Short Communication CAL 27 is an oral adenosquamous carcinoma cell line Lu Jiang a,1 , Ning Ji a,b,1 , Yu Zhou a , Jing Li a,b , Xianting Liu a,b , Zhi Wang a , Qianming Chen a, * , Xin Zeng a, * a State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, No. 14, Sec. 3, Renminnan Road, Chengdu, Sichuan 610041, China b Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, Sichuan University, Chengdu, China article info Article history: Received 18 April 2009 Received in revised form 4 June 2009 Accepted 4 June 2009 Available online 23 July 2009 Keywords: CAL 27 Adenosquamous carcinoma Cell line summary CAL 27 is one of the most frequently used cell lines in the field of oral squamous cell carcinoma (OSCC) studying. However, our recent studies showed that the growth pattern of CAL 27 xenograft did not seem like typical OSCC. In this study, we verified the identity of CAL 27 cells by using by short tandem repeat analysis. Then, we performed tumor formation assay, HE staining and immunohistochemistry assay to further study the growing characteristics and histopathological diagnosis of CAL 27 xenografts. Our results showed that CAL 27 xenografts grew slowly in vivo with vesicle formation at both the surfaces and deeper areas of the tumors. The CAL 27 xenografts were then diagnosed as oral adenosquamous carcinomas. Thus CAL 27 appears to be an oral adenosquamous carcinoma cell line. Ó 2009 Elsevier Ltd. All rights reserved. Introduction Continuous oral squamous cell carcinoma (OSCC) cell lines have been established and become important research tools for studies of better understanding of the pathogenesis of this disease and searching for efficient therapeutic methods. Up till now, there have been a variety of OSCC cell lines derived from tissues of patients with OSCC. However, since the growth patterns of cell lines are dif- ferent, not all of them fit for building OSCC models for studies in vitro and in vivo. Therefore, the growing characteristics and his- topathological diagnoses of these OSCC cell lines are very impor- tant considerations when selecting cell lines to build OSCC models. In 1982, Gioanni and his colleagues established a new OSCC cell line CAL 27 from the tumor tissue of a 56 year old Caucasian male with poorly differentiated squamous cell carcinoma at the middle of the tongue. 1 At the time CAL 27 cell line was built, its tumorige- nicity in athymic nude mice had been confirmed. The histological examination of CAL 27 xenografts showed that they were well dif- ferentiated squamous cell carcinomas at varying degrees of matu- rity. In the following decades after CAL 27 cell line was established, it has been widely used to build OSCC models for studies in vitro and in vivo, 3–14 and thus regarded as a representative cell line for OSCC studying. In one of our recent studies, CAL 27 cells were selected to build OSCC models for studies both in vitro and in vivo. The studies in vitro went on well with CAL 27 cells. However, the studies in vivo were severely bothered by the growth pattern of CAL 27 xenografts, showing slow growing speed with vesicles formation at both the surface and the deeper areas of the tumors. Since the growth pattern of CAL 27 xenografts showing in our study did not seem like typical OSCC, and this issue has not been reported in previous literatures, we performed the current study to further explore this problem. Materials and methods Cell culture Human tongue squamous cell carcinoma cell line CAL 27 was bought from American Tissue Culture Collection (ATCC). Cells were grown in DMEM (Gibco) with 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 lg/ml streptomycin in a humidified 37 °C incubator with 5% CO 2 . DNA extraction, amplification and DNA fingerprinting DNA was extracted from CAL 27 cells and eluted in TE buffer (1 mM Tris, 0.1 mM EDTA, pH 8). The extracted DNA was amplified for 16 different genetic loci by the PowerPlex 16 System kit (Prome- ga, Crop. Madison, Wisconsin, USA) following the manufacturer’s recommendations. PCR was performed according to the standard protocol supplied with the PowerPlex 16 System kit. Capillary elec- trophoresis was carried out on an ABI PRISM 3100 Genetic Analyzer. The 16 short tandem repeat (STR) loci were Amelogenin, D3S1358, 1368-8375/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.oraloncology.2009.06.001 Abbreviations: OSCC, oral squamous cell carcinoma; STR, short tandem repeat; HE staining, hematoxylin-eosin staining; IHC, immunohistochemistry. * Corresponding authors. Tel.: +86 138 80535268; fax: +86 028 85405251. E-mail addresses: [email protected](Q. Chen), [email protected](X. Zeng). 1 These authors contributed equally to this work. Oral Oncology 45 (2009) e204–e207 Contents lists available at ScienceDirect Oral Oncology journal homepage: www.elsevier.com/locate/oraloncology
CAL 27 is an oral adenosquamous carcinoma cell line
Lu Jiang a,1, Ning Ji a,b,1, Yu Zhou a, Jing Li a,b, Xianting Liu a,b, Zhi Wang a, Qianming Chen a,*, Xin Zeng a,*
a State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, No. 14, Sec. 3, Renminnan Road, Chengdu, Sichuan 610041, Chinab Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, Sichuan University, Chengdu, China
a r t i c l e i n f o
Article history:Received 18 April 2009Received in revised form 4 June 2009Accepted 4 June 2009Available online 23 July 2009
Keywords:CAL 27Adenosquamous carcinomaCell line
1368-8375/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.oraloncology.2009.06.001
CAL 27 is one of the most frequently used cell lines in the field of oral squamous cell carcinoma (OSCC)studying. However, our recent studies showed that the growth pattern of CAL 27 xenograft did not seemlike typical OSCC. In this study, we verified the identity of CAL 27 cells by using by short tandem repeatanalysis. Then, we performed tumor formation assay, HE staining and immunohistochemistry assay tofurther study the growing characteristics and histopathological diagnosis of CAL 27 xenografts. Ourresults showed that CAL 27 xenografts grew slowly in vivo with vesicle formation at both the surfacesand deeper areas of the tumors. The CAL 27 xenografts were then diagnosed as oral adenosquamouscarcinomas. Thus CAL 27 appears to be an oral adenosquamous carcinoma cell line.
� 2009 Elsevier Ltd. All rights reserved.
Introduction
Continuous oral squamous cell carcinoma (OSCC) cell lines havebeen established and become important research tools for studiesof better understanding of the pathogenesis of this disease andsearching for efficient therapeutic methods. Up till now, there havebeen a variety of OSCC cell lines derived from tissues of patientswith OSCC. However, since the growth patterns of cell lines are dif-ferent, not all of them fit for building OSCC models for studiesin vitro and in vivo. Therefore, the growing characteristics and his-topathological diagnoses of these OSCC cell lines are very impor-tant considerations when selecting cell lines to build OSCC models.
In 1982, Gioanni and his colleagues established a new OSCC cellline CAL 27 from the tumor tissue of a 56 year old Caucasian malewith poorly differentiated squamous cell carcinoma at the middleof the tongue.1 At the time CAL 27 cell line was built, its tumorige-nicity in athymic nude mice had been confirmed. The histologicalexamination of CAL 27 xenografts showed that they were well dif-ferentiated squamous cell carcinomas at varying degrees of matu-rity. In the following decades after CAL 27 cell line was established,it has been widely used to build OSCC models for studies in vitroand in vivo,3–14 and thus regarded as a representative cell line forOSCC studying.
In one of our recent studies, CAL 27 cells were selected to buildOSCC models for studies both in vitro and in vivo. The studies
ll rights reserved.
a; STR, short tandem repeat;ohistochemistry.fax: +86 028 [email protected] (X. Zeng).
in vitro went on well with CAL 27 cells. However, the studiesin vivo were severely bothered by the growth pattern of CAL 27xenografts, showing slow growing speed with vesicles formationat both the surface and the deeper areas of the tumors. Since thegrowth pattern of CAL 27 xenografts showing in our study didnot seem like typical OSCC, and this issue has not been reportedin previous literatures, we performed the current study to furtherexplore this problem.
Materials and methods
Cell culture
Human tongue squamous cell carcinoma cell line CAL 27 wasbought from American Tissue Culture Collection (ATCC). Cells weregrown in DMEM (Gibco) with 10% fetal bovine serum (FBS; Gibco),100 U/ml penicillin, and 100 lg/ml streptomycin in a humidified37 �C incubator with 5% CO2.
DNA extraction, amplification and DNA fingerprinting
DNA was extracted from CAL 27 cells and eluted in TE buffer(1 mM Tris, 0.1 mM EDTA, pH 8). The extracted DNA was amplifiedfor 16 different genetic loci by the PowerPlex 16 System kit (Prome-ga, Crop. Madison, Wisconsin, USA) following the manufacturer’srecommendations. PCR was performed according to the standardprotocol supplied with the PowerPlex 16 System kit. Capillary elec-trophoresis was carried out on an ABI PRISM 3100 Genetic Analyzer.The 16 short tandem repeat (STR) loci were Amelogenin, D3S1358,
L. Jiang et al. / Oral Oncology 45 (2009) e204–e207 e205
TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820,D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA.
In vivo tumor formation assay
The animal studies were conducted following the US PublicHealth Service’s policy on humane care and use of laboratoryanimals. Seven 6-week-old female BALB/c-nu/nu nude mice werepurchased from the Animal Center of Sichuan University. 2 � 106
log-growing CAL 27 cells were harvested by trypsinization, washedtwice with 1 � PBS, suspended in 100 ll of PBS, and injected subcu-taneously into the right flank site of each mouse. The mice were keptin pathogen-free environments, and the xenografts were measuredby caliper every 3 days for nearly two months. The tumor volumewas calculated by the following formula: tumor volume = p/6 � longer diameter � shorter diameter2. All the mice were sacri-ficed after the xenografts seeded about two months.
Histology and immunohistochemistry
All specimens of the CAL 27 xenografts were fixed in 10% buf-fered formalin, processed, and embedded in paraffin. Sections of3 lm thickness were prepared and evaluated by hematoxylin-eo-sin (HE) staining and immunohistochemistry (IHC) assay. TheIHC assay was performed as described elsewhere.2 The antibodyto MUC1 mucin (4538) was obtained from Cell Signaling Technol-ogy (Beverly, MA), and diluted 1:50 following the manufacturer’srecommendations. The histopathological diagnoses were made bythree different pathologists at the Department of Histopathology,Hospital of West China College of Stomatology.
Figure 1 STR profiles of CAL 27 cells. The DNA derived from the CAL 27 cells was analyzeABI PRISM 3100 Genetic Analyzer. The DNA extraction, amplification, and the detection
Table 1STR profiles of CAL 27 cells.
Cellline
Percenmatch
STR profile results
Amelc D3S1358 TH01 D21S11 D18S51 PentaE
D5S818 D
CAL 27a X 6, 9.3 11,12 1CAL 27b 100 X 16 6, 9.3 28,29 13 7 11,12 1
a The STR profiles of CAL 27 cells published by ATCC.b The STR profiles of CAL 27 cells used in our study.c Amel, amelogenin.
Results
DNA fingerprinting profiles of CAL 27 cells
To verify the identity of CAL 27 cells used in our study, and torule out the possibility of cell line cross-contamination and misi-dentification, the DNA fingerprints analysis using probes for thegender-specific amelogenin and 15 STRs was performed (Fig. 1).The results showed that the CAL 27 cells we used were accuratesince they had more than 7 loci same with the standard STR pro-files of CAL 27 cells (Table 1).
CAL 27 xenografts grow slowly in vivo with vesicles formation
The in vivo tumor formation assay was performed to study thegrowth pattern of CAL 27 xenografts in vivo. According to the tu-mor growth curve (Fig. 2A), the first palpable xenograft arose atabout 7 days after seeding, and the growth speed of the CAL 27xenografts was slow in the following 20 days. At the end of the firstmonth, the average volume of the CAL 27 xenografts reached about100 mm3. In the process of CAL 27 xenografts growth, small vesi-cles came into being at the surface of some xenografts at the endof the third week after seeding. In the fifth week, vesicles with dif-ferent volumes could be found at the surface of all the xenografts.From then on, the volumes of the solid tumors increased slowly,and the change of the average tumor volume depended mainlyon the change of vesicle volumes (Fig. 2A and B). At the end ofthe second month, all the mice were sacrificed and the CAL 27xenografts were collected for further analysis. The results of tumoranatomy showed that vesicles formed not only at the surfaces of
d by using the STR profile multiplex system: PowerPlex� 16 kit (Promega) with theof DNA fingerprints were performed as described in Section 2.
Figure 2 CAL 27 xenografts belong to adenosquamous carcinoma. (A) Tumor growth curve of the CAL 27 xenografts. Results represent the Means ± SE (n = 7 tumors). The greyarea indicates the period when the change of tumor volume depended mainly on the vesicles. (B) CAL 27 xenografts with vesicle formation. Arrows indicate vesicles. (a)Unbroken vesicle; (b) broken vesicle. (C) HE staining of CAL 27 xenograft tissues. Arrows indicate the glandular differentiated area. (D) MUC1 mucin positively expressed inthe glandular structures of the CAL 27 xenograft. Arrows indicate the MUC1 mucin-positive glandular differentiated areas. All micrographs were taken with a Leica DMI 6000B inverted fluorescence microscope (Leica Microsystems). Original magnification, �100 (a) and �200 (b). (For interpretation of the references in colour in this figure legend,the reader is referred to the web version of this article.)
Table 2Selected studies using CAL 27 to set up in vivo OSCC model.
Studies Site Seeding cell number Tumor growing time (days) Tumor volume (mm3)
Lee et al. (2007)10 Anterior tongue 5 � 105 30 –a
LoTempio et al. (2005)11 Flant 1 � 106 22 85Alderson et al. (2002)12 Flant 2 � 106 42 50Azemar et al. (2000)13 Flant 3 � 106 22 600Simons et al. (2007)8 Flant 8 � 106 18 600
a Data not presented in the paper.
e206 L. Jiang et al. / Oral Oncology 45 (2009) e204–e207
the xenografts but also in the deeper areas of the tumor tissues.During tumor anatomy, no sign of local tumor aggressivenesswas observed since continuous and integrate basement membranecould be found in every xenograft. Moreover, no lymph nodemetastasis was detected. Therefore, the CAL 27 xenografts do notseem to be invasive in nude mice model.
CAL 27 xenografts belong to oral adenosquamous carcinoma
It is notable that vesicles formed both at the surface and in thedeeper areas of the CAL 27 xenografts. Since this phenomenonhasn’t been reported in previous literatures, we performed HEstaining to further study this issue. Our results showed that CAL27 xenografts had two distinct histological components (Fig. 2C).One is the usual squamous cell carcinoma, with moderately differ-entiation degree. The other is glandular-like structures, which dis-played in the deeper areas of the tumor. Therefore, the diagnosisseemed to be either adenosquamous carcinoma or adenoid squa-mous cell carcinoma. Since the results of HE staining is not suffi-cient for differential diagnosis of these two kinds of diseases, IHCstaining of mucin was performed. As shown in Fig. 2D, MUC1 mu-cin was positively expressed in the glandular-like structures of thexenografts. Therefore, the CAL 27 xenografts were diagnosed asadenosquamous carcinoma with two distinct histological compo-nents, one is the squamous cell carcinoma, and the other is theglandular differentiated areas.
Discussion
OSCC cell line CAL 27 was established by Gioanni in 1982. At thetime it was built, Gioanni and his colleagues had tested its tumor-igenicity in athymic nude mice.1 Their results showed that 6 weeksafter s.c. injection of 2 � 106 CAL 27 cells in both flanks of 10 mice,solid tumor developed in 8 of the 20 injection sites. The tumorgrowth speed of CAL 27 xenografts was rapid, about 3–5 mm perweek. In their study, no phenomenon about vesicle formation inthe process of tumor growth were reported, and the histologicalexamination of the CAL 27 xenografts showed that they were welldifferentiated squamous cell carcinoma with varying degree ofmaturity. However, no photomicrographs of this histology werepresented in their paper. In the following decades after CAL 27 cellline was established, it has been used as a representative OSCC cellline to build models for studies in vitro and in vivo.3–10 From 2000to 2009, there are 7 studies we searched in the PubMed databaseusing CAL 27 cell line to build OSCC models for studies in vivo.8–
14 Since the numbers of the initial seeded CAL 27 cells in thesestudies were different from each other, the growth speeds of theCAL 27 xenografts were different (Table 2). In these studies,although the tumor growing periods varied from 20 to 55 days,no vesicles formation phenomenon was mentioned. Also, none ofthese studies present adequate information about CAL 27 xeno-graft histology except Lee’s study,10 which has presented photomi-crographs of this histology in their published article. However,
L. Jiang et al. / Oral Oncology 45 (2009) e204–e207 e207
since adenosquamous carcinoma has two distinct histologicalcomponents, the squamous cell carcinoma component and theglandular differentiated areas, the magnification of the figures pre-sented in Lee’s article seems to be too large to completely rule outthe possibility that glandular differentiated areas exist in their CAL27 xenografts. In our study, 2 � 106 CAL 27 cells were seeded onthe right flank of 7 mice to build OSCC xenografts. Solid tumordeveloped in all the injection sites, and the xenografts grew slowlywith vesicle formation. During the process of tumor anatomy, ves-icles were found not only at the surfaces but also in the deeperareas of the tumor tissues. By histology and immunohistochemis-try examination, squamous cell carcinoma components and glan-dular differentiated areas were found to coexist in CAL 27xenograft tissues. Therefore, the CAL 27 xenografts were diagnosedto be adenosquamous carcinoma. Though adenosquamous carci-noma belongs to a subtype of OSCC, its specific growing patternand histological characteristics may affect the results of studies.Therefore, it is our suggestion that researchers should take this intoconsideration before they use CAL 27 cells to build OSCC modelsfor studies in vivo.
In the field of OSCC study, up till now, there are numerous OSCCcell lines established. However, since the growth patterns of thesecell lines are different from each other, not all of them fit for build-ing up OSCC models for studies in vitro and in vivo. Therefore, it isimportant for researchers to get enough information about thecharacteristics of the cell lines established. Otherwise, time and re-sources will be wasted.
Given the importance of cell line selection for studies, it is ouropinion that information about the characteristics of cell growthin vitro and in vivo should be published. Therefore, researches willbe more efficient, and less resource will be wasted. Notably, withthe widespread problem of cell line cross-contamination and misi-dentification,15–17 it is necessary to confirm the identifications of celllines by STR before starting a new project, or drawing conclusions.18
Conflict of interest statement
None declared.
Acknowledgements
We acknowledge Dr. Jinsheng Yu, at the School of public health,Center for Molecular Biology of Oral Diseases, College of Dentistry,University of Illinois at Chicago, IL, USA, for critical reading of themanuscript. This work was supported by grants from the NationalScience Funds for Talented Professionals of China (No. 30725041),the National Basic Research Program of China (2008CB517307), theNational Natural Science Foundation of China (Nos. 30471891,
30672323), and the New Century Talents Support Program ofMOE (NCET-04-0865).
References
1. Gioanni J, Fischel JL, Lambert JC, Demard F, Mazeau C, Zanghellini E, et al. Twonew human tumor cell lines derived from squamous cell carcinomas of thetongue: establishment, characterization and response to cytotoxic treatment.Eur J Cancer Clin Oncol 1988;24(9):1445–55.
2. Jiang L, Zeng X, Yang H, Wang Z, Shen J, Bai J, et al. Oral cancer overexpressed 1(ORAOV1): a regulator for the cell growth and tumor angiogenesis in oralsquamous cell carcinoma. Int J Cancer 2008;123(8):1779–86.
3. Aznavoorian S, Moore BA, Alexander-Lister LD, Hallit SL, Windsor LJ, Engler JA.Membrane type I-matrix metalloproteinase-mediated degradation of type Icollagen by oral squamous cell carcinoma cells. Cancer Res2001;61(16):6264–75.
4. Simoens C, Korst AE, De Pooter CM, Lambrechts HA, Pattyn GG, Faircloth GT,et al. In vitro interaction between ecteinascidin 743 (ET-743) and radiation, inrelation to its cell cycle effects. Brit J Cancer 2003;89(12):2305–11.
5. Herak Bosnar M, Bago R, Konjevoda P, Pavelic J. Gene expression profiling ofNm23-H2 overexpressing CAL 27 cells using DNA microarray. Neoplasma2008;55(5):447–54.
6. Tseng MY, Liu SY, Chen HR, Wu YJ, Chiu CC, Chan PT, et al. Serine proteaseinhibitor (SERPIN) B1 promotes oral cancer cell motility and is over-expressedin invasive oral squamous cell carcinoma. Oral Oncol 2009;45(9):771–6.
7. Lo Muzio L, Pannone G, Staibano S, Mignogna MD, Serpico R, Fanali S, et al.P120(cat) delocalization in cell lines of oral cancer. Oral Oncol2002;38(1):64–72.
8. Simons AL, Fath MA, Mattson DM, Smith BJ, Walsh SA, Graham MM, et al.Enhanced response of human head and neck cancer xenograft tumors tocisplatin combined with 2-deoxy-D-glucose correlates with increased 18F-FDGuptake as determined by PET imaging. Int J Radiat Oncol Biol Phys 2007;69(4):1222–30.
9. Rosenthal EL, Vidrine DM, Zhang W. Extracellular matrix metalloproteaseinducer stimulates fibroblast-mediated tumor growth in vivo. Laryngoscope2006;116(7):1086–92.
10. Lee TK, Poon RT, Wo JY, Ma S, Guan XY, Myers JN, et al. Lupeol suppressescisplatin-induced nuclear factor-kappaB activation in head and neck squamouscell carcinoma and inhibits local invasion and nodal metastasis in an orthotopicnude mouse model. Cancer Res 2007;67(18):8800–9.
11. LoTempio MM, Veena MS, Steele HL, Ramamurthy B, Ramalingam TS, CohenAN, et al. Curcumin suppresses growth of head and neck squamous cellcarcinoma. Clin Cancer Res 2005;11(19 Pt 1):6994–7002.
12. Alderson R, Gohari-Fritsch S, Olsen H, Roschke V, Vance C, Connolly K. In vitroand in vivo effects of repifermin (keratinocyte growth factor-2, KGF-2) onhuman carcinoma cells. Cancer Chemother Pharmacol 2002;50(3):202–12.
13. Azemar M, Schmidt M, Arlt F, Kennel P, Brandt B, Papadimitriou A, et al.Recombinant antibody toxins specific for ErbB2 and EGF receptor inhibit thein vitro growth of human head and neck cancer cells and cause rapid tumorregression in vivo. Int J Cancer 2000;86(2):269–75.
14. McLaughlin PJ, Levin RJ, Zagon IS. The opioid growth factor receptor in humanhead and neck squamous cell carcinoma. Int J Mol Med 2000;5(2):191–6.
15. Masters J. False cell lines. Carcinogenesis 2002;23(2):371.16. Nardone RM. Eradication of cross-contaminated cell lines: a call for action. Cell
Biol Toxicol 2007;23(6):367–72.17. MacLeod RA, Dirks WG, Matsuo Y, Kaufmann M, Milch H, Drexler HG.
Widespread intraspecies cross-contamination of human tumor cell linesarising at source. Int J Cancer 1999;83(4):555–63.
18. Azari S, Ahmadi N, Tehrani MJ, Shokri F. Profiling and authentication of humancell lines using short tandem repeat (STR) loci: report from the National CellBank of Iran. Biologicals 2007;35(3):195–202.
