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Mesothelioma /
Asbestos Cancer |
Characterization
of human Malignant Mesothelioma cell
lines orthotopically implanted in the
pleural cavity of immunodeficient mice for
their ability to grow and form metastasis
Daniele Martarelli1, Alfonso Catalano2, Antonio Procopio2, Sara Orecchia3, Roberta Libener3
and Giorgio Santoni1
1Department of Experimental
Medicine and Public Health, University of
Camerino, 62032 Camerino, Italy,
2Department of Molecular
Pathology and Innovative Therapies,
Polytechnic University of Marche, 60100,
Ancona, Italy and Center of Cytology,
Italian National Research Centers on Aging (INRCA
– IRCCS), Ancona, Italy,
3Pathology Unit, Dept. Of
Oncology, A.S.O. Alessandria, Italy,
BMC Cancer 2006, 6:130 doi:10.1186/1471-2407-6-130
© 2006 Martarelli et al; licensee BioMed
Central Ltd. This is an Open Access
article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the
original work is properly cited. |
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Abstract
Background
Malignant
pleural
mesothelioma (MPM) is a tumor
known to be resistant to conventional
therapies. Thus, an in vivo model can
represent an important tool for assessing
the efficacy of novel approaches in the
treatment of Malignant Pleural Mesothelioma.
Presently, human
Malignant Pleural Mesothelioma cells have been grown orthotopically in
mice upon transplantation of tumor masses or
tumor cell suspensions following surgery. In
these models however, surgery can interfere
with the tumor growth and the early stages
of tumor development cannot be easily
explored. Finally, results may not be so
accurate due to implantation of potentially
different tumor samples in different
experimental groups.
Our work aimed
at establishing a nude mouse model
xenotransplanted with human Malignant Pleural
Mesothelioma cell lines
in which tumor progression exhibits some
features of the human disease.
Method
Three distinct
human Malignant Pleural
Mesothelioma cell lines previously established from
Malignant Pleural Mesothelioma
patients displaying two different phenotypes,
biphasic (MM-B1 and IST-Mes3) and epithelioid
(IST-Mes2), were directly injected into the pleural
cavity of nude mice. At different times, mice were
sacrificed for autopsy, tumor nodules were counted
and then removed for histology. Presence of
metastases in visceral organs was also monitored.
Results
IST-Mes2 cells
were unable to grow in nude mice. MM-B1 and
IST-Mes3 cells were capable of growing in
nude mice and formed tumor nodules in the
pleura. Post-mortem examination showed that
Malignant Pleural
Mesothelioma
cells progressively colonized the parietal
and visceral pleura, the diaphragm, the
mediastinum and, lastly the lung parenchyma.
No pneumo-thorax was evidenced in the mice.
Pleural effusions as well as
lymph node
metastases were observed only at later
times.
Conclusion
This model
mimics the progression of human malignant
mesothelioma
and it is easy to perform and reproducible;
therefore it can be useful to study human
Malignant Pleural Mesothelioma (MPM)
biology and evaluate the efficacy of novel
therapies.
Background
Malignant
pleural mesothelioma is a tumor of the
pleura mainly caused by exposure to asbestos
fibers. Malignant Pleural Mesothelioma diagnosis regards about 2500
persons every year in the United States [1]
and the prognosis is poor despite the
therapies currently used, including surgery,
radiotherapy and
chemotherapy.
Because of the
inefficacy of the conventional treatments,
novel therapeutic strategies are under
investigation, with particular attention
devoted to agents capable of inhibiting the
angiogenesis or inducing tumor cell
apoptosis [2-7].
Tumor
angiogenesis, apoptosis and metastasis,
strictly depend on the site of tumor
development; therefore a human-like animal
model is an important tool for studying new
approaches for Malignant Pleural Mesothelioma treatment.
A number of
evidences indicate that orthotopic models of
tumor growth are more valuable as compared
to those in which the tumor mass grows
subcutaneously [8].
In regard to Malignant Pleural
Mesothelioma,
subcutaneous implantation of human cancer
cells in immunodeficient mice results in
tumor growth at the injection site and no
metastatic dissemination, whereas human Malignant Pleural Mesothelioma
growth in humans is associated with regional
tumor spreading and lung invasion [9].
Presently, human
Malignant Pleural Mesothelioma cells have been grow orthotopically in
mice upon transplantation of tumor masses or
tumor cell suspensions following surgery [10].
In these models however, surgery can
interfere with the tumor growth [8]
and the early stages of tumor development
cannot be easily explored. Finally, results
may be not so accurate due to implantation
of potentially different tumor samples in
different experimental groups [9].
Overall, the aim of this work was to
establish a new orthotopic tumor model by
injecting human Malignant Pleural Mesothelioma cells directly into the
pleural cavity of nude mice. This model does
not need surgical operations, can be easily
performed and more importantly can mimic
tumor development in humans. Thus it can
represent an useful tool for studying human
Malignant Pleural Mesothelioma biology and assessing the efficacy of
novel therapies.
Methods
Cell lines
Human malignant
pleura mesothelial cell lines were
established as previously described [11,12].
Three distinct cell lines with two different
phenotypes, biphasic (MM-B1 and IST-Mes3)
and epithelioid (IST-Mes2) were used between
the eighth and twelfth passage in culture.
Cells were maintained in RPMI-1640 medium
and 10% fetal bovine serum, 1% L-glutamine
and 1% penicillin-streptomycin, (Euroclone,
Devon, UK) at 37°C in a humidified incubator
in an atmosphere of 5% CO2 in air.
Animals
Athymic male
nude mice nu/nu (Harlan, Italy), 6 week-old
were used. Mice were kept in laminar-flow
cages in standardized environmental
conditions. Sterilized food (Harlan, Italy)
and water were supplied ad libitum.
Subcutaneous implantation of Malignant
Pleural Mesothelioma cells in nude
mice
Cells were
harvested at near confluence with trypsin/EDTA
solution. Only cell suspensions with a
viability of >90% as assessed by trypan blue
exclusion assay, were used. Two × 106
MPM cells in 100
μl of Ca++ and Mg++ free Hank's
balanced salt solution (HBSS) were injected
subcutaneously on the left lateral chest
wall near to the axilla. Tumor growth was
monitored twice a week using a caliper.
Tumor volume was calculated using the
formula: V (mm3) = (D × d2)/2, where d (mm)
and D (mm) are the smallest and largest
perpendicular tumor diameter, respectively.
Orthotopic implantation of Malignant Pleural
Mesothelioma cells in nude mice
Mice were
anaesthetized with Tiletamine chlorohydrate
and Zolazepam chlorohydrate and placed in
position of right lateral decubitus. A 27
gauge needle of a 1000
μl syringe was advanced approximately
through the fourth intercostal space for
about 5 mm, into the left pleural cavity,
and two × 106 tumor cells
suspended in 100 μl
of HBSS were injected. The site of injection
in the chest and the precise depth of the
needle tip required to reach the pleural
space were previously determined by
injecting cresyl violet. A primary tumor
cell line IST-Mes3/2P (IST-Mes3/2nd Passage)
was established from a IST-Mes3 tumor grown
orthotopically. Tumors were grounded into
small pieces in RPMI-1640 and digested with
trypsin/EDTA solution. The cell suspension
was then plated in a 75 ml culture flask and
the following day, non adherent cells were
removed. Cells were maintained in culture,
and 2 × 106 cells were injected
into the pleural cavity of nude mice when
devoid of fibroblasts or endothelial cells.
Therapeutic procedures
Four groups of
animals were used. The first group of
animals were used to analyze the
tumorigenicity of Malignant Pleural
Mesothelioma
cell lines after orthotopic or subcutaneous
implantation in nude mice. Animals were then
injected with Malignant Pleural
Mesothelioma cells as reported in Table
1.
The second,
third and fourth groups were used to analyze
the growth rate of IST-Mes3, IST-Mes3/2P and
MM-B1 cells respectively after orthotopic
implantation in mice.
Autopsy and histology
Mice were
sacrificed at different times following
tumor cell injection as specified.
Tumor nodules
were counted, measured with the caliper,
removed, immediately snap frozen in liquid
nitrogen and stored at -80°C. The presence
of metastasis in the visceral organs was
macroscopically checked.
Immunohistochemistry
Microvessel
density (MVD) was determined by using the
endothelial cell marker CD31. Tumors were
placed in OTC compound and snap frozen in
liquid nitrogen and stored at -80°C. Frozen
section (10–20 μm)
were fixed with cold acetone (5 min),
acetone/chlorophorm 1/1 (5 min) and cold
acetone (5 min). Samples were then rinsed
with PBS/Triton 1%, and treated with 3%
hydrogen peroxide in methanol (vol/vol).
Slides were incubated in a blocking solution
and then overnight at 4°C in a humidified
chamber with a rat anti-mouse CD31
monoclonal antibody (BD Biosciences
Pharmingen, NJ, USA). Thereafter, slides
were rinsed with PBS and incubated, first
with the blocking solution for 20 min and
then with a biotin-conjugated goat anti-rat
antibody (Santa Cruz Biotechnology, CA, USA)
for 1 h. Slides were then rinsed with PBS
and incubated for 30 min with the Vector
Vectastain ABC Kit (Vinci-Biochem, Vinci, FI).
After 3 washes with PBS, positive reactions
were visualized by incubating the slides for
5 min with stable DAB (Sigma, Italy). Slides
were dried and mounted with Universal Mount.
For
mesothelioma
markers, 3–4 μm
paraffin tumor sections were float mounted
on poly-L lysine coating slides,
deparaffinized in xylene and rehydrated in a
descending ethanol series. Anti-Ber-EP4
monoclonal antibody required enzymatic
pre-treatment of the slides with 0.1%
trypsin (Sigma, St. Louis, MO) for 10 min at
37°C; for the other antibodies, slides were
not enzimatically pre-treated, but they were
placed in 0.1 M citrate buffer (pH 6.0) and
boiled for 10 min in a microwave oven at 750
W to enhance antigenicity. Slides were
washed in phosphate buffer and incubated for
10 min in 0.3% hydrogen peroxide to quench
endogenous peroxidase activity. Slides were
then loaded onto a LabVision automated
immunostainer (NeoMarkers, Fremont, CA) and
sequentially incubated with intervening
washes in PBS for 5 min, with 10% ovalbumin
for 15 min to reduce non-specific background
staining, primary antibody for 60 min at
room temperature, the appropriate
biotinylated linking antibody (NeoMarkers,
Fremont, CA) for 10 min, peroxidase-labeled
streptavidin (NeoMarkers, Fremont, CA) for
10 min, and finally with
3,3'-diaminobenzidine chromogen substrate
for 10 min. Slides were then thoroughly
rinsed in distilled water and counterstained
with Mayer's haematoxylin, dehydrated,
cleared in xylene and finally mounted in
Entellan. Appropriate positive and negative
controls were included for each marker. A
panel of eight markers was used. Mouse
anti-human antibodies included anti-CEA
(dilution 1:100; NeoMarkers, Fremont, CA),
polyclonal anti-calretinin (dilution 1:1000;
NeoMarkers, Fremont, CA), monoclonal
anti-CD15 (Leu-M1) (dilution 1:4; Becton
Dickinson, NJ), monoclonal anti-Ber-EP4
(dilution 1:100; NeoMarkers, Fremont, CA),
monoclonal anti-cytokeratin 8 and 18
(dilution 1:50; Biomeda, Foster City, CA),
monoclonal anti-EMA (dilution 1:500;
NeoMarkers, Fremont, CA). monoclonal anti-mesothelioma
(HBME-1) (dilution 1:25; NeoMarkers,
Fremont, CA), monoclonal anti-podoplanin
(dilution 1:50; Serotec, Dusseldorf,
Germany).
Hematoxylin-and-eosin staining
Tumors were
placed in OTC compound and snap frozen in
liquid nitrogen and stored at -80°C. Frozen
section (10–20 μm)
were fixed with cold acetone (5 min),
acetone/chlorophorm 1/1 (5 min), cold
acetone (5 min). Slides were then rinsed
with water for10 min, stained with Mayer's
Hematoxylin Solution for 10 min (Sigma, St.
Louis, MO), rinsed with water for 10 min and
then stained with eosin 0.5% for 30 sec
(Sigma, St. Louis, MO).
Slides were then
dehydrated through 95% alcohol (5 min.) and
2 changes of absolute alcohol, 5 min each,
cleared in 2 changes of xylene (5 min) and
finally mounted with xylene based mounting
medium.
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Result
for
Growth of
human Malignant Pleural Mesothelioma cells
in nude mice
Immunological Analysis
of human Malignant Pleural Mesothelioma
Discussion about the
Malignant Pleural Mesothelioma test results
Conclusion based on
the Malignant Pleural Mesothelioma test
Immune
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Table 1
[1]
[2]
[3] |
Tumorigenicity of Malignant Pleural
Mesothelioma cell lines after
orthotopic and subcutaneous implantation
in nude mice.
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Cell line |
Histology |
Tumorigenicity |
Implantation method |
Survival(days) (mean ± SD) |
Survival (days) (Min/Max) |
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IST-Mes2 |
Epithelioid |
0/15 |
Orthotopic |
- |
- |
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Epithelioid |
0/15 |
Subcutaneous |
- |
- |
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IST-Mes3 |
Biphasic |
15/15 |
Orthotopic |
81,5 ± 21,7 |
60/122 |
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Biphasic |
15/15 |
Subcutaneous |
Sacrificed after 90 days |
- |
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IST-Mes3/2P |
Biphasic |
15/15 |
Orthotopic |
69,6 ± 14,5 |
54/89 |
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MM-B1 |
Biphasic |
15/15 |
Orthotopic |
72,5 ± 6,4 |
68/77 |
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Biphasic |
0/15 |
Subcutaneous |
- |
- |
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2 × 106
Malignant Pleural Mesothlioma
cells were injected into the pleural
cavity of nude mice. The experiment
ended when mice were moribund. |
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