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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 of Human malignant
mesothelioma cell tests |
Background for the
human Malignant Pleural Mesothelioma tests
The human Malignant
Pleural Mesothelioma test methods
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Results
Growth of human Malignant Pleural
Mesothelioma cells in nude mice
Human Malignant Pleural
Mesothelioma (MPM) cell
lines were tested for their ability to form
tumors after intrapleural and subcutaneous
injection in nude mice. Table
I summarizes the tumor take rates of
these cell lines. Tab.
II, and
III summarize the growth rate of these
cell lines after orthotopic implantation,
whereas Fig.
1. shows their growth rate following
subcutaneous implantation. IST-Mes2 cells
were unable to grow in nude mice. By
contrast, 2 � 106 IST-Mes3 or
MMB-1 tumor cells had a take-rate of 100 %
when injected into the left pleural cavity.
The mean survival time was of 72.5 � 6.4
days for mice injected with MM-B1 cells;
81.5 � 21.7 days for mice injected with the
IST-Mes3 cells and 69.6 � 14.5 days for mice
injected with the IST-Mes3/2P cells. These
mice were sacrificed when moribund, or at
different times after cell injection as
specified in Tab.
I,
II,
III and
IV. Animals injected with the IST-Mes3
cells were analyzed when they became
dyspneic because of the tumor growth rate
varies from animal to animal. These
variations were not observed upon in vitro
culture of IST-Mes3 tumor as shown by the
analysis of the growth rate of IST-Mes3/2P
cells. Tumor cells grew on both sides of
parietal and visceral pleura, diaphragm and
mediastinum; at later times, tumor invasion
into lung parenchyma was observed (Fig.
2,
3). No pneumothorax was evidenced in the
mice. Pleural effusions as well as
lymph
node metastasis were noted only in the later
stages of orthotopic growth of MM-B1,
IST-Mes3 and IST-Mes3/2P cells. MM-B1 and
IST-Mes2 cells did not grow when injected
subcutaneously. IST-Mes3 cells started to
grow only at forty days after tumor cell
injection. This lag phase was independent on
the number of cells injected (Fig.
1).
Immunohistochemical analysis
on Malignant Pleural Mesothlioma
cells
Malignant Pleural
Mesothelioma (MPM) cells were
injected into pleura cavity of nude mice and
tumor samples were collected for
immunohistochemical analysis. CD31 staining
revealed that tumor mass was highly
vascularized (Fig.
3). For the phenotypic characterization
of tumor mass, a panel of eight markers was
used (Tab.
5). Recent findings showed that
Mesothelin and TTF-1 have a limited value in
assisting in the
diagnosis of
Mesothelioma and therefore were not used [13,14].
CEA and BerEP4 are adenocarcinoma specific
markers and therefore are nor expressed by
mesothelioma. Calretinin and HBME-1 are
positive markers shared by both epithelioid
and biphasic variants of mesothelioma,
whereas CD15 is a negative marker. Surface
expression of the epithelial membrane
antigen (EMA) is a marker that discriminates
between reactive proliferation of
mesothelial cells and
malignant mesothelioma.
The epithelia of biphasic mesotheliomas show
strong reactivity for Cytokeratin 8/18,
whereas Podoplanin is the most recently
recognized marker for epithelioid mesotheliomas.
Discussion
New strategies
for Malignant Pleural
Mesothelioma treatment, which include inhibition,
of angiogenesis, induction of tumor cell
apoptosis, gene therapy and vaccines [15],
need clinically representative animal models
to test new drugs and explore tumor biology.
In this work we
established a non invasive orthotopic model
of human Malignant Pleural Mesothelioma by injecting the cancer cells
directly into the pleural cavity of nude
mice. The characteristics of tumor growth
resemble those observed in tumor-bearing
patients, with colonization of parietal and
visceral pleura, diaphragm, mediastinum and,
at later stages, lung parenchyma. We
analyzed the tumorigenicity of three
different human Malignant Pleural Mesothelioma cell lines, which
exhibited different biological behaviors in
vivo. Interestingly, the growth of MM-B1
cells seems to be site-specific as they did
not form tumor masses when injected
subcutaneously. Differently, the IST-Mes3
cells grow subcutaneously with a latency
which does not depend on the number of cells
injected (Fig.
1). This resembles some human situations
in which cancer cells start to grow
exponentially and form manifest tumor masses
only after a long period of time from the
acquisition of the tumorigenic phenotype.
As previously
reported, neo-angiogenesis is an important
process in Malignant Pleural Mesothelioma and protocols to inhibit this
process are currently under investigation.
Our model can be useful to study
angiogenesis in Malignant Pleural Mesothelioma as tumors displayed high
density of micro-vessels. IST-Mes3 tumor
grew with a latency of 60 days and this
period remained substantially constant after
tumor passage in mice. All the cell lines
tested were unable to form metastases in the
visceral organs, likely due to lack of time
for tumor dissemination as the rapid
spreading of the tumor cells into the
pleural cavity dramatically resulted in
mouse death. Among the cell lines tested,
the MM-B1 cells seem to be the more
appropriate for drug evaluation. In fact, as
shown in Tab.
II, the IST-Mes3 cells grow very rapidly
to follow the progression of the disease,
colonizing the entire pleural cavity and
dramatically affecting the physical
conditions of the mice which become
cachectic and dyspneic, and the latency of
tumor takes varies from animal to animal.
The results obtained with the IST-Mes3/2P
cell show that these variations are not
observed when ex vivo tumor cells are
cultured in vitro.
The growth rate
of MM-B1 cells is reproducible. Tumors
develop in all animals with a similar
latency and the rate of tumor growth is slow
enough to follow the progression of the
disease. In this context, a noninvasive
imaging methodology would be a useful tool
to follow tumor development.
IST-Mes2 cells
did not grow in nude mice, but, since the
epithelioid phenotype is the most common
sub-type of mesothelioma, we plan to set up
an orthotopic model using different
ephitelioid cells.
Immunohistochemistry of tumors growing in
the nude mice showed that MM-B1 and IST-Mes3
cells maintained the mesothelioma-specific
characteristics, thus confirming the
validity of the orthotopic model here
established.
Conclusion
Overall, this
work describes the biological behavior of
human Malignant Pleural Mesothelioma cells injected in the pleural
cavity of nude mice. Because of the
similarity of this orthotopic model with the
human disease, the simplicity of execution
and the reproducibility of the results, we
propose this model as an useful tool for in
vivo Malignant Pleural Mesothelioma studies.
Competing
Interests
The author(s)
declare that they have no competing
interests.
Authors'
Contributions
DM performed the
orthotopic and subcutaneous implantation of
cancer cells in nude mice, the autopsy and
the immunoassays. AC carried out the cells
culture and participated in the immunohistochemical
analysis. GS designed and coordinated the
study.
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PA participated
in the coordination of the study and helped
to draft the manuscript. SO and RL performed
the phenotypic characterization on Malignant Pleural
Mesothelioma cells
orthotopically implanted in nude mice. All
the authors read and approved the final
manuscript.
Acknowledgement
We thanks
Roberta Lucciarini, Consuelo Amantini and
Maria Rita Rippo for technical advices and
help. This work was supported and approved
by the Italian Ministry of Health and the
University of Camerino (Italy).
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Resources
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13. |
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King JE, Thatcher N, Pickering CA,
Hasleton PS: Sensitivity and
specificity of immunohistochemical
markers used in the diagnosis of
epithelioid
mesothelioma: a detailed
systematic analysis using published
data.
Histopathology 2006, 48:223-232. |
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Return to citation in text: [1] |
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14. |
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Ordonez NG: Application of mesothelin
immunostaining in tumor diagnosis.
Am J Surg Pathol 2003, 27:1418-1428. |
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Return to citation in text: [1] |
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15. |
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Nowak AK, Lake RA, Kindler HL, Robinson
BWS: New approaches for mesothelioma:
biologics, vaccines, gene therapy and
other novel agents.
Semin Oncol 2002, 29:82-96. |
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Return to citation in text: [1] |
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Table 2
[1]
[2] [3] |
Growth progression of IST-Mes3 cell
line after orthotopic implantation in
nude mice.
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Days after implantation |
<56 |
60 |
65 |
71 |
73 |
80 |
87 |
94 |
98 |
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N� of animals with tumor nodules1 |
0/5 |
1/5 |
2/5 |
1/5 |
1/5 |
0/5 |
1/5 |
1/5 |
2/5 |
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N� of tumor nodules detected in each
animal |
- |
>20 |
>20 |
>20 |
>20 |
- |
>20 |
>20 |
>20 |
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2 � 106
IST-Mes3 cells were injected into the
pleural cavity of nude mice. Five
animals were sacrificed when they became
dyspneic or at indicated times and tumor
nodules were counted, measured, removed
and frozen for immunohistochemical
analysis. Data shown report tumor
development in individual animals. |
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Table 3
[1]
[2] |
Growth
progression of IST-Mes3/2P cell line
after orthotopic implantation in nude
mice.
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Days after implantation |
54 |
61 |
68 |
75 |
82 |
89 |
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Mediastinum nodules |
< 101 |
<102 |
<103 |
<103 |
10�203 |
10�203 |
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Pericardium nodules |
< 101 |
<102 |
<103 |
<103 |
10�203 |
10�203 |
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Parietal Pleura nodules |
< 101 |
<102 |
<103 |
<103 |
10�203 |
10�203 |
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Lungs nodules |
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- |
- |
- |
<102 |
<102 |
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Diaphragms nodules |
- |
- |
- |
- |
<102 |
<102 |
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2 � 106
IST-Mes3/2P cells were injected into the
pleural cavity of nude mice. At
indicated times mice were sacrificed and
tumor nodules were counted, measured,
removed and frozen for
immunohistochemical analysis. Data shown
are mean of 5 mice.
1 Nodule diameter ≤ 1 mm;
2 Nodule diameter = 2�3 mm;
3 Nodule diameter ≥ 3. No
nodules were found before the days
indicated in the table. |
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Table 4
[1] |
Growth
progression of MM-B1 cell line after
orthotopic implantation in nude mice.
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Days after implantation |
38 |
45 |
52 |
59 |
66 |
73 |
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Mediastinum nodules |
< 101 |
10�201 |
10�202 |
10�202 |
>203 |
>203 |
|
Pericardium nodules |
< 101 |
10�201 |
10�202 |
10�202 |
>203 |
>203 |
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Parietal Pleura nodules |
- |
- |
- |
10�202 |
>203 |
>203 |
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Lungs nodules |
- |
- |
- |
10�202 |
>203 |
>203 |
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Diaphragms nodules |
- |
- |
- |
- |
- |
- |
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2 � 106
MM-B1 cells were injected into the
pleural cavity of nude mice. At
indicated times, mice were sacrificed
and tumor nodules were counted,
measured, removed and frozen for
immunohistochemical analysis. Data shown
are mean of 5 mice.
1 Nodule diameter ≤ 1 mm;
2 Nodule diameter 2�3 mm;
3 Nodule diameter ≥ 3 mm. No
nodules were found before the days
indicated in the table. |
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Table 5
[1] |
Phenotypic characterizzation of MM-B1
and IST-Mes3 cells after orthotopic
implantation in nude mice.
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Cell lines |
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Marker |
IST-Mes3 |
MM-B1 |
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CEA |
- |
- |
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Calretinin |
+ |
+ |
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CD15 (LeuM1) |
- |
- |
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Ber-EP4 |
- |
- |
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Cytokeratin 8/18 |
+ |
+ |
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EMA |
+ (membrane) |
+ (membrane) |
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HBME-1 |
+ |
+ |
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Podoplanin |
+ |
+ |
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IST-Mes3 and MM-B1
cells were injected into the pleural
cavity of nude mice. At indicated times
the mice were sacrificed and tumor
nodules were removed and frozen for
immunohistochemical analysis of
mesothelioma-specific markers. |
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Figure 1
[1]
[2] [3] |
Resolution: standard /
high
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 |
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Subcutaneous growth of Malignant
Pleural Mesothelioma cells.
Subcutaneously growth of Malignant
Pleural
Mesothlioma cells in nude mice.
◆ = IST-Mes3 (2 � 106 cells
injected), ■ = IST-Mes3 (4 � 106
cells injected), ▲ = IST-Mes2 (2 �
106 cells injected), ● =
MM-B1 (2 � 106 cells
injected). 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 diameters,
respectively. |
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Figure 1
[1] |
Resolution: standard /
high |
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 |
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Subcutaneous growth of Malignant
Pleural Mesothelioma cells.
Subcutaneously growth of Malignant
Pleural Mesothelioma cells in
nude mice. ◆ = IST-Mes3 (2 � 106
cells injected), ■ = IST-Mes3 (4 �
106 cells injected), ▲ =
IST-Mes2 (2 � 106 cells
injected), ● = MM-B1 (2 � 106
cells injected). 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 diameters,
respectively. |
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Figure 2
[1] |
Resolution: standard /
high
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 |
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Pleural Malignant Pleural
Mesothelioma nodules. IST-Mes3
cells (2 � 106 in 100
μl of HBSS)
were injected into the left pleural
cavity of nude mice. Tumor masses
completely invaded the thoracic and
visceral pleural and the lungs. The
irregular growth of this cell line is
evidenced by a major bulk of tumor in
the right chest. |
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Figure 2 |
Resolution: standard /
high |
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 |
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Pleural Malignant Pleural
Mesothelioma nodules. IST-Mes3
cells (2 � 106 in 100
μl of HBSS)
were injected into the left pleural
cavity of nude mice. Tumor masses
completely invaded the thoracic and
visceral pleural and the lungs. The
irregular growth of this cell line is
evidenced by a major bulk of tumor in
the right chest. |
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Figure 3
[1]
[2] |
Resolution: standard /
high
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Orthotopic Malignant Pleural
Mesothelioma histology. Fig. 3a.
Frozen sections of tumors grown into the
pleural cavity of nude mice injected
with IST-Mes3 cells were stained with an
anti-mouse CD31 antibody. Tumors were
intensively stained revealing an
intricate network of microvessels. Bar =
500 μm. Fig.
3b. H&E staining of tumor invading the
lung. T: tumor, L: lung. Bar = 100
μm. |
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Orthotopic Malignant Pleural
Mesothelioma histology. Fig. 3a.
Frozen sections of tumors grown into the
pleural cavity of nude mice injected
with IST-Mes3 cells were stained with an
anti-mouse CD31 antibody. Tumors were
intensively stained revealing an
intricate network of microvessels. Bar =
500 μm. Fig.
3b. H&E staining of tumor invading the
lung. T: tumor, L: lung. Bar = 100
μm. |
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