Institute of Development, Aging and Cancer, Tohoku University


Department of Project Programs

Professor Ryuta KAWASHIMA
Associate Professor Masashi TAKAO


Detection of Circulating Tumor Cells (CTC).

We started the project to detect and analyze CTC from peripheral blood since 2009. CTC have become an important tumor maker to monitor the cancer development. Distant metastasis actually takes place via CTC. Although tumor markers of proteins or nucleic acids have utilized for monitoring tumor progression, they monitor a trace of degraded molecules from cancerous cells or from their primary tissue site. In contrast, CTC itself contains the tumor cell information and it could be more informative marker to tell us the quality and quantity.

Refining rare CTC detection strategy.

It is important challenge to detect rare CTC from blood in cancer patients, while usually no CTC in healthy donors. There are two major methods to detect CTC. One is immunomagnetic CTC-enrichment based on an epithelial surface marker such as EpCAM (CD326). The other is size filtration depending on the knowledge that CTC shows larger than blood cells. In any case, the current applications do not exceed the prognostic value. For cancer therapy, we aimed to monitor the quantity of CTC, and to expand their genomic or expressional characteristics. To do so, a novel blood processing and CTC detection method is under development, named iCeap (intact CTC enumeration and analysis procedure). A prominent feature of iCeap is that there are no steps to cell-fixation and/or membrane-permeabilization. Therefore, the detected live CTC can be examined by many downstream molecular analysis such as gene-expression profiling and mutation analysis.

Current progress of iCeap: a new version named as iCeap2

One problem of the rare cell detection from the blood sample is the high background noise, such as nonspecific binding (NSB) of blood cells. Five milliliter blood contains 10^10-order blood cells or 10^7-order PBMC, while the supposed CTC is less than hundreds. Exclusion of NSB of PBMC is achieved by use of new EpCAM antibody-conjugated super-paramagnetic beads. The contamination of PBMC in the final specimen is less than hundred.
Another problem is phenotypic change of tumor. We assumed that carcinoma of epithelial origin may keep some traits of the epithelial cells, such as EpCAM and cytokeratins, even though epithelial-mesenchymal transition (EMT) occurs. To examine the detection efficiency, we use PC3 cell line (known as EpCAM-low expressing cancer cell line) and obtain satisfactory detection in spike-in test.
We also examine the exact detection and enumeration of CTC aggregates. The CTC aggregates, also called as circulating tumor microemboli (CTM), have been underlined as an indicator of the tumor progression as well as a cause of the distant metastasis. Unlike the preexisting immunomagnetic enrichment method, iCeap can detect CTC aggregates (Figure 1). In the original iCeap, we used flow cytometry as the CTC detector (Publication list #2), but it turned out to be very poor recognition for CTM. Now we are developing the next generation of iCeap (iCeap2) utilizing imaging cytometry in order to capture such CTC aggregates.

Figure 1 CTC aggregates (test CTC cell line in spike-in examination) enriched by EpCAM- immunomagnetic particles, followed by labeling with another EpCAM antibody recognizing the different epitope (red), and DNA (green).





Future of iCeap2

Firstly, CTC have information more than the value for prognosis. The number of CTC, and live/dead ratio will be reflecting the cancer treatment. Monitoring such numbers/ratios would provide therapeutic indicators. Thus, a periodic monitoring from patients will give information for appropriate treatment. Secondary, live CTC contains the genomic information. Since iCeap2 is designed to keep CTC integrity, several molecular analyses can be performed from the detected live CTC. Lastly, iCeap2 may be beneficial for diagnostic tool to detect early cancer. Many researchers have claimed that CTC are observed in early stage of cancer. Considering aggressive growth of tumor cells with chaotic angiogenesis, it would be of benefit to those who are unaware of cancer symptom.

Masashi Takao Ph.D.

Background of the Chief Scientist

Physics, Biophysics, Photobiology, Molecular biology, Cell biology, Biochemistry.

Publication list
  1. Takao M and Kasuya R: Flow cytometric detection of circulating tumor cells (CTC): Intact CTC enumeration and analysis. Cytometry Research 2011 21 (1): 51-56,
  2. Takao M and Takeda K: Fenumeration, Characterization, and Collection of Intact Circulating Tumor Cells by Cross Contamination-Free Flow Cytometry. Cytometry PartA 2011 79A: 107-117
  3. Takao M, Oohata Y, Kitadokoro,K, Kobayashi K, Iwai S, Yasui A, Yonei S, Zhang QM: Human Nei-like protein NEIL3 has AP lyase activity specific for single-stranded DNA and confers oxidative stress resistance in Escherichia coli mutant. Genes to Cells 2009 Feb;14(2):261-70
  4. Parker AR, Sieber OM, Shi C, Hua L, Takao M, Tomlinson IP, Eshleman JR: Cells with pathogenic biallelic mutations in the human MUTYH gene are defective in DNA damage binding and repair. Carcinogenesis. 2005 Nov;26(11):2010-8.
  5. Zhang QM, Yonekura S, Takao M, Yasui A, Sugiyama H, Yonei S: DNA glycosylase activities for thymine residues oxidized in the methyl group are functions of the hNEIL1 and hNTH1 enzymes in human cells. DNA Repair (Amst). 2005 Jan 2;4(1):71-9.
  6. Takao M, Yasui A: DNA repair initiated by glycosylases in the nucleus and mitochondria of mammalian cells; how our cells respond to a flood of oxidative DNA damage. J. Dermatol. Sci. suppl. 2005 1, S9-S19
  7. Lan L, Nakajima S, Oohata Y, Takao M, Okano S, Masutani M, Wilson SH, Yasui A: In situ analysis of repair processes for oxidative DNA damage in mammalian cells. Proc Natl Acad Sci U S A. 2004 Sep 21;101(38):13738-43.
  8. Nakajima S, Lan L, Kanno S, Takao M, Yamamoto K, Eker AP, Yasui A: UV light-induced DNA damage and tolerance for the survival of nucleotide excision repair-deficient human cells. J Biol Chem. 2004 Nov 5;279(45):46674-7.
  9. Lan L, Hayashi T, Rabeya RM, Nakajima S, Kanno S, Takao M, Matsunaga T, Yoshino M, Ichikawa M, Riele H, Tsuchiya S, Tanaka K, Yasui A: Functional and physical interactions between ERCC1 and MSH2 complexes for resistance to cis-diamminedichloroplatinum(II) in mammalian cells. DNA Repair (Amst). 2004
  10. Takao M, Kanno S, Kobayashi K, Zhang QM, Yonei S, van der Horst GT, Yasui A: A back-up glycosylase in Nth1 knock-out mice is a functional Nei (endonuclease VIII) homologue. J Biol Chem. 2002 Nov 1;277(44):42205-13.
  11. Miyabe I, Zhang QM, Kino K, Sugiyama H, Takao M, Yasui A, Yonei S: Identification of 5-formyluracil DNA glycosylase activity of human hNTH1 protein. Nucleic Acids Res. 2002 Aug 1;30(15):3443-8.
  12. Takao M, Iwasa T, Yamamoto H, Takeuchi T, Tokunaga F: Anti-bovine rhodopsin monoclonal antibody recognizing light-dependent structural change. Zoolog Sci. 2002 Jun;19(6):651-9.
  13. Takao M, Kanno S, Shiromoto T, Hasegawa R, Ide H, Ikeda S, Sarker AH, Seki S, Xing JZ, Le XC, Weinfeld M, Kobayashi K, Miyazaki J, Muijtjens M, Hoeijmakers JH, van der Horst G, Yasui A: Novel nuclear and mitochondrial glycosylases revealed by disruption of the mouse Nth1 gene encoding an endonuclease III homolog for repair of thymine glycols. EMBO J. 2002 Jul 1;21(13):3486-93. Erratum in: EMBO J 2002 Aug 15;21(16):4391
  14. Matsumoto Y, Zhang QM, Takao M, Yasui A, Yonei S: Escherichia coli Nth and human hNTH1 DNA glycosylases are involved in removal of 8-oxoguanine from 8-oxoguanine/guanine mispairs in DNA. Nucleic Acids Res. 2001 May 1;29(9):1975-81.
  15. Takao M, Zhang QM, Yonei S, Yasui A: Differential subcellular localization of human MutY homolog (hMYH) and the functional activity of adenine:8-oxoguanine DNA glycosylase. Nucleic Acids Res. 1999 Sep 15;27(18):3638-44.
  16. Kanno S, Iwai S, Takao M, Yasui A: Repair of apurinic/apyrimidinic sites by UV damage endonuclease; a repair protein for UV and oxidative damage. Nucleic Acids Res. 1999 Aug 1;27(15):3096-103.
  17. van der Horst GT, Muijtjens M, Kobayashi K, Takano R, Kanno S, Takao M, de Wit J, Verkerk A, Eker AP, van Leenen D, Buijs R, Bootsma D, Hoeijmakers JH, Yasui A: Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature. 1999 Apr 15;398(6728):627-30.
  18. Yoon JH, Swiderski PM, Kaplan BE, Takao M, Yasui A, Shen B, Pfeifer GP: Processing of UV damage in vitro by FEN-1 proteins as part of an alternative DNA excision repair pathway. Biochemistry. 1999 Apr 13;38(15):4809-17.
  19. Okano S, Kanno S, Takao M, Eker AP, Isono K, Tsukahara Y, Yasui A: A putative blue-light receptor from Drosophila melanogaster. Photochem Photobiol. 1999 Jan;69(1):108-13.
  20. Kobayashi K, Kanno S, Smit B, van der Horst GT, Takao M, Yasui A: Characterization of photolyase/blue-light receptor homologs in mouse and human cells. Nucleic Acids Res. 1998 Nov 15;26(22):5086-92.
  21. Hayashi T, Takao M, Tanaka K, Yasui A: ERCC1 mutations in UV-sensitive Chinese hamster ovary (CHO) cell lines. Mutat Res. 1998 Jun;407(3):269-76.
  22. Takao M, Aburatani H, Kobayashi K, Yasui A: Mitochondrial targeting of human DNA glycosylases for repair of oxidative DNA damage. Nucleic Acids Res. 1998 Jun 15;26(12):2917-22.
  23. Aburatani H, Hippo Y, Ishida T, Takashima R, Matsuba C, Kodama T, Takao M, Yasui A, Yamamoto K, Asano M: Cloning and characterization of mammalian 8-hydroxyguanine-specific DNA glycosylase/apurinic, apyrimidinic lyase, a functional mutM homologue. Cancer Res. 1997 Jun 1;57(11):2151-6.
  24. Otrin VR, McLenigan M, Takao M, Levine AS, Protić M: Translocation of a UV-damaged DNA binding protein into a tight association with chromatin after treatment of mammalian cells with UV light. J Cell Sci. 1997 May;110 ( Pt 10):1159-68.
  25. Yonemasu R, McCready SJ, Murray JM, Osman F, Takao M, Yamamoto K, Lehmann AR, Yasui A: Characterization of the alternative excision repair pathway of UV-damaged DNA in Schizosaccharomyces pombe. Nucleic Acids Res. 1997 Apr 15;25(8):1553-8.
  26. Tohda H, Takao M, Kikuchi A, Yasumoto T, Yasui A: Unstable expression of the multi-drug-resistant phenotype in Chinese hamster ovary cells resistant to okadaic acid. Biochem Biophys Res Commun. 1997 Mar 17;232(2):398-402.
  27. van der Spek PJ, Kobayashi K, Bootsma D, Takao M, Eker AP, Yasui A: Cloning, tissue expression, and mapping of a human photolyase homolog with similarity to plant blue-light receptors. Genomics. 1996 Oct 15;37(2):177-82.
  28. Yajima H, Takao M, Yasuhira S, Zhao JH, Ishii C, Inoue H, Yasui A: A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light. EMBO J. 1995 May 15;14(10):2393-9.
  29. Yajima H, Takao M, Yasuhira S, Zhao JH, Ishii C, Inoue H, Yasui A: A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light. EMBO J. 1995 May 15;14(10):2393-9.
  30. Takao M, Abramic M, Moos M Jr, Otrin VR, Wootton JC, McLenigan M, Levine AS, Protic M: A 127 kDa component of a UV-damaged DNA-binding complex, which is defective in some xeroderma pigmentosum group E patients, is homologous to a slime mold protein. Nucleic Acids Res. 1993 Aug 25;21(17):4111-8.
  31. Takao M, Yasui A, Oikawa A: Unique characteristics of superoxide dismutase of a strictly anaerobic archaebacterium Methanobacterium thermoautotrophicum. J Biol Chem. 1991 Aug 5;266(22):14151-4.
  32. Iwasa T, Nakajima K, Takao M and Tokunaga F: Light-dependent proton muvement and photointermediates ofn bacteriorhodopsin photocycle Sci. Rep. Tohoku University 1991 Vol11, No2/3 229-237
  33. Takao M, Oikawa A, Yasui A: Characterization of a superoxide dismutase gene from the archaebacterium Methanobacterium thermoautotrophicum. Arch Biochem Biophys. 1990 Nov 15;283(1):210-6.
  34. Kobayashi T, Takao M, Oikawa A, Yasui A: Increased UV sensitivity of Escherichia coli cells after introduction of foreign photolyase genes. Mutat Res. 1990 Jul;236(1):27-34.
  35. Takao M, Kobayashi T, Oikawa A, Yasui A: Tandem arrangement of photolyase and superoxide dismutase genes in Halobacterium halobium. J Bacteriol. 1989 Nov;171(11):6323-9.
  36. Takao M, Oikawa A, Eker AP, Yasui A: Expression of an Anacystis nidulans photolyase gene in Escherichia coli; functional complementation and modified action spectrum of photoreactivation. Photochem Photobiol. 1989 Nov;50(5):633-7.
  37. Kobayashi T, Takao M, Oikawa A, Yasui A: Molecular characterization of a gene encoding a photolyase from Streptomyces griseus. Nucleic Acids Res. 1989 Jun 26;17(12):4731-44.
  38. Tokunaga F, Iwasa T, Takao M, Sato S and Takeuchi T: Preparation andproperties of monoclonal antibodies against bovine rhodopsin. Zoolog Sci. 1989 6, 167-171
  39. Yasui A, Takao M, Oikawa A, Kiener A, Walsh CT, Eker AP: Cloning and characterization of a photolyase gene from the cyanobacterium Anacystis nidulans. Nucleic Acids Res. 1988 May 25;16(10):4447-63.
  40. Takao M, Yasui A, Tokunaga F: Isolation and sequence determination of the chicken rhodopsin gene. Vision Res. 1988;28(4):471-80.
  41. Iwasa T, Takao M, Tsujimoto K, Tokunaga F: Photochemical properties of naphtylbacteriorhodopsins differing in their protein-chromophore interactions. Biochemistry 1988 27 2416-2419
  42. Ishikawa M, Takao M, Washioka H, Tokunaga F, Vatanabe H, Tonosaki A: Demonstration of rod and cone photoreceptors in the lamprey retina by freeze-replication and immunofluorescence. Cell Tissue Res 1987 249, 241-246
  43. Tokunaga F, Takao M, Iwasa T, Tsujimoto K: Two kinds of bacteriorhodopsin anaologes synthesized from naphthylretinal. Primary processes in photobiology (Ed. Kobayashi T), Springer Processdings in Physics 1987, 20,154-163
  44. Tokunaga F, Takao M, Iwasa T, Koike S, Sato S, Takeuchi T, Kitani H, Horiuchi S, Koshida Y: Anti-bovine rodhopsin monoclonal antibodies. Retinal proteins. VNU Science Press 1987 145-152.
  45. Iwasa T, Takao M, Yamada M, Tsujimoto K, Tokunaga F: Properties of an analogue pigment of bacteriorhodopsin synthesized with naphthylretinal.. Biochemistry 1984 27 2416-2419

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