Original Article
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd 39
Keywords
bioluminescent imaging ,
canine lymphoma , humoral
hypercalcaemia of
malignancy , PTHrP ,
xenograft
Correspondence address:
Dr Thomas J. Rosol
Department of Veterinary
Biosciences
College of Veterinary
Medicine
The Ohio State University
1925 Coffey Road
Columbus, OH 43212
USA
e-mail: rosol.1@osu.edu
Introduction
Non-Hodgkin ’ s lymphoma (NHL) includes sev-
eral malignant lymphoproliferative diseases that
originate from lymphocytes and represents the
fi fth most common cause of cancer-related deaths
in humans in the USA. 1 Lymphoma is the third
most common neoplasm in dogs, 2 with an esti-
mated annual incidence of approximately 33 per
10 0000 dogs. 3 In the absence of chemotherapy,
survival beyond 1 month after diagnosis of lym-
phoma is uncommon. 4 The most common anatomic
presentation of canine lymphoma is the multicentric
NOD/SCID mouse model of canine T-cell
lymphoma with humoral hypercalcaemia
of malignancy: cytokine gene expression
profi ling and in vivo bioluminescent
imaging
M. V. P. Nadella 1 , W. C. Kisseberth 2 , K. S. Nadella 3 , N. K. Thudi 1 , D. H. Thamm 4 ,
E. A. McNiel 5 , A. Yilmaz 1 , K. Boris-Lawrie 1 and T. J. Rosol 1
1 Department of Veterinary Biosciences, The Ohio State University, Columbus, OH, USA
2 Department of Veterinary Clinical Sciences, The Ohio State University, Columbus, OH, USA
3 Human Cancer Genetics, The Ohio State University, Columbus, OH, USA
4 Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State
University, Fort Collins, CO, USA
5 Department of Veterinary Clinical Sciences, University of Minnesota, St. Paul, MN, USA
Abstract
Lymphoma is a malignant neoplasm arising from B or T lymphocytes. In dogs, one-third of lympho-
mas are highly aggressive multicentric T-cell lymphomas that are often associated with humoral
hypercalcaemia of malignancy (HHM). There are no cell lines or animal models to investigate the
pathogenesis of T-cell lymphoma and HHM in dogs. We developed the fi rst xenograft model by
injecting lymphoma cells from an Irish Wolfhound intraperitoneally into NOD/SCID mice. The mice
developed multicentric lymphoma along with HHM and increased parathyroid hormone-related
protein (PTHrP) as occurs in dogs with T-cell lymphoma. Using cytokine complementary DNA arrays,
we identifi ed genes that have potential implications in the pathogenesis of T-cell lymphoma. Quan-
titative reverse transcriptase-polymerase chain reaction (RT-PCR) of T-cell lymphoma samples from
hypercalcaemic canine patients showed that PTHrP likely plays a central role in the pathogenesis of
HHM and that hypercalcaemia is the result of a combinatorial effect of different hypercalcaemic
factors. Finally, we monitored in vivo tumour progression and metastases in the mouse model by
transducing the lymphoma cells with a lentiviral vector that encodes a luciferase - yellow fl uorescent
protein reporter and showed that in vivo traffi cking patterns in this model were similar to those seen
in dogs. This unique mouse model will be useful for translational research in lymphoma and for
investigating the pathogenesis of T-cell lymphoma and HHM in the dog.

40 M. V. P. Nadella et al.
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd, Veterinary and Comparative Oncology, 6, 1, 39–54
form, which usually presents as superfi cial lymph-
adenopathy 5 with or without hepatosplenomegaly.
About 26 – 38% of the lymphomas in dogs 6,7 and
15% of the lymphomas in humans 8 are T cell in
origin. Humoral hypercalcaemia of malignancy
(HHM) is a frequent paraneoplastic syndrome
seen in approximately 40% of the dogs with T-cell
lymphoma 9 and in 15% of humans with NHL. 10 In
addition, 70% of human patients with adult T-cell
leukaemia/lymphoma caused by human T-cell
lymphotropic virus type-1 11 develop HHM. HHM
is a multifactorial syndrome caused by the action
of multiple factors produced by neoplastic cells
affecting bone, kidney and intestine that disrupt
normal calcium homeostasis. 12,13 The primary
mechanism for hypercalcaemia in patients with
lymphoma is increased osteoclastic bone resorp-
tion induced by humoral mediators. 14,15 Factors
produced by lymphoma implicated in the patho-
genesis of HHM include parathyroid hormone-
related protein (PTHrP), calcitriol, transforming
growth factors (TGFs), tumour necrosis factors
(TNF), interleukin-1 (IL-1), interleukin-6 (IL-6),
macrophage infl ammatory protein-1 alpha (MIP-
1
) and granulocyte colony-stimulating factor. 16 – 21
Calcitriol, the active product of vitamin D metabo-
lism, enhances gastrointestinal calcium absorption
and also mobilizes calcium from bone. Increased
calcitriol production was reported in human NHL
patients with hypercalcaemia and was implicated
as a major humoral mediator in the pathogenesis
of HHM in lymphoma; however, dysregulated cal-
citriol production was also observed in normocal-
caemic patients with NHL. 10,19 Firkin et al. reported
that NHL patients with hypercalcaemia had ele-
vated circulating levels of PTHrP, with no increase
in the levels of calcitriol. 22
PTHrP originally was isolated from specifi c tu-
mours as the primary cause of HHM 23 and is over-
expressed by many different types of neoplasms. 24
Studies over the past several years have shown that
PTHrP plays a primary role in HHM 25 and hyper-
calcaemia in tumour-bearing animals could be
corrected using a neutralizing antibody to PTHrP. 26
Amino-terminal peptides of PTHrP have been
shown to exert PTH-like actions in bone and kid-
ney by binding to a common receptor for PTH/
PTHrP (PTH-1 receptor), resulting in hypercal-
caemia. 27,28 Our laboratory previously reported
that dogs with lymphoma and hypercalcaemia have
elevated levels of plasma PTHrP but that these lev-
els were lower than in dogs with carcinomas and
hypercalcaemia. Moreover, there was no signifi -
cant correlation between serum calcium and
PTHrP concentrations in dogs with lymphoma
and hypercalcaemia, suggesting a role for other cy-
tokines in this syndrome. 29 Factors such as TGF
,
IL-1, IL-6 and TNF have been shown to enhance
the hypercalcaemic effects of PTHrP. 30 Further-
more, TGF  , TNF
and IL-1 have been reported
to upregulate PTHrP gene expression in a variety
of nonlymphoid cell lines and tissues. 31,32 We hy-
pothesized that PTHrP plays a central role in the
pathogenesis of HHM in dogs with T-cell lym-
phoma and acts synergistically with other cytokines
produced by the tumour cells.
Canine lymphoma is a spontaneous disease that
has a clinical presentation and biologic behaviour
that closely resembles the human disease. 33 Fur-
thermore, canine lymphoma is a useful transla-
tional model to study the pathogenesis and
treatment of lymphoma because dogs share exten-
sive genome homology and a common environ-
ment with humans. 34,35 The value of the canine
model also depends on the availability of rodent
models that can reproduce the disease as it occurs
in dogs. Development of animal models that reca-
pitulate the natural history of cancers and their
clinical response to therapy is an important prereq-
uisite for rapid bench-to-bedside translation of an-
ticancer therapies. 36 Moreover, the pathogenesis of
HHM in dogs with T-cell lymphoma has not been
investigated because of the lack of relevant in vivo
models and little is known about PTHrP expres-
sion and its interrelationship with other cytokines.
In this study, we report the development and char-
acterization of a NOD/SCID mouse model of ca-
nine T-cell lymphoma with HHM that closely
resembles the disease as it occurs in dogs and
humans.
The study of animal models has been limited by
the diffi culty of accurately assessing disease burden
and response to therapy. Measurement of tumour
volume using callipers is limited to tumours that
occur at accessible sites. 37 Some of the available
in vivo models of haematological malignancies do not