Persistent use of ‘‘false’’ cell lines

MINI REVIEW

Persistent use of‘‘false’’cell lines

Marc Lacroix*

InTextoResearch,Baelen,Wallonia,Belgium

From HeLa and its multiple identities,to MDA-MB-435,errone-ously and widely used as breast cancer cells,the history of cancer cell lines is rich in misidenti?cation and cross-contamination events.Despite the fact that these problems were regularly sig-naled during the last decades,many actors of research still seem to ignore them.A never-ending story?Solutions exist,notably based on recent technical advances in cell line authentication (short tandem repeat analysis).However,a collaborative action involving users of cell lines,cell banks,journals and funding agen-cies is needed to achieve success.

'2007Wiley-Liss,Inc.

Key words:cell lines;misidenti?cation;cross-contamination; authentication;short tandem repeat;HeLa;MDA-MB-435;misuse

Review

Examination of the current scienti?c literature indicates that a large percentage of papers reporting on experimental cancer research use human cell lines.Indeed,cell lines are expected to provide an unlimited source of speci?c self-replicating material, free of contaminating cells and often easily cultured in simple standard media.Alas,since the establishment of the?rst cancer cell lines,problems with misidenti?cation and cross-contamina-tion have occurred and seriously compromised research.These problems were regularly brought to light during the past deca-des,1–14but have received few audience until cell banks(Amer-ican Tissue Culture Collection,ATCC;Deutsche Sammlung von Mikroorganismen und Zellkulturen,DSMZ;European Collection of Cell cultures,ECACC;Japanese Collection of Research Biore-sources,JCRB)decided to act by informing their clients or even by withdrawing the false cell lines from their catalogue.It must be noted that the DSMZ has been a pioneer and is still a major actor in that process.

Various recent studies have shown that between18and36%of cell lines were incorrectly designated.5,6,14It is likely that new false cell lines continue to be established without the knowledge of their originators.At the same time,detection of false cell lines is rendered increasingly dif?cult as numbers and varieties of circu-lating cell lines increase.Even more worrisome is the fact that many cell lines that have been proven false,sometimes since years,are still used by researchers who seem to ignore their true identity or who act as if they were ignoring it.

This is notably illustrated by Table I,which presents a nonex-haustive list of misidenti?ed or cross-contaminated cell lines that have been cited during the?rst semester of2007by scientists apparently not aware of their exact identity.The search was per-formed using the HighWire database(http://highwire.stanford. edu)including PubMed journals.

A signi?cant part of these cell lines have been contaminated with HeLa cells,31which,indeed,are frequently used in the labo-ratories,are robust,and multiply rapidly.32In a recent(2004)sur-vey of483mammalian cell culturists,it was shown that32%of respondents used HeLa cells and9%well-known HeLa contami-nants(including Hep-2,KB,WISH,Chang Liver,INT407).Only about a third of respondents were testing their lines for cell iden-tity.33Thus,it is not surprising that many researchers are still using HeLa contaminants without apparent awareness of their true identity.

Some of the cell lines mentioned in the Table I are intensively used under their false identity.This is notably observed for Chang Liver,ECV304,KB,SK-N-MC,MCF-7/ADR,MDA-MB-435 cells...In some cases,the incriminated articles are from research-ers not always familiar with the world of tumor cell lines,for instance toxicologists or chemists who wanted to test natural or modi?ed compounds on a well-known cell line,which they there-fore considered as highly representative.

For6of the cell lines listed in Table I,a more detailed High-Wire database search was performed to identify the number of articles mentioning them under their false identity during the last years(Table II).From the Table,it appears that:(i)WISH and Hep-2/Hep2cell lines are still used under their false identity by several researchers,despite the fact that their misidenti?cation was shown in197615or1988,16respectively;(ii)the misuse of DAMI(identi?ed as HEL erythroleukemia cells in199727)and ECV-304/ECV304(identi?ed as T24bladder carcinoma in 199922)cell lines does not appear to rapidly decrease over years, and the incertitude on the exact origin of HBL-100cells(presence of Y chromosome mentioned before2003)is apparently not a problem for dozens of research teams.

The misuse of several cell lines appears to be relatively more frequent in works originating from various emerging countries (South Corea,India,...),and particularly from China.For instance,45on102(44%)papers published in2006and presented ECV-304/ECV304cells under their false identity were from China,as there were21on66(32%)articles describing Hep-2/ Hep2cells as laryngeal cells and5on22(23%)articles in which WISH cells were used as amnion-derived cells.China was cultur-ally isolated a long time,what could explain why so many chinese researchers seem not to be aware of cell line cross-contamination. Paradoxically,it can arrive that false cell lines are exactly appreciated because they have a characteristic that distinguish them from other cell lines of the same supposed(and actually erro-neous)origin.For instance,one of the most recently unmasked cell lines,the putative‘‘breast cancer’’cell line MDA-MB-435 had gained a great popularity due to its unrivaled metastatic ef?-ciency in nude mice.34,35Contrasting with most breast cancer cell lines,which have an epithelial-like aspect,MDA-MB-435cells express a mesenchymal-like portrait.35This feature has favoured the use of MDA-MB-435cells,since it was previously widely believed that most breast cancer cells should undergo an epithelial-to-mesenchymal phenotype transition(EMT)to be able to meta-stasize.36MDA-MB-435cells were for that reason considered as very advanced in the process of metastasization.It is now estab-lished that EMT is in fact rarely seen in breast cancer progres-sion.37The melanocytic nature of MDA-MB-435cells was?rst suspected following micro-array studies,where these cells were found to cluster with melanoma cells,rather than with other breast cancer cell lines.38Afterward,MDA-MB-435cells were found to express several genes commonly transcribed in melanocytes,such as RXRG,TYR,ACP5and DCP,but which are not found in vari-

*Correspondence to:InTextoResearch,4chemin de Hoevel,B-4837 Baelen,Wallonie,Belgium.Fax:1132-87-762861.

E-mail:itr@https://www.wendangku.net/doc/6f8601879.html,

Received25July2007;Accepted after revision19September2007 DOI10.1002/ijc.23233

Published online24October2007in Wiley InterScience(www.interscience. https://www.wendangku.net/doc/6f8601879.html,).

Int.J.Cancer:122,1–4(2008)

'2007Wiley-Liss,Inc.

Publication of the International Union Against

Cancer

ous commonly used breast cancer cell lines.39Expression of mela-nocyte proteins tyrosinase and melan-A by MDA-MB-435cells was also shown.40However,these published observations were not followed by a decrease in the use of MDA-MB-435as breast cancer cells (see Table II).MDA-MB-435cells are in fact derived from the melanoma cell line M14.The misidenti?cation is likely to have occurred prior to 1982and therefore,nearly all of the existing literature using the MDA-MB-435cell line describes the M14melanoma cell line,which has been far less studied under its true name.30

Of note,another cell line,LCC15-MB,which has not been men-tioned in 2007,was recently identi?ed as being MDA-MB-435,41thus in reality M14melanoma cells.LCC15-MB had drawn atten-tion due to its invasive and metastatic phenotype.Moreover,as these cells were believed to originate from a bone metastase in a breast cancer patient,they seemed to constitute a useful model for studying molecular mechanisms important for breast cancer me-tastasis to bone.42

In a recent white paper,43Dr.Roland Nardone proposed cell line authentication as a condition for the award of research grants

TABLE I –NONEXHAUSTIVE LIST OF MISIDENTIFIED OR CROSS-CONTAMINATED CELL LINES THAT HAVE BEEN CITED DURING THE FIRST SEMESTER

OF 2007BY SCIENTISTS APPARENTLY NOT AWARE OF THEIR EXACT IDENTITY

Cell line

Putative origin

True identity

Reference(s)identifying cross-contamination or misidenti?cation

Chang liver

Liver cells

HeLa cells (glandular cancer of the cervix)15Girardi heart Atrial myoblast cells HeLa cells 15Hep-2(or Hep2)

Larynx carcinoma cells HeLa cells 16INT407(or INT-407,or Intestine 407)Embryonic intestine cells HeLa cells 15J111Monocytic leukemia cells

HeLa cells 15

KB Oral epidermoid carcinoma cells HeLa cells 2,3,17,18L132

Embryonic lung epithelium cells HeLa cells 15MT-1(or MT1)Breast cancer cells HeLa cells 6NCTC2544

Skin epithelium cells (keratinocytes)HeLa cells

15WISH

Amnion cells

HeLa cells 15Wong-Kilbourne

Conjunctiva-derived cells HeLa cells 15RPMI-8402(or RPMI8402)T cell leukemia

Unknown

19IM-9(or IM9)Multiple myeloma cells Epstein-Barr virus-transfected B cell lymphoblastoid line 20

HBL-100(or HBL100)

Breast transformed but non-tumorigenic cells Unknown,and not female (found to contain Y chromosome)ATCC website (http://

https://www.wendangku.net/doc/6f8601879.html,/common/cultures/probline.cfm)TSU-Pr1(or TSUPr1)Prostate cancer cells

T24cells (bladder cancer)21ECV-304(or ECV304)‘‘Spontaneously transformed’’umbilical cord endothelial cells

T24cells

22–24

EJ138

Bladder cancer cells T24cells 14and ECACC website EJ-1(or EJ1)Bladder cancer cells T24cells

14and ECACC website PPC-1(or PPC1)

Prostate cancer cells PC-3cells (prostate cancer)25ALVA-31(or ALVA31)Prostate cancer cells PC-3cells 25ALVA-41(or ALVA41)Prostate cancer cells PC-3cells

25SK-N-MC Neuroblastoma cells Ewing family tumor cells 26DAMI Megakaryocyte

HEL cells (erythroleukemia)27HS-Sultan

Plasma cell line (multiple myeloma)

Jijoye cells (Burkitt’s lymphoma)

20ARH-77(or ARH77)Plasma cells from a multiple myeloma patient Epstein-Barr virus-transfected B cell lymphoblastoid line 19

WiDr

Colon cancer cells HT-29cells (colon carcinoma)28

SNB-19(or SNB19)Glioblastoma cells U-373MG cells (glioblastoma)14and ATCC website U251

Glioblastoma cells U-373MG cells

14and ATCC website MCF-7ADR (re-designated NCI/ADR-RES)

Breast cancer cells OVCAR-8cells (ovarian cancer)29MDA-MB-435(or MDA-MB-435S,or MDA-MB435,or MDA-435)

Breast cancer cells

M14cells (melanoma)

30

ATCC,American tissue culture collection;ECACC,European collection of cell cultures.

TABLE II –NUMBER OF ARTICLES CITING SEVERAL CELL LINES UNDER THEIR FALSE IDENTITY

Cell line

Year

1990

1995

2000

2001

2002

2003

2004

2005

2006

20071

ECV-304or ECV304115101124132111120109102>53DAMI

228191620151597>7HBL-100or HBL100221957595148473140>16Hep-2or Hep2

182552584865588766>53MDA-MB-435or MDA-MB-435S or MDA-MB435or MDA-435533101141164173276276272>140WISH

6722193130232322>11

1

Search performed in August 2007.

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and for the publication of research?ndings.Clearly,resolution of the problem of misidenti?cation and cross-contamination requires the conscientization and the collaboration of all involved actors: users(including originators)of cell lines,cell banks,journals and funding agencies.

Users normally do not wish to use false cell lines that are the basis of misleading publications,which can potentially have a very high cost in terms of invalid hypotheses and paradigms,mis-spent effort and protracted development of patient treatments. Indeed,only in a very few investigations is the exact origin of a cell line devoid of any importance.However,most(new)cell lines are freely exchanged between laboratories,rarely having their identities checked.To avoid cross-contamination of these lines, periodic reauthentication of cell lines is advisable.In addition, working from validated freeze-downs,where cells are maintained in culture,and ideally separated from other cell lines,should mini-mize the risk of cross-contamination.12,44

All reputable cell banks now employ methods to con?rm the identity and origin of the cell lines they distribute.This is notably because distribution of misidenti?ed or cross-contaminated cell lines,even when supplied in good faith,may later be the subject of costly and embarassing recall actions.Moreover,cell banks may facilitate de novo detection of cross-contamination by identi-fying untoward matches between new and existing cell lines.Most cell banks may also test,to a low cost,cell lines provided by their users or originators.While various techniques,not described here, have been used in the past,recent technical advances have led to the development of short tandem repeat(STR)analysis.STRs are repetitive sequences characterized by a variable number of repeated short sequence elements of2–7bp in length as a unit (e.g.,di-,tri-,tetra-nucleotide sequences),also known as microsa-tellites or simple sequence repeats.They are highly polymorphic, the repeat sizes are small and can be easily ampli?ed by the poly-merase chain reaction method.Furthermore,when the sizes of the products(accurate to1base pair)are determined,a series of num-bers are generated,which can be used as a bar code for that DNA source.A registry of bar codes would make it easy to compare DNA samples and thus allow ef?cient cell line authentication,as notably shown by an international consortium.45The STR method, although not perfect,46is easy,reliable,inexpensive and can be done‘‘in house’’or analyzed by a commercial laboratory.14,45,47–50 The peer review process carried out by many(but not all)jour-nals and funding agencies still fails to consider the authenticity of the cell lines used.Editors of journals and heads of agencies should be encouraged to examine such issues and,in?ne,to reject papers from authors unable to substantiate the authenticity of the cell lines they have used.Along the same line,publication of new cell lines by originators,or the funding of their production should be conditional upon these lines being made freely available to other investigators,for instance by reposition in cell banks.

It is now time for a concerted action.Otherwise,days and costly resources will continue to be wasted,as a result of spurious experi-mental results,and some scienti?c reputations will continue to face the risk of being compromised.

Acknowledgements

Many thanks to‘‘Fondation Fornarina’’and SciMedWeb.This article is dedicated to the memory of my father,Mr.Albert Lacroix(1935–2006).

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