Annals of RSCB
Vol. XVI, Issue 2/2011
DEVELOPMENT OF THE B16 MURINE MELANOMA MODEL
Dorina Gheorgheosu1, 2, Cristina Dehelean1*, Mirabela Cristea3,
Danina Muntean2*
“VICTOR BABES” UNIVERSITY OF MEDICINE AND PHARMACY, TIMISOARA:
1
DEPARTMENT OF TOXICOLOGY, FACULTY OF PHARMACY;
2
– DEPARTMENT OF PATHOPHYSIOLOGY, FACULTY OF MEDICINE;
3
– COUNTY HOSPITAL NO. 1, TIMISOARA AND NATIONAL CENTER FOR
IMMUNOLOGY AND TRANSPLANTATION
Summary
Cutaneous malignant melanoma (CMM) is an aggressive type of skin cancer with
significantly high morbidity and mortality and an increased incidence in the last years. The
aim of the present study was to develop a spontaneously - arising murine melanoma model
employing the B16 murine melanoma cell line using C57BL/6J mice in order to observe the
process of solid-tumor formation and the following metastatic process. In the day 20 after
inoculation of B16 cells at C57BL/6J mice, histological studies revealed the presence of
malignant melanoma and metastasis in liver, lungs and spleen. Our data show that the B16
mouse melanoma model is an easy to reproduce in vivo model of carcinogenesis that will be
further used in order to test therapeutical agents against skin carcinoma.
Keywords: melanoma, B16 cell line, mouse, metastasis
cadehelean@umft.ro; daninamuntean@umft.ro
Greek physician Hippocrates in the fifth
century B.C (Bennet and Hall, 1994; Chin
et al., 1998).
A large body of evidence with
respect to animal models of melanoma was
provided in the last decade due to the
increased incidence and high mortality of
melanoma. Historically, the animals
models studied in most detail have been
the Xiphophorus hybrid fish and the South
American opossum (Kusewitt and Ley,
1996; Dlugosz et al., 2002, Ley, 2002).
Other melanoma models were developed
on hamster (Della Porta et al., 1995),
guinea pig (Pawlowski et al., 1980) and
angora goat (Green et al., 1996). The
problem with all such models, however,
was that arising melanocytic tumors do not
resemble
human
CMM
from
histopathological point of view, and
extensive genetic manipulation is not
possible. The mouse represents an
especially attractive system because of the
availability of an extensive genetic base
Introduction
Cutaneous malignant melanoma
(CMM) is the most lethal form of skin
cancer and its incidence has been steadily
increasing worldwide, resulting in a major
public health problem (Markovic et al.,
2007). Melanoma is a malignant tumor
derived from epidermal melanocytes and
can occur in any tissue that contains these
cells, including noncutaneous sites such as
the oral mucosa, nasopharynx, paranasal
sinuses, tracheobronchial tree, vulva,
vagina, anus, urinary tract, central nervous
system, and eye (Cummins et al., 2006).
The first description of melanoma
as a disease entity was provided in 1806 by
Rene Laennec, the inventor of the
stethoscope, in his presentation to the
Faculté de Médicine in Paris and the first
published use of the word “melanoma”
appeared in 1812 in Laennec manuscript,
but references to “black cancer” and “fatal
black tumors with metastasis” can be
traced back to the writings of the legendary
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Vol. XVI, Issue 2/2011
upon which to build (Dlugosz et al., 2002).
The Sinclair swine and dogs also
developed melanoma tumors, but the
murine models were by far the most
widely employed animal model of
melanoma and included the induction of
tumors via application of physical agents
or transgenic manipulations as well as the
inoculation of mice with tumor-cell line
such as the spontaneously occurring
Harding-Passey, Cloudman and B16 cell
lines (Carson and Walker, 2002).
The aim of the present study was to
develop a spontaneously - arising murine
melanoma model employing the B16
murine melanoma cell line using
C57BL/6J mice in order to observe the
process of solid-tumor formation and the
following metastatic process.
Histology. Histological samples were
prepared from skin samples and tumors
from each group of mice and for each
treatment. Samples tissue were fixed in 4%
formalin solution which was improved
with a silver colloidal solution (1/1 v/v).
The histologically prepared samples were
stained with hematoxylin and eosin (H&E)
and examined by electron microscopy.
Results
Melanoma model obtained by inoculation
of B16 murine melanoma cell line. The
mice treated with B16 murine melanoma
cell line suspension developed tumors
rapidly in different organs, such as: skin,
lung, liver and spleen. Lungs were the first
organs affected by metastasis (Figure 1).
Material and methods
Cell preparation. B16 melanoma 4A5
(ECACC and Sigma Aldrich, origin Japan
stored UK ) cells were grown in DMEM
media, supplemented with 10% fetal
bovine serum, penicillin (100 U/ml) and
streptomycin (100 mg/ml) in specific cells
culture plates. Cells were incubated with 1
ml of media at 37°C in an incubator for
cell culture with 95% air and 5% CO2.
Animals. C57BL/6J mice of eight weeks
were purchased from Charles River
(Sulzfeld, Germany). The experimental
protocol followed all NIAH (National
Institute of Animal Health) rules: animals
were kept on a 12h/12h light/dark cycle, at
a normal (24 ºC) animal house
temperature, humidity above 55%, were
fed ad libitum and had free access to water.
The experiments were approved by
University of Medicine and Pharmacy
Timisoara Bioethical Committee.
Experimental design. Melanoma model
obtained by inoculation of B16 murine
melanoma cell line. C57BL/6J mice
(n=4/group) were shaved dorsally once a
week with an electric razor, or as needed to
remove hair. B16 cells (1x106cells in 0.5
ml in saline) were administered IP and in
day 20, mice were killed.
Figure 1. B16 xenograft on C57BL/6J mouse
on day 20 of experiment
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Histopathology of the skin lesions and
tumors. Histological analysis of skin
samples
revealed
dermic
tumoral
proliferation consisting of round and oval
tumor cells with nuclei at the periphery
(Figure 2).
A.
A.
B.
Figure 3. A. Lung tumor proliferation with
blood vessels (HE x 400); B. Pulmonary stasis
combined with tumor proliferation (HE X
200).
Histological analysis of spleen samples
indicated a tumor proliferation similar with
the lungs one characterized by oval tumor
cells with pale eosinophilic cytoplasm and
vesicular nuclei (Figure 4).
B.
C.
Figure 2. A. Wide area of skin ulceration in
the surface (HE x 200); B. Dermic tumor
proliferation (HE x 400); C. Round and oval
tumor cells with nuclei at the periphery (HE x
400).
A.
The histological lung samples showed
pulmonary stasis, tumor proliferation with
a large number of blood vessels and
inflammation of bronchial wall. Tumor
proliferation consisted in beaches and
lobules of round and oval cells with
vesicular nuclei and a small number of
pleomorphic nuclei (Figure 3).
B.
Figure 4. A. Splenic tumor proliferation (HE x
400); B. Oval tumor cells with pale
eosinophilic cytoplasm and vesicular nuclei
(HE x 400).
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tumorigenic line in 1954. After s.c.
implantation, the tumor spontaneously
developed in a C57BL/6J mouse at the
Jackson Laboratories in Maine.
The initial neoplastic lesion arose
in the skin at the base of the ear and the
tumor is described as metastatic to lung,
liver and spleen (Green, 1968; Alvarez,
2002).
In 1970, Dr. Isaiah Fidler carefully
documented the final disposition of the
melanoma cells after IV injection in mice.
In order to develop this study B16 cells
125
were
labeled
with
I-5-iodo-2deoxyuridine. At 14 days after injection
only the lungs contained labeled cells that
could be observed as tumor nodules in that
moment.
Liver, spleen, kidneys and blood all
showed the early presence of the labeled
cells, but none of these tissues show the
establishment of tumors at 14 days
postinjection. Intravenously-injected B16
melanoma cells in mice could lead to rapid
accumulation of cells in the pulmonary
tissue, lead to early high levels of tumor
cells in circulation or ultimately produce a
very low rate of tumor formation in the
animals (Fidler, 1970, Alvarez 2002).
Starting from his initial studies, Fidler
obtained new subtypes of B16 cells, some
of them with a higher metastatic potential
(B16-F1 and B16-F10) (Fidler, 1973).
Another organ affected by metastasis
was the liver. The liver presented a
moderate stasis and a spot-type tumor
proliferation, characterized by tumor cells
with vesicular nuclei and blood vessels similar with lung tumor proliferation
(Figure 5).
A.
B.
Figure 5. A. Liver tumor proliferation with
blood vessels (HE x 400); B. Tumor cells with
vesicular nuclei (HE x 400).
Discussion
The use of animal models in the
study of malignant melanoma represented
an important source of information about
the disease mechanisms, the molecular
events in cells and their progress towards
malignancy. Inoculation of B16 cells
determined the rapid development of
melanoma, followed by metastasis in lung,
liver and spleen, in less than a month, thus
confirming the results obtained by Waheed
Roomi M. (2008).
The use of the B16 murine
melanoma line as a model for both solidtumor formation and metastasis was an
important development in oncology
research. Although the introduction of this
model for metastasis research can be traced
back to the 70s, the cell line itself had been
identified and characterized as a
Conclusion
In vivo melanoma models represent
a great step in oncology research due to
their great similarity to clinical metastasis
and we could easily reproduce the B16
mouse melanoma model in our Labs. View
the relative short delay of time needed to
reproduce it, we hope to get further
insights not only in the steps of
carcinogenesis but also in assessing the
anti-melanoma effects of different natural
or chemical agents.
Acknowledgements
PhD fellowship POSDRU/88/1.5/S/63117.
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