Animal models of multiple myeloma: an overview
Karin Vanderkerken
Department Hematology and Immunology,
Free University Brussels (VUB)
Kos, June 26 2007
· Presence of MM cells in
the BM
·Secretion of
of monoclonal
monoclonal
paraprotein
·Induction
·
of
angiogenesis
·Induction of bone
disease
Bommert K et al. Eur J Cancer (2006) 15741580
Experimental approach of these interactions
· In vitro: two-dimensional interactions
· In vivo: three-dimensional interactions
experimental mouse models
Tools to investigate the different processes
in-
in vivo and ex
ex-vivo
Tumor burden
Bone disease
Ai
Angiogenesis
Isolation of the different cellular compartments
Tumor burden
Measurement of plasmacytosis in the bone marrow
Flow cytometry: anti-idiotype antibodies, anti CD138
Measurement of serum M spike
Elisa (human
(human or murine IgG)
Serum electrophoresis
Tracing
Tracing of
of tumor
tumor burden
Tracing of tumor burden
Stably transduce tumor cells with viral
constt
tructs
t
con i
a ning :
Optical imaging genes
e.g. eGFP, dsRed, ...
CCD Camera
Optical
e.g. Firefly & Renilla
CCD Camera
bioluminescence genes luciferases
Transportergenes
e.g. Sodium Iodide
PET / SPECT
Symporter (NIS)
Enzymatic based
e.g. HSV1-tk
PET / SPECT
reporter genes
Case Study : 5T33MM -
Bioluminescence
5T33vtFLuc cells ; 21 days after
injection
Measurement of
of proliferating
proliferating fraction
fraction
Injection of BrdU and measurement of BrdU incorporation
immunocytochemistry
flow cytometry
yy
Immunohistochemistry for proliferating markers
ex Ki67, PCNA
Measurement of bone disease
Assessing the
number of osteolytic bone
lesions
Assessing the trabecular
bone area
Assessing the number
of osteoclasts
Assessing calcein
uptake
Micro CT
Measurement of angiogenesis
Microvessel density
Radiological techniques
CD31immuno
Circulating EC
Isolation of
of different
different cellular compartments
compartments
Different cell isolation techniques (MACS, FACS,
density gradient
gradient centrifugation)
centrifugation)
Laser capture microdissection
To evaluate the differential gene expression after
in vivo inoculation and interaction with the bone marrow
microenvironment
Experimental mouse models
·Plasmacytoma
· Syngeneic mouse models:
·Cell lines s.c.
·5TMM modl
dels, bone marrow
· SCID models:
·Tumor development in different sites
· SCID-hu models:
·Fetal implanted bone
· Fl
Fetal chip il
impl
d
ante
· Different transgene models
· Hollow fibre models
Plasmacytoma in Balb/c mice
i p
. . injection of
of mineral
mineral oil (ex
(ex. Pristane)
Proliferation of plasma cells
Limitation
Not the typical characteristics of myeloma disease
Pt
Po t
tter M
t
e al. JNCI
1983
: 71
: 391
5
Syngeneic models
Murine cells injected
Advantages
ex. B38, C11C1 cell lines
(derived from P3X63Ag8
Limitation
(derived from
Limitation
cell line) in Balb/c mice
Easy
Bone marrow independent
Assessment of tumor size
size and blood vessel
vessel density
No limitations in material
Mouse model
Syngeneic
yg
Immunocompetent
Sainz IM et al; Cancer Immunol Immunother 2006; 55:797807
5TMM models
· Spontaneous development in old C57BlKaLwRij mice
· The lines are maintained b
y in vivo
f
trans er in young
syngeneic mice
· Preferential localization in
bone
marrow
· Ig serum concentration is correlated to tumor mass
· Each 5TMM line has a different growth rate
· Development of osteolytic lesions
Rd
Ra l
dl J t
e al, J Il
Immunol. 122
, 609
613, 1979
Terminally diseased animal
In vitro assays
In vivo assays
Isolation and
Serum paraproti
tein
it
mon
i
purification of
oring
purification
Preclinical assays
5TMM cells from
·
long bones
homing
·
tumor burden
·
bone disease
·
angiogenesis
Intravenous injection
of 5TMM
cells in naïve mice
MIP-1
MMP9
SDF-1
CCR5
CXCR4
BMSC
IGF-I
IGF-1R
in-1 67kD LR
CD44v6
Lamin
HGF
D44v1OC
E
VEGF
MM
RANKL
PDGF
BON
bFGF
Ang-1
MIP-1
Osteoclast
IL-1
MCP-1
DKK1
RANK
67kD
6
LR
CCR2
RANKL
Osteoblast
BMEC
Vanderkerken et al. Immunological Reviews, 2003, 194:196206
5TMM models
Advantages
Syngeneic system
Limitations involving tumor-bone marrow interactions
Features typical
Each
of
model
of myeloma
rep
myeloma disease
resents only
disease (bone
one typ
(bone disease,
e of p
disease, angiogenesis
atients
Immunocompetent
Exclusive in system
vivo model-no in vitro growth
Murine cells
SCID models
SCID mice: point mutation in chromosome 16
of CB-17 inbred mice leading to disturbance of the lymphocyt
tt
matura i
tion
d
an d f
e i
fi i
c ency in B
d
an T
l
ce llls
NOD/SCID mice:
additional immunological deficiencies
NOD/SCID/ null:
no B, T cells, NK cells and
c
disturbed dendritic cells
SCID Xenografts
MM cells from blood of MM patients
NOD/SCID mice
MM growth in BM, induction of osteolytic bone disease,
Presence of human paraprotein in serum
Pilarski et al. Blood 2000: 95: 105665
Pilarski, L. M. et al. Blood 2000;95:10561065
Pilarski, L. M. et al. Blood 2000;95:10561065
LAGlambda model: injection of human MM samples
jp
in hind leg
muscle of SCID mice
s.c. local tumors
iv
i.v. lytic
lesions
Limitation
Campbell et
Some al. Int J Oncol 2006; 28:
cytokines/adhesion
140917
molecules might not be
compatible between the two compartments of the
xenogeneic systems
SCID-hu model
Human fetal bone is implanted in SCID mice followed
by implantation
yp
of "fresh" human MM cells in the fetal bone
MM disease in the fetal bone
Bl
Bone l
i
es ons
Human Ig
Angiogenesis
gg
Yaccoby S,
Ya Blood
ccoby 1998;
S et
al.92:
2908
Blood 13
1998, 92: 29082913
SCID-hu model
SCID mice with human fetal implanted bone
GFP+ INA-6 cells are injected
Tassone P et al. Blood 2005, 106: 713716
SCID-hu model
Advantages
Limitations
Syngeneic
Reduced system
number of
system
mice that can be
be used
used
Patients MM cells
Immunoincompetent
Interaction
Irradiation with the bone marrow microenvironment
Angiogenesis,
Ethical issues Bone lesions
SCID-rab model
SCID mice + rabbit bones + fresh human MM cells
Yata K et al. Leukemia 2004: 18; 18917
Radiographs (a) before and (b) 12 weeks after myeloma
PCs injection show severe
resorption of the myelomatous rabbit bone.
Transgene models
·Double transgene Myc/Bcl-X (L) mice develop plasma tumors
Pd
·Producti
tion f
o
l
monoc onal Ig
·Infiltration in the bone marrow
·Enhanced expression of Myc(His) and Bcl-X(L) proteins
compared to normal plasma
plasma cells
cells
·Some features of MM
Cheung et al J Clin Invest. 2004 113(12):176373.
Hollow fibre
fibre model
Screening and short term in vivo pharmacodynamic
studies (NCI developed)
s.c. in Balb/c mice
Tumor cells can be retrieved and analyzed
Temminck OH. Et al Br J Canc 2007; 96: 6166
Conclusion
Several models have been developed for the study
of multiple myeloma
Each model has its advantages/limitations
Depending on the biological and preclinical question
a specific experimental
pp
model has to be selected.
Dr. Peter Croucher (Sheffield University, UK)
Dr. Helena Jernberg-Wiklund
(Uppsala
pp
University,
y, Sweden)
Dr. Rik De Raeve (UIA, Belgium)
Free University Brussels Thomas Andersen (South Danish University)
Dr. Selina Chen-Kiang (Cornell, NY, USA)
Prof. Dr. Ben Van Camp
Prof. Dr.
Dr Ivan Van
Va Riet
Riet
Dr. Marleen Bakkus
Dr. Els Van Valckenborgh
Dr. Eline Menu
Elke De Bruyne
Tomas Bos
Dr. Isabelle Vanden Broek
Jinsong Hu
Angelo Willems
Carine Seynaeve
Rl
Rona d
ld De Zanger
Nicole Arras