Abstract Background The Sézary syndrome is an aggressive leukemic form of cutaneous T cell lymphoma and there is no cure of this disease. Until now there is no true animal model for Sézary syndrome, by which new drugs against the disease could be tested. Methods Immune deficient CB-17 SCID beige mice were injected subcutaneously with HUT78 cells, a cell line, derived from a Sézary syndrome patient. Developing tumors were analyzed by immunohistochemistry. Results Injected HUT78 cells formed tumors at the site of injection. In contrast to the Sézary syndrome in man, no malignant cells were observed in the blood of tumor bearing CB-17 SCID beige mice. The tumors appeared 44-62 days after injection and tumor bearing mice survived further 25 - 62 days until they had to be euthanized according to the guidelines of the Swiss animal protection law, since the tumors had reached the maximal allowed size. Conclusion Although the mouse model does not exactly match the human disease, it will be suited for tests of new substances for the treatment of the Sézary syndrome. The formation of an isolated tumor on the skin has the advantage that the effect of a potential drug can be directly monitored without the use of invasive methods.</p

A Dalloul

A Ito

A Tun Kyi

AH Sarris

E Olsen

EJ Kim

F Trautinger

HS Zackheim

J-Z Qin

M Girardi

MR Charley

S Thaler

U Döbbeling

Udo Doebbeling

English

PubMed

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A mouse model for the Sézary syndrome

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Abstract Background The Sézary syndrome is an aggressive leukemic form of cutaneous T cell lymphoma and there is no cure of this disease. Until now there is no true animal model for Sézary syndrome, by which new drugs against the disease could be tested. Methods Immune deficient CB-17 SCID beige mice were injected subcutaneously with HUT78 cells, a cell line, derived from a Sézary syndrome patient. Developing tumors were analyzed by immunohistochemistry. Results Injected HUT78 cells formed tumors at the site of injection. In contrast to the Sézary syndrome in man, no malignant cells were observed in the blood of tumor bearing CB-17 SCID beige mice. The tumors appeared 44-62 days after injection and tumor bearing mice survived further 25 - 62 days until they had to be euthanized according to the guidelines of the Swiss animal protection law, since the tumors had reached the maximal allowed size. Conclusion Although the mouse model does not exactly match the human disease, it will be suited for tests of new substances for the treatment of the Sézary syndrome. The formation of an isolated tumor on the skin has the advantage that the effect of a potential drug can be directly monitored without the use of invasive methods.</p

Doebbeling Udo

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RESEARCH Open Access
A mouse model for the Sézary syndrome
Udo Doebbeling
Abstract
Background: The Sézary syndrome is an aggressive leukemic form of cutaneous T cell lymphoma and there is no
cure of this disease. Until now there is no true animal model for Sézary syndrome, by which new drugs against the
disease could be tested.
Methods: Immune deficient CB-17 SCID beige mice were injected subcutaneously with HUT78 cells, a cell line,
derived from a Sézary syndrome patient. Developing tumors were analyzed by immunohistochemistry.
Results: Injected HUT78 cells formed tumors at the site of injection. In contrast to the Sézary syndrome in man, no
malignant cells were observed in the blood of tumor bearing CB-17 SCID beige mice. The tumors appeared 44-62
days after injection and tumor bearing mice survived further 25 - 62 days until they had to be euthanized
according to the guidelines of the Swiss animal protection law, since the tumors had reached the maximal allowed
size.
Conclusion: Although the mouse model does not exactly match the human disease, it will be suited for tests of
new substances for the treatment of the Sézary syndrome. The formation of an isolated tumor on the skin has the
advantage that the effect of a potential drug can be directly monitored without the use of invasive methods.
Background
Cutaneous T cell lymphomas (CTCL) are a class of non-
Hodgkin’s lymphomas characterized by clonal prolifera-
tion of neoplastic T-lymphocytes [1-3]. The two main
forms are Mycosis fungoides (MF) and its leukemic
counterpart, the Sézary syndrome (SzS). MF remains
confined to the skin and often presents with patches
and plaques or in more advanced forms with tumors
and a generalized erythema (erythroderma). Sometimes
MF proceeds to SzS. Sézary Syndrome patients show
generalized erythroderma, leukemic T cells in the blood
and a reduced life expectancy compared to MF patients
with only approximately 30% of patients surviving
beyond 5 years after diagnosis. This is probably due to
the circulating malignant T cells producing various
immunosuppressant molecules such as IL-10, which can
lead to down regulation of the immunological tumor
surveillance.
Sézary syndrome patients are treated with psoralen
and UVA (PUVA) in combination with interferon alpha,
locally applied cytostatic substances such as BCNU
(1,3-bis(2-chloroethyl)-1-nitrosourea), or low dose
methotrexate or radiation therapy [4-6]. These therapies
show often complete remission for several months, but
the patients relapse. Currently no cure for SzS has been
found.
An animal model is a prerequisite for testing newly
developed drugs for their efficiency and potential
adverse side effects. Animal experiments help to sort
out inefficient or harmful compounds that could threa-
ten the health of patients of phase I trials. To study the
effects of potential anti-cancer agents often immune
deficient mouse strains are used that accept xenotrans-
plants from human tumors or human tumor cell lines.
Until now, no true mouse model for the Sézary syn-
drome is available. Experiments to induce SzS tumors
or leukemia in immune deficient mouse strains as athy-
mic nude mice by injecting cells from SzS patients or
SzS cell lines have failed. This may be either due to the
thin skin of these mice, which may not be ale to deliver
the necessary growth factors for the SzS cells, or to the
fact that athymic nude mice still possess functional
B and NK cells. Here I show that one can induce
tumors in CB-17 SCID beige mice, which have no T, B,
and NK cells, by injecting cells of the SzS cell line
under the skin of these mice.
Correspondence: Doebbeling@usz.ch
Department of Dermatology, University Hospital Zurich, Zurich, Switzerland
Doebbeling Journal of Experimental & Clinical Cancer Research 2010, 29:11
http://www.jeccr.com/content/29/1/11
© 2010 Doebbeling; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Methods
Cells and cell culture
The cell line Hut78 (Sézary syndrome) was obtained
from ECACC. MyLa 2059 and SeAx cells were kindly
provided by Keld Kaltoft, University of Aarhus, Den-
mark. The cell lines were grown in HEPES buffered
RPMI 1640 medium supplemented with 2 mM gluta-
mine, 10% fetal calf serum (FCS), 0.25 mg/ml amphoter-
icin B, 100 U penicillin G, 100 U streptomycin and
1 mM pyruvate (all from Invitrogen, the Netherlands).
Mice and tumor formation
CB-17 beige mice (CB-17/lcr.Cg-Prkdc scid Lyst bg/Crl)
were obtained from Taconic (Lille Skensved, Denmark).
The mice kept under sterile conditions in the central ani-
mal laboratory of the University Hospital Zurich. 3 × 106
Hut78 cells were injected subcutaneously into the right
flank of the mice. Tumors were visible after 5-9 weeks
and were grown until the maximal allowed size of 1 cm3.
The experiments were performed following the ethic
guidelines for animal experiments of the Swiss National
Fund and were approved by the Veterinary Authorities of
the Kanton of Zurich, Switzerland (license no. 53/2005).
Immunohistochemistry
Tumors were excised and fixed in formaldehyde and
small tumor pieces were embedded in paraffin. Tumor
sections were stained by haematoxylin and eosin (HE).
For immune histochemistry the slides were probed with
antibodies against human CD3 (DAKO, no. A0452,
Glostrup, Denmark) and FLIP (Abcam no. 15319).
Staining of this antibody was detected using an alkaline
phosphatase anti-alkaline phosphatase (APAAP)-immu-
nohistochemistry technique (reagents from DAKO,
Glostrup, Denmark).
Results
Tumor growth of SzS cells lines on immune deficient CB-
17 SCID beige mice
To obtain tumors two groups of seven CB-17 SCID
beige immune deficient mice were injected subcuta-
neously with 3 × 106 cells of the SzS cell lines HUT78
and SeAx. The injected mice were observed for three
months for tumor formation. During this time two
tumors were observed in the group that had been
injected with HUT78 cells, whereas no tumors were
seen in the group that had been injected with SeAx
cells. As a positive control two CB-17 SCID beige mice
were injected with 3 × 106 MyLa 2059 cells, which have
been shown form tumors on immune deficient athymic
nude mice [7,8]. One tumor was observed during the
given time span on these animals. Compared to other
mouse tumor systems the take on rate of the malignant
cells was quite low (28.3% (2/7) for Hut78 cells and 0%
(0/7)for SeAx cells).
Since malignant cells derived from tumors that had
already grown on mice are more effective in tumor for-
mation, cells derived from these two tumors were cul-
tured in vitro and 3 × 106 cells of the culture were
injected again subcutaneously into 8 further CB-17
SCID beige mice. This time the formation of 6 tumors
was observed corresponding to a take on rate of 75%
(6/8). The growth of the individual tumors differed
markedly (Figure. 1A). They appeared 5 - 9 weeks after
injection. 5 tumors grew continuously and three tumors
showed a transient reduction of tumor volume, which
was due to the formation of a necrotic area in the cen-
ter and involution of the central area of the tumor. The
growth of the tumor did not cause hair loss in the
tumor area and the area had to shaved make the tumors
better visible. A clinical picture of a tumor bearing
mouse is given in Figure 1B.
The blood of human SzS patients contains malignant
T cells. Since the blood of CB-17 SCID beige mice con-
tains no mature lymphocytes they should be easily
detected. However, no malignant SzS cells were detected
in the blood of the tumor bearing animals, indicating
that the malignant human T cells cannot grow in the
blood of CB-17 SCID beige mice. The inspection of the
inner organs of the tumor bearing mice showed no
signs of metastasis formation.
Morphology of the SzS tumors on CB-17 SCID beige mice
The inspection of excised tumors under the microscope,
showed that larger tumors contained a necrotic inner
center that was covered by zone of living cells. These
cells were surrounded by areas that contained atypical
blood vessels (Figure 2A), which had mostly only an
incomplete endothelium. The tumors consisted of two
populations of cells. One population consisted of malig-
nant T cells with large spongiform nuclei. Their identity
as malignant T cells (Hut78 cells) was confirmed by
staining with an antibody against CD3 (Figure 2B). The
Hut78 cells in the tumor appeared as plasma rich malig-
nant T cells, whose plasma membrane stained strongly
by the CD3 antibody, confirming the presence of the
T cell receptor on these cells. Malignant T cells also
infiltrated the dermis and epidermis and caused in some
tumors the formation of a visible necrotic area in the
center of the tumor.
The other cell population consisted of tumor infiltrat-
ing granulocytes, which were easily identified by their
segmented and more condensed nuclei (Figure 2c). The
granulocytes reacted strongly with an antibody against
the anti-apoptotic FLIP protein, whereas the Hut78 only
weakly stained with this antibody.
Doebbeling Journal of Experimental & Clinical Cancer Research 2010, 29:11
http://www.jeccr.com/content/29/1/11
Page 2 of 5
Discussion
Subcutaneous injection of malignant SzS cells under the
skin of CB-17 SCID beige mice led to the formation of
isolated tumors at the sites of injection. In contrast to
the Sézary syndrome in man, no leukemic T cells were
detected in the blood of the injected mice. No metas-
tases were observed.
In contrast to other malignancies, it has been difficult
to establish mouse models for CTCLs as mycosis fun-
goides and the Sézary syndrome [7-10]. For MF a
mouse model has been developed [7] that has already
been used for pharmaceutical studies [8], however a true
model for SzS was still missing. It has been shown that
grafts from SzS patients can survive on CB-17 SCID
beige mice [9], but these experiments have never been
repeated. Successful experiments with grafts from SzS
patients and athymic nude mice have not yet been
reported. Thus CB-17 SCID beige mice seem to be bet-
ter hosts for sensitive tumor cells.
Recently Ito et al. [10] reported that they obtained
tumors by injecting cells of the SzS cell line HH under
the skin of immune deficient NOD/Shi-scid, IL-
2Rgamma(null) mice. The injected cells induced extre-
mely fast growing tumors that reached a size of 1 - 3
cm3 within 10-15 days and also infested the liver within
this time. This behaviour is in total contrast to the slow
growth of SzS tumors and does not represent the patho-
biology of SzS and MF. The cells were only character-
ized by CD30 staining, an antigen that is only expressed
by a minority of MF and SzS tumors, but that is indica-
tive for anaplastic large cell lymphoma (ALCL) cells,
which can indeed induce fast growing tumors in
immune deficient mice.
It is supposed that MF and SzS cells depend on sev-
eral growth factors that have to be delivered by the host
skin or blood [11-13]. These and other still unknown
growth factors in turn activate different signalling path-
ways that stimulate the expression of survival and
growth promoting genes as bcl-2 and c-myc [14-16].
Since immune deficient mice lack functional lympho-
cytes they are unable to deliver growth factors that are
produced by these cells. The lack of these growth
Figure 1 Tumor formation and tumor growth on CB-17 SCID beige mice injected with 3 × 106 Hut78 cells. A) Tumor growth on 8 CB-17
SCID beige mice injected with Hut78 cells (animal 1-8). MyLa indicates a control mouse that had been injected with the same number of MyLa
2059 cells. The tumor volume is indicated by the y axis (in mm3). The number of days after the injection is indicated by the x axis. B) Clinical
picture of a tumor bearing CB-17 SCID beige mouse injected with Hut78 cells.
Doebbeling Journal of Experimental & Clinical Cancer Research 2010, 29:11
http://www.jeccr.com/content/29/1/11
Page 3 of 5
factors could be an explanation why “Sézary cells” can-
not grow in the blood of CB-17 SCID beige mice. It has
also to be taken in account that sometimes a murine
growth factor cannot substitute the homologous human
growth factor, as the differences in the amino acid
sequence is too big, so that a murine cytokine cannot
bind to the homologous human receptor. In contrast to
HUT78 cells, the injection of SeAx cells under the skin
of CB-17 SCID beige mice did not induce tumors. The
reason for this is unclear. The HUT78 cell line has been
established before more than 30 years and there is evi-
dence that HUT78 cells have become independent of
several growth factors [8,14] during their long propaga-
tion time in vitro. The SeAx cell line has been estab-
lished approximately 15 years later and it may still
depend of additional growth factors that can not be sup-
plied by a murine host, precluding its growth on
immune deficient mice.
Conclusion
Here I report a mouse system for the Sézary syndrome
that is reproducible and reliable. Although this mouse
model does not exactly match the human disease, since
no malignant T cells were found in the blood, it will
allow testing new substances for the treatment of the
Sézary syndrome. The formation of an isolated tumor
on the skin has the advantage that the effect of a poten-
tial drug can be directly monitored without the use of
invasive methods. Besides oral and intravenous applica-
tions one can also test the directly the effect of a locally
applied substance.
Acknowledgements
The author wants to thank C. Dudli for the processing of the histological
samples and M. Bär for the preparation of the photographs. This work has
been supported by Anna Feddersen Wagner Fonds, and the Kanton of
Zurich, Switzerland.
Authors’ contributions
All mouse experiments were done by UD. The tumors were isolated and
minced by UD and passed to the histology lab. The author read and
approved the final manuscript.
Competing interests
The author declares that he has no competing interests.
Received: 3 November 2009 Accepted: 11 February 2010
Published: 11 February 2010
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doi:10.1186/1756-9966-29-11
Cite this article as: Doebbeling: A mouse model for the Sézary
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Edelson RL: Cutaneous T-cell lymphoma: pathogenesis and treatment. Oncology

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A mouse model for the Sézary syndrome

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