2010/01/20).PubMedGoogle Scholar
  17. 17.
    Kim TH, Mount CW, Dulken BW, Ramos J, Fu CJ, Khant HA, et al. Filamentous, mixed micelles of triblock copolymers enhance tumor localization of indocyanine green in a murine xenograft model. Mol Pharm. 2012;9(1):135–43.PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Mitsunaga M, Ogawa M, Kosaka N, Rosenblum LT, Choyke PL, Kobayashi H. Cancer cell–selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules. Nat Med. 2011;17(12):1685–91.PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Otake M, Nishiwaki M, Kobayashi Y, Baba S, Kohno E, Kawasaki T, et al. Selective accumulation of ALA-induced PpIX and photodynamic effect in chemically induced hepatocellular carcinoma. Br J Cancer. 2003;89(4):730–6.PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Ogawa M, Kosaka N, Choyke PL, Kobayashi H. In vivo molecular imaging of cancer with a quenching near-infrared fluorescent probe using conjugates of monoclonal antibodies and indocyanine green. Cancer Res. 2009;69(4):1268–72.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Fox IJ, Wood EH. Applications of dilution curves recorded from the right side of the heart or venous circulation with the aid of a new indicator dye. Proc Staff Meet Mayo Clinic. 1957;32(19):541–50. (Epub 1957/09/18).Google Scholar
  22. 22.
    Iijima T, Aoyagi T, Iwao Y, Masuda J, Fuse M, Kobayashi N, et al. Cardiac output and circulating blood volume analysis by pulse dye-densitometry. J Clin Monit. 1997;13(2):81–9. (Epub 1997/03/01).PubMedCrossRefGoogle Scholar
  23. 23.
    Cherrick GR, Stein SW, Leevy CM, Davidson CS. Indocyanine green: observations on its physical properties, plasma decay, and hepatic extraction. J Clin Investig. 1960;39:592–600. (Epub 1960/04/01).PubMedCrossRefGoogle Scholar
  24. 24.
    Hochheimer BF. Angiography of the retina with indocyanine green. Arch Ophthalmol. 1971;86(5):564–5. (Epub 1971/11/01).PubMedCrossRefGoogle Scholar
  25. 25.
    van Gemert MC, Welch AJ. Clinical use of laser-tissue interactions. IEEE Eng Med Biol Mag. 1989;8(4):10–3. (Epub 1989/01/01).PubMedCrossRefGoogle Scholar
  26. 26.
    Hock C, Villringer K, Muller-Spahn F, Wenzel R, Heekeren H, Schuh-Hofer S, et al. Decrease in parietal cerebral hemoglobin oxygenation during performance of a verbal fluency task in patients with Alzheimer’s disease monitored by means of near-infrared spectroscopy (NIRS)—correlation with simultaneous rCBF-PET measurements. Brain Res. 1997;755(2):293–303. (Epub 1997/05/02).PubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 2013

Authors and Affiliations

  • Junichi Kaneko
    • 1
  • Yoshinori Inagaki
    • 1
    • 2
  • Takeaki Ishizawa
    • 1
  • Jianjun Gao
    • 1
    • 3
  • Wei Tang
    • 1
  • Taku Aoki
    • 1
  • Yoshihiro Sakamoto
    • 1
  • Kiyoshi Hasegawa
    • 1
  • Yasuhiko Sugawara
    • 1
  • Norihiro Kokudo
    • 1
  1. 1.Hepato-Biliary-Pancreatic Surgery Division and Artificial Organ and Transplantation Division, Department of Surgery, Graduate School of MedicineThe University of TokyoTokyoJapan
  2. 2.Laboratory of Microbiology, Graduate School of Pharmaceutical SciencesThe University of TokyoTokyoJapan
  3. 3.Department of Pharmacology, School of Pharmaceutical SciencesShandong UniversityJinanChina

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