ARTICLE |
Correspondence to: Hilmar Quentmeier, DSMZ, German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1 B, D-38124 Braunschweig, Germany. E-mail: hqu@dsmz.de
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Summary |
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Antibodies recognizing tissue-specific antigens are widely used to identify the histological origin of tumors. Here we tested the fidelity of selected tissue markers on all 167 solid tumor-derived continuous cell lines in the DSMZ cell lines bank. Most lines had an intermediate filament content consistent with the tumor type from which they were derived. Thus, 93% of all carcinoma cell lines expressed keratin filaments. With certain antibodies, some subclassification was possible. For example, the CK7 keratin 7 antibody can differentiate between colon and pancreas-derived carcinoma cell lines. Cell lines derived from non-carcinomas, in general, did not express keratin but were vimentin-positive. Four of 10 glioma/astrocytoma cell lines expressed GFAP, five of six neuroblastoma cell lines expressed neurofilaments, and the TE-671 rhabdomyosarcoma cell line expressed desmin. When other tissue markers were tested, 12/16 melanoma-derived cell lines expressed HMB-45, while PSA, CA125, and thyroglobulin were less useful. These results demonstrate that cell lines retain some but not all markers typical of the original tumor type and identify certain markers useful in characterizing the histological origin of cell lines. Our data question the identity of some cell lines submitted to the bank in the past. The immunoprofiles of 167 solid tumor-derived and 131 hematopoetic cell lines can be found at www.dsmz.de.
(J Histochem Cytochem 49:13691378, 2001)
Key Words: keratin, intermediate filaments, vimentin, carcinoma, cell lines, immunocytochemistry, melanoma, sarcoma
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Introduction |
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THE AIM of a cell line bank is to provide well-characterized cell lines that recapitulate as faithfully as possible the properties of the differentiated tissue or tumor from which the cell line was derived. Usually, a cell line bank contains multiple cell lines derived from a given tumor. Detailed characterization of the properties of such cell lines can therefore be of use in confirming their origin and can help investigators choose the most appropriate cell line for their purpose. The DSMZ cell line bank is the major reference center for cell lines in Germany. Currently it has 298 continuous human cell lines in its collection, of which 131 are hematopoetic and 167 are solid tumor-derived cell lines.
Recently, using DNA fingerprinting and chromosome analysis, we have shown that 18% of the cell lines obtained directly from the originators were falsely named or misidentified before arrival at the DSMZ (
All hematopoetic cell lines submitted to the bank are routinely tested for the expression of cell surface markers with a panel of well-characterized monoclonal antibodies (
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Materials and Methods |
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Cell Culture
The continuous cell lines were taken from the DSMZ cell lines bank (
Antibodies
Sources.
The following monoclonal antibodies were obtained from DAKO (Hamburg, Germany): the broad specific keratin antibodies MNF-116, 34ßE12, and AE1/AE3; the human keratin 17 monoclonal antibody (clone E3); the keratin 18 monoclonal antibody (clone DC10); antibodies against melanoma (HMB-45), CD31 (JC/70A), vimentin (V9), desmin (D33), neurofilament (2F11), thyroglobulin (DAK-Tg6), CA125 (M11), and prostate specific antigen (ER-PR8). Anti-GFAP monoclonal antibodies (N358 and GF5) were purchased from Amersham (Braunschweig, Germany) and DPC Biermann (Bad Nauheim, Germany), respectively. The anti-GFAP (GA5), neurofilament (NN18 and NR4), broad specificity keratin (Lu-5), keratin 7 (CK7), and keratin 8 (a4.1) monoclonal antibodies are also commercially available from a variety of sources.
Specificity.
MNF-116 is reported as showing a broad spectrum of reactivity with human epithelial tissues (
Immunocytochemical Analysis
Adherent cells were detached from the flask using trypsin/EDTA. Alternatively, cells were grown on slides before acetone fixation. Cytospin preparations were air-dried for 218 hr before acetone fixation (4C, 10 min). The preparations were allowed to dry for 20 min and were stored at -80C. After thawing, the cells were fixed again with acetone. Staining was performed in a two-step procedure. The antibodies were added to the cells at the appropriate concentrations in a volume of 40 µl (24C, 30 min). The cytospin preparations were washed thoroughly and stained with fluorescein iso-thiocyanate (FITC)-conjugated anti-mouse Ig antibodies (24C, 30 min). After a further wash, they were mounted either in PBS/glycerol (1:1) or, more recently, in Mowiol 4-88 (Hoechst; Frankfurt, Germany). Antigen expression was analyzed by fluorescence microscopy.
A carcinoma cell line was defined as keratin-negative when less than 2% of the cells were positive in immunofluorescence analysis and as positive when more than 90% expressed the antigen. Only 3/121 carcinoma cell lines exhibited percentages in between these borders (all three had more than 25% keratin-positive cells and were also evaluated as keratin-positive). In some cases, scored as negative for a given antigen, occasional single cells stained brightly positive.
Western Blotting Analysis
A total of 4 x 106 cells were lysed in 100 µl SDS lysis buffer containing 15% glycerol, 125 mM Tris-HCl, pH 6.8, 5 mM EDTA, 2% SDS, 1% ß-mercaptoethanol, and 0.1% bromophenol blue. The samples (10 µl/lane) were separated on a 9% gel and blotted onto nitrocellulose membranes by electroblotting (Trans-Blot; Biorad, München, Germany). Equal protein loading of the lanes was confirmed by Ponceau S staining. The membranes were labeled overnight with the appropriate antibodies and specific bands were visualized with the biotinstreptavidinhorseradish peroxidase system (Amersham) in combination with the "Renaissance Western Blot Chemoluminescence Reagent" protocol (NEN; Bad Homburg, Germany).
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Results |
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Carcinoma-derived Cell Lines
A Total of 93% of All Carcinoma-derived Cell Lines Express Keratin.
Epithelial cells contain keratins as intermediate filaments. Therefore, keratin antibodies can be used to identify tumors of epithelial origin (
Of the carcinoma-derived cell lines, 93% (113/121) stained positively with this antibody (Fig 1). One hundred percent of the breast carcinoma cell lines (14/14), 100% of the colon carcinoma cell lines (12/12), and 100% of the squamous cell carcinoma cell lines (19/19), as well as the vast majority of cell lines derived from carcinomas at other sites, stained positively with the MNF-116 antibody (Table 1). Those cell lines evaluated here as positive for keratin have an intermediate filament content consistent with their derivation from carcinomas.
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A total of 7% (8/121) of the carcinoma-derived cell lines were keratin-negative. They included one bladder cell line, one endometrium carcinoma cell line, one kidney cell line, three lung carcinoma cell lines, one ovary carcinoma cell line, and one thyroid carcinoma cell line (Table 1 and Table 2).
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Further Analysis of Keratin-negative Cell Lines Originally Reported as Carcinoma-derived.
The car-cinoma cell lines that did not express keratin in our first analysis were reexamined with MNF-116, by both immunofluorescence and Western blotting assays (Fig 2; Table 2). Again, all eight cell lines were negative in immunofluorescence with MNF-116 as well as with two other broad-specificity keratin antibodies (AE1/AE3 and 34ßE12). The seven cell lines that were tested by Western blotting were keratin-negative also in this assay. In addition, the cell lines were tested with antibodies specific for single keratin polypeptides. Antibodies specific for either keratin 7, 8, 17, or 18, the keratins usually expressed in simple epithelia, were available. Six of eight of the cell lines that were negative with the pan-keratin antibodies were negative also with the single-chain specific keratin antibodies (Table 2). Only the bladder carcinoma cell line TCC-SUP and the kidney carcinoma cell line BFTC-909 displayed a weak dotted staining pattern, but not the usual filament pattern when the keratin 8 and 18 antibodies were applied, so we concluded that these cell lines do not express intact keratin filaments. It has been reported that AN3-CA cells do not express keratin 8 (
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We next tried to gain further information on the origin of these cell lines by testing them with a variety of additional markers, including antibodies against other intermediate filament proteins, i.e., with antibodies against desmin, GFAP, neurofilaments, and vimentin. In addition, they were tested with antibodies against CD31 and HMB-45 (Table 2). Two of the keratin-negative cell lines (COLO-668 and COLO-699) stained positively with the melanoma marker HMB-45 and were also vimentin-positive. This strongly suggests that they are melanoma-derived (see below) and that the cells were mistakenly listed as being derived from carcinomas. Six cell lines remained that were vimentin-positive and negative not only for keratins but also for desmin, GFAP, neurofilaments, and CD31.
HeLa Subclones Show Variable Keratin Expression Patterns.
Because the DSMZ cell line bank holds several HeLa subclones from different sources, we used these to check whether cells can downregulate keratin expression in vitro. The HeLa cell line has been reported as containing keratins 7, 8, 17, 18, and only trace amounts of keratin 19 (
Characterization of Keratin-positive Carcinoma Cell Lines Using Additional Antibodies.
Antibodies specific either for particular keratin polypeptides (e.g.,
34ßE12.
34ßE12 is an antibody reported to recognize squamous cell carcinomas (SCCs) of lung, skin, nasopharynx and cervix, as well as some but not all adenocarcinomas in situ (
Antibodies Specific for Keratin 8.
Examination of keratins isolated from human tumors has shown that although keratin 8 expression is abundant in adenocarcinomas it is also found in certain types of squamous cell carcinoma, e.g., those from lung or cervix (
Antibodies Specific for Keratin 7.
Pancreatic ducts in normal tissue as well as certain pancreatic tumors stain positively with the CK7 antibody, which is specific for keratin 7 in immunoblotting. Other gastrointestinal tumors, such as adenocarcinomas from stomach or colon, are negative when tested with the CK7 antibody (
In the pancreas, keratin 7 expression is restricted to the pancreatic duct (
HMB-45. This marker was negative on all 20 keratin positive carcinoma-derived cell lines on which it was tested (data not shown).
Other Antibodies. Twenty-one selected epithelial cell lines were tested with antibodies against prostate-specific antigen (PSA), CA125 (ovarian carcinoma), and thyroglobulin (thyroid carcinoma). The results of these tests suggested that these markers are not useful to further subdivide carcinoma cell lines (data not shown).
Vimentin. A total of 55% of the cell lines listed as carcinoma-derived co-expressed vimentin.
Non-carcinoma-derived Cell Lines
The non-carcinoma cell lines in the DSMZ collection are listed in Table 1. Again, we started by testing all cell lines in this group with the broad-specificity keratin antibody MNF-116. Among the non-carcinoma-derived cell lines in the collection, 80% (37/46) were keratin-negative when tested with the MNF-116 antibody (Table 1). These included all melanoma-derived cell lines (16/16). Both retinoblastoma cell lines were keratin-negative, as were 6/7 of the neuroblastoma cell lines and 8/10 of the glioma/astrocytoma cell lines. Altogether, nine cell lines in this group were keratin-positivethe seven cell lines listed in Table 3, CADO-ES1, and CAL-72. For the latter two cell lines no filamentous staining was seen. Whereas the seven lines listed in Table 3 were positive also with the broad-specificity Lu-5 antibody, CADO-ES1 and CAL-72 were not. In addition, CADO-ES1 has been described as keratin-negative (
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We face a similar situation with the fibrosarcoma cell line HT-1080 and the thyroid sarcoma cell line S-117. Keratin-positive fibrosarcoma cells have been described (
Most Melanoma Cell Lines, but None of the Carcinoma Cell Lines, Express the Antigen Detected by Antibody HMB-45.
A total of 12/16 (75%) melanoma cell lines were positive when tested with the HMB-45 antibody (Table 4). In contrast, 0/20 carcinoma cell lines (data not shown) and 0/7 cell lines reported as being non-carcinoma/non-melanoma-derived (Table 3, CADO-ES1 and CAL-72) were HMB-45-pos-itive. To our knowledge, only one of the melanoma cell lines tested here has been analyzed previously with HMB-45. IGR-39 was found to be HMB-45 negative (
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Intermediate Filament Typing of Rhabdomyosarcoma and Neuroblastoma Cell Lines.
The results of the analysis of rhabdomyosarcoma and neuroblastoma cell lines with antibodies against different intermediate filament proteins are shown in Table 5. Both human and rat rhabdomyosarcoma in situ are distinguished by the presence of the intermediate filament protein desmin. Vimentin positivity has been reported in 550% of the tumor cells in approximately half the cases studied (
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The human neuroblastoma cell lines are also of interest. Of the six cell lines shown in Table 5, five are positive with antibodies against neurofilaments and vimentin, consistent with a derivation from neuroblastoma (
Other Markers. A total of 4/10 glioma/astrocytoma cell lines expressed GFAP. A total of 98% of the cell lines listed as non-carcinoma-derived were positive with vimentin antibodies. The only vimentin-negative cell line in this group was the retinoblastoma cell line WERI-RB-1.
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Discussion |
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The first major conclusion from this study is that the vast majority of carcinoma-derived cell lines express keratin filaments. In this study, 93% (113/121) of the cell lines listed in the DSMZ catalogue as carcinoma-derived were positive in immunofluoresence with broad-specificity pan-keratin antibodies (Table 1). This result is consistent with data in the literature on carcinoma cell lines (e.g.,
The finding that carcinomas and cell lines derived from them are in general keratin-positive calls into question the origin of keratin-negative cell lines that are listed as carcinoma-derived. Such cell lines should be screened for the expression of markers associated with tissues other than epithelia in an attempt to understand the true origin of the cell line in question. In our study 7% (8/121) of such cell lines did not display true keratin filaments when tested with a panel of broad-specificity and single chain-specific keratin antibodies. By testing the eight cell lines for other markers, we were able to show that two were positive with the melanoma antibody HMB-45 and were vimentin-positive. Therefore, these two cell lines are most likely melanoma-derived. DNA fingerprinting enabled us to identify one of these cell lines as the melanoma cell line COLO-679, which is also HMB-45-positive and keratin-negative and which was provided to the DSMZ by the same depositor as the other two cell lines. A further four cell lines were vimentin-positive and negative for other intermediate filament proteins as well as for HMB-45 and for CD31. These cell lines might be derived from the stromal cells rather than tumor cells in the carcinoma, i.e., a selection for the stromal cells took place during early passages in culture. A complete loss of keratin filaments during in vitro passage has been reported in the literature only under very special circumstances (
To assess the feasibility of a further subclassification of carcinoma cell lines, we tested a panel of antibodies also used for classification of carcinomas. This effort was only partially successful. The expression of PSA (prostate carcinoma), CA125 (ovarian carcinoma), thyroglobulin (thyroid carcinoma), and keratin polypeptides that are typical for squamous cell carcinoma did not correlate sufficiently with the respective cell types. Interestingly, however, 8/9 pancreatic carcinoma cell lines but 0/12 colon cell lines reacted positively with the CK7 antibody. Therefore, as in primary tumors (
The results of testing non-carcinoma-derived cell lines in the DSMZ collection showed that 85% (39/46) were negative when tested with broad-specificity keratin antibodies. Keratin positivity of most of the seven keratin-positive cell lines in Table 3 can be explained because they originate from tumors such as Ewing's tumor or epitheloid sarcoma, which are known to contain keratin-positive tumor cells. The analysis of the immunoprofile of the melanoma cell lines shows that all cell lines are vimentin-positive and that 75% of the melanoma cell lines stained positively for HMB-45. Neither carcinoma cell lines nor other solid tumor-derived cell lines were HMB-45-positive, confirming that this marker effectively detects melanoma-derived cells. However, our data showed that a substantial fraction (25%) of cell lines reported as being derived from melanoma did not express the antigen recognized by HMB-45 (cf.
The combined data show that tissue marker analysis is a very useful technique for verification of the histological origin not only of histological and cytological tumor specimens but also of cell lines. However, the panel of antibodies applicable for characterization of cell lines is relatively small. Useful for the histological characterization of cell lines are pan-keratin antibodies (carcinoma vs non-carcinoma), the CK7 antibody raised against keratin 7 (pancreatic carcinoma vs colon carcinoma), desmin antibodies (rhabdomyosarcoma), neurofilament antibodies (neuroblastoma), and HMB-45 (melanoma vs non-melanoma).
It is very important to draw attention to the fact that not all tissue markers show the same expression pattern in cell lines as in the original tumors. For example, vimentin, which is primarily expressed in mesenchymal tumors (see
Better methods for cell line identification and characterization are needed, as shown in this study and in a previous study in which a minimum of 18% wrong cell lines designated for cell bank entry were identified by other techniques (
Received for publication February 14, 2001; accepted June 5, 2001.
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