Dear Sir,

This letter aims at emphasizing the importance of using certified continuous cell lines in cancer research. The editors' growing demand to submit articles analyzing already certified cell lines is a praiseworthy request as it may have a very positive impact on the achievement of sound results.

Using human continuous cell lines is a key element in cancer stem cell (CSC) studies. Considering the scarcity of fresh samples and the consequent need to have access to a wider sampling of available material for biochemical and in vivo assays, the use of cell line models able to recapitulate as faithfully as possible the properties of the tumor from which they have been derived plays a pivotal role in scientific research. Furthermore, the use of CD133 antibodies and flow cytometry shows that the analysis of cell markers may be a very useful technique for splitting the putative stem cell fraction in cancer cell line specimens.1

Several articles have recently reported divergent data about CD133 expression on human colon cancer-derived HCT-116 cell line.1–5 In fact, it has been observed that HCT-116 cells are described as either lacking2, 3 or fully1, 4, 5 expressing CD133 glycoprotein.

These results have spurred us to purchase two different certified batches of HCT-116 cell line from American Type Culture Collection (ATCC; Manassas, VA) and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ; Braunschweig, Germany) cell banks. As for cell line practice, we have complied with the criteria of the United Kingdom Coordinating Committee on Cancer Research,6 moreover we used the well-characterized CD133 monoclonal antibody AC133-1 from Miltenyi, which has been used to correctly identify CSC in fresh colon carcinoma samples.7, 8 Of note, in one research study, it has been demonstrated that AC133 epitope is present on CSC but lost during differentiation.7

Both ATCC- and DSMZ-derived HCT-116 cells included in our study turned out to be more than 90% CD133 positive, as shown in Figure 1.

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Figure 1. (a) Unlabelled negative control and labeled HCT-116 from ATCC. CD133 positive cells (on the right of the marker) were 94%. (b) HCT-116 from DSMZ. CD133 positive cells were 90%.

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Some authors are aware that their findings are different from data reported elsewhere.2 They usually claim that inconsistent findings might be due to the presence, in various laboratories, of different HCT-116 clones showing divergent features probably acquired over long periods of cell culture.2

If on the one hand we do agree on the above-mentioned explanation, on the other we believe it is worthwhile recalling that cross-contamination between different cell lines can be considered as another cause of divergent data. Dirks et al.9 have recently evidenced the growing perception in scientific community that cross-contamination of mammalian cell lines represents a major risk of generating discordant scientific data. Freshney10 has also evidenced that 15–20% of cell lines currently in use may be cross contaminated, which has led to the production of articles, discussions at scientific meetings as well as recommended procedures for anyone employing cell lines to assess in vitro aspects related to biological research.

For the above reasons, we are firmly convinced that the authentication of cell lines upon submission of articles required by Scientific Journals has to be fulfilled because cross-contamination of cell lines is a widespread problem that is likely to invalidate many experimental observations and consequent clinical results in basic and oncologic research.

Yours sincerely,


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  • 1
    Botchkina IL, Rowehl RA, Rivadeneira DE, Karpeh MS Jr, Crawford H, Dufour A, Ju J, Wang Y, Leyfman Y, Botchkina GI. Phenotypic subpopulations of metastatic colon cancer stem cells: genomic analysis. Cancer Genomics Proteomics 2009; 6: 1929.
  • 2
    Kai K, Nagano O, Sugihara E, Arima Y, Sampetrean O, Ishimoto T, Nakanishi M, Ueno NT, Iwase H, Saya H. Maintenance of HCT116 colon cancer cell line conforms to a stochastic model but not a cancer stem cell model. Cancer Sci 2009; 100: 227582.
  • 3
    Yi JM, Tsai HC, Glöckner SC, Lin S, Ohm JE, Easwaran H, James CD, Costello JF, Riggins G, Eberhart CG, Laterra J, Vescovi AL, et al. Abnormal DNA methylation of CD133 in colorectal and glioblastoma tumors. Cancer Res 2008; 68: 8094103.
  • 4
    Dittfeld C, Dietrich A, Peickert S, Hering S, Baumann M, Grade M, Ried T, Kunz-Schughart LA. CD133 expression is not selective for tumor-initiating or radioresistant cell populations in the CRC cell lines HCT-116. Radiother Oncol 2009; 92: 35361.
  • 5
    Song B, Wang Y, Xi Y, Kudo K, Bruheim S, Botchkina GI, Gavin E, Wan Y, Formentini A, Kornmann M, Fodstad O, Ju J. Mechanism of chemoresistance mediated by miR-140 in human osteosarcoma and colon cancer cells. Oncogene 2009; 28: 406574.
  • 6
    United Kingdom Coordinating Committee on Cancer Research guidelines for the use of cell lines in cancer research. Br J Cancer 2000; 82: 1495509.
  • 7
    Kemper K, Sprick MR, de Bree M, Scopelliti A, Vermeulen L, Hoek M, Zeilstra J, Pals ST, Mehmet H, Stassi G, Medema JP. The AC133 epitope, but not the CD133 protein, is lost upon cancer stem cell differentiation. Cancer Res 2010; 70: 71929.
  • 8
    Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R. Identification and expansion of human colon-cancer-initiating cells. Nature 2007; 445: 1115.
  • 9
    Dirks WG, MacLeod RA, Nakamura Y, Kohara A, Reid Y, Milch H, Drexler HG, Mizusawa H. Cell line cross-contamination initiative: an interactive reference database of STR profiles covering common cancer cell lines. Int J Cancer 2010; 126: 3034.
  • 10
    Freshney RI. Database of misidentified cell lines. Int J Cancer 2010; 1: 302.

Marica Gemei, Peppino Mirabelli, Rosa Di Noto, Giuliana Fortunato, Luigi Del Vecchio