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
a r t i c l e i n f o
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
s u m m a r y
CAL 27 is one of the most frequently used cell lines in the eld 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 veried 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.
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 efcient 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 t 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 conrmed. 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,
314
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 humidied
37 C incubator with 5% CO
2
.
DNA extraction, amplication and DNA ngerprinting
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 amplied
for 16 different genetic loci by the PowerPlex 16 Systemkit (Prome-
ga, Crop. Madison, Wisconsin, USA) following the manufacturers
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 PRISM3100 Genetic Analyzer.
The 16 short tandem repeat (STR) loci were Amelogenin, D3S1358,
1368-8375/$ - see front matter 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: qmchen@scu.edu.cn (Q. Chen), zengxin22@163.com (X. Zeng).
1
These authors contributed equally to this work.
Oral Oncology 45 (2009) e204e207
Contents lists available at ScienceDirect
Oral Oncology
j our nal homepage: www. el sevi er . com/ l ocat e/ or al oncol ogy
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 Public
Health Services policy on humane care and use of laboratory
animals. Seven 6-week-old female BALB/c-nu/nu nude mice were
purchased from the Animal Center of Sichuan University. 2 10
6
log-growing CAL 27 cells were harvested by trypsinization, washed
twice with 1 PBS, suspended in 100 ll of PBS, and injected subcu-
taneously into the right ank site of eachmouse. The mice were kept
in pathogen-free environments, and the xenografts were measured
by caliper every 3 days for nearly two months. The tumor volume
was calculated by the following formula: tumor volume = p/
6 longer diameter shorter diameter
2
. All the mice were sacri-
ced after the xenografts seeded about two months.
Histology and immunohistochemistry
All specimens of the CAL 27 xenografts were xed in 10% buf-
fered formalin, processed, and embedded in parafn. Sections of
3 lm thickness were prepared and evaluated by hematoxylin-eo-
sin (HE) staining and immunohistochemistry (IHC) assay. The
IHC assay was performed as described elsewhere.
2
The antibody
to MUC1 mucin (4538) was obtained from Cell Signaling Technol-
ogy (Beverly, MA), and diluted 1:50 following the manufacturers
recommendations. The histopathological diagnoses were made by
three different pathologists at the Department of Histopathology,
Hospital of West China College of Stomatology.
Results
DNA ngerprinting proles of CAL 27 cells
To verify the identity of CAL 27 cells used in our study, and to
rule out the possibility of cell line cross-contamination and misi-
dentication, the DNA ngerprints analysis using probes for the
gender-specic amelogenin and 15 STRs was performed (Fig. 1).
The results showed that the CAL 27 cells we used were accurate
since they had more than 7 loci same with the standard STR pro-
les 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 the
growth pattern of CAL 27 xenografts in vivo. According to the tu-
mor growth curve (Fig. 2A), the rst palpable xenograft arose at
about 7 days after seeding, and the growth speed of the CAL 27
xenografts was slow in the following 20 days. At the end of the rst
month, the average volume of the CAL 27 xenografts reached about
100 mm
3
. In the process of CAL 27 xenografts growth, small vesi-
cles came into being at the surface of some xenografts at the end
of the third week after seeding. In the fth 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 mainly
on the change of vesicle volumes (Fig. 2A and B). At the end of
the second month, all the mice were sacriced and the CAL 27
xenografts were collected for further analysis. The results of tumor
anatomy showed that vesicles formed not only at the surfaces of
Figure 1 STR proles of CAL 27 cells. The DNA derived from the CAL 27 cells was analyzed by using the STR prole multiplex system: PowerPlex

16 kit (Promega) with the

ABI PRISM 3100 Genetic Analyzer. The DNA extraction, amplication, and the detection of DNA ngerprints were performed as described in Section 2.
Table 1
STR proles of CAL 27 cells.
Cell
line
Percen
match
STR prole results
Amel
c
D3S1358 TH01 D21S11 D18S51 Penta
E
D5S818 D13S317 D7S820 D16S539 CSF1PO Penta
D
vWA D8S1179 TPOX FGA
CAL 27
a
X 6, 9.3 11,12 10,11 10 11,12 10,12 14,17 8
CAL 27
b
100 X 16 6, 9.3 28,29 13 7 11,12 10,11 10 11,12 10,12 9,10 14,17 13,15 8 25
a
The STR proles of CAL 27 cells published by ATCC.
b
The STR proles of CAL 27 cells used in our study.
c
Amel, amelogenin.
L. Jiang et al. / Oral Oncology 45 (2009) e204e207 e205
the xenografts but also in the deeper areas of the tumor tissues.
During tumor anatomy, no sign of local tumor aggressiveness
was observed since continuous and integrate basement membrane
could be found in every xenograft. Moreover, no lymph node
metastasis was detected. Therefore, the CAL 27 xenografts do not
seem 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 the
deeper areas of the CAL 27 xenografts. Since this phenomenon
hasnt been reported in previous literatures, we performed HE
staining to further study this issue. Our results showed that CAL
27 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 diagnosis
seemed to be either adenosquamous carcinoma or adenoid squa-
mous cell carcinoma. Since the results of HE staining is not suf-
cient for differential diagnosis of these two kinds of diseases, IHC
staining of mucin was performed. As shown in Fig. 2D, MUC1 mu-
cin was positively expressed in the glandular-like structures of the
xenografts. Therefore, the CAL 27 xenografts were diagnosed as
adenosquamous carcinoma with two distinct histological compo-
nents, one is the squamous cell carcinoma, and the other is the
glandular differentiated areas.
Discussion
OSCC cell line CAL 27 was established by Gioanni in 1982. At the
time it was built, Gioanni and his colleagues had tested its tumor-
igenicity in athymic nude mice.
1
Their results showed that 6 weeks
after s.c. injection of 2 10
6
CAL 27 cells in both anks of 10 mice,
solid tumor developed in 8 of the 20 injection sites. The tumor
growth speed of CAL 27 xenografts was rapid, about 35 mm per
week. In their study, no phenomenon about vesicle formation in
the process of tumor growth were reported, and the histological
examination of the CAL 27 xenografts showed that they were well
differentiated squamous cell carcinoma with varying degree of
maturity. However, no photomicrographs of this histology were
presented in their paper. In the following decades after CAL 27 cell
line was established, it has been used as a representative OSCC cell
line to build models for studies in vitro and in vivo.
310
From 2000
to 2009, there are 7 studies we searched in the PubMed database
using 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 these
studies were different from each other, the growth speeds of the
CAL 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 of
these studies present adequate information about CAL 27 xeno-
graft histology except Lees study,
10
which has presented photomi-
crographs of this histology in their published article. However,
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 grey
area 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 in
the glandular structures of the CAL 27 xenograft. Arrows indicate the MUC1 mucin-positive glandular differentiated areas. All micrographs were taken with a Leica DMI 6000
B inverted uorescence microscope (Leica Microsystems). Original magnication, 100 (a) and 200 (b). (For interpretation of the references in colour in this gure legend,
the reader is referred to the web version of this article.)
Table 2
Selected studies using CAL 27 to set up in vivo OSCC model.
Studies Site Seeding cell number Tumor growing time (days) Tumor volume (mm
3
)
Lee et al. (2007)
10
Anterior tongue 5 10
5
30
a
LoTempio et al. (2005)
11
Flant 1 10
6
22 85
Alderson et al. (2002)
12
Flant 2 10
6
42 50
Azemar et al. (2000)
13
Flant 3 10
6
22 600
Simons et al. (2007)
8
Flant 8 10
6
18 600
a
Data not presented in the paper.
e206 L. Jiang et al. / Oral Oncology 45 (2009) e204e207
since adenosquamous carcinoma has two distinct histological
components, the squamous cell carcinoma component and the
glandular differentiated areas, the magnication of the gures pre-
sented in Lees article seems to be too large to completely rule out
the possibility that glandular differentiated areas exist in their CAL
27 xenografts. In our study, 2 10
6
CAL 27 cells were seeded on
the right ank of 7 mice to build OSCC xenografts. Solid tumor
developed in all the injection sites, and the xenografts grew slowly
with vesicle formation. During the process of tumor anatomy, ves-
icles were found not only at the surfaces but also in the deeper
areas 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 27
xenograft tissues. Therefore, the CAL 27 xenografts were diagnosed
to be adenosquamous carcinoma. Though adenosquamous carci-
noma belongs to a subtype of OSCC, its specic growing pattern
and histological characteristics may affect the results of studies.
Therefore, it is our suggestion that researchers should take this into
consideration before they use CAL 27 cells to build OSCC models
for studies in vivo.
In the eld of OSCC study, up till now, there are numerous OSCC
cell lines established. However, since the growth patterns of these
cell lines are different from each other, not all of them t for build-
ing up OSCC models for studies in vitro and in vivo. Therefore, it is
important for researchers to get enough information about the
characteristics of the cell lines established. Otherwise, time and re-
sources will be wasted.
Given the importance of cell line selection for studies, it is our
opinion that information about the characteristics of cell growth
in vitro and in vivo should be published. Therefore, researches will
be more efcient, and less resource will be wasted. Notably, with
the widespread problem of cell line cross-contamination and misi-
dentication,
1517
it is necessarytoconrmtheidentications of cell
lines by STR before starting a newproject, or drawing conclusions.
18
Conict 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 the
manuscript. This work was supported by grants from the National
Science Funds for Talented Professionals of China (No. 30725041),
the National Basic Research Program of China (2008CB517307), the
National Natural Science Foundation of China (Nos. 30471891,
30672323), and the New Century Talents Support Program of
MOE (NCET-04-0865).
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