Mouse Model in Tumor
Microenvironment and
Bond Disease
X International Myeloma Workshop
Sydney, 2005
Contributors Past and Present
Shmuel Yaccoby
James Chen
John Freeman III
Ryuichi Fujii
Jonathan Stringer
Kenichiro Yata
Hiroyuki Hata
John Shaughnessy
M. Teresa Petrucci
Xavier Thomas
MIRT Faculty and Staff
Susan Mahaffey
Bart Barlogie
Hannah Pratt
Michele Wezeman
Aminah Henderson
NCI, MMRF, IMF, CRFA
Ling Liu
Lei Yin
Primary Myeloma Plasma Cells
in SCID-hu Host
Pre PC
Final
Control
Myeloma Plasma Cells Proliferate
in the SCID-hu Host
Plasma Cell-Depleted
Purified Plasma Cells
Myeloma Bone Marrow Cells
Bone Marrow Cells
IgA Myeloma
IgA Myeloma
IgA Myeloma
800
800
800
Ig
Ig
Ig
Ig
Ig
Ig
)
)
600
IgG
IgG
)
600
600
IgG
ml
IgA
ml
IgA
ml
IgA
g/
g/
g/
µ
µ
µ
(
(
(
400
400
400
Ig
Ig
Ig
man
man
man
u
u
u
200
200
H
200
H
H
0
0
0
0246
8 10 12 14 16 18 20 22 24 26
0
2
4
6
8
1012
1416
1820
0
2
4
6
8 101214 1618 2022 2426
Weeks From Inoculation
Weeks From Inoculation
Weeks From Inoculation
Proliferative Myeloma Cell
Myeloma growth is restricted to the human
microenvironment
Control
Myeloma
Sustained Proliferation of Primary Myeloma Plasma
Cells.
1000
1st passage
l)
2nd passage
m
3rd passage
100
µg/
in(
hac 10
th
lig
dab 1
m
La
0.1
0
3
6
9
12
1518212427
Weeks from inoculation of myeloma cells
Myeloma Changes the Bone Marrow Microenvironment
Osteoblast apoptosis
Osteoclastogenesis
Angiogenesis
Are these changes required
for myeloma cell survival?
Prevent myeloma-induced changes
Osteoclast Activity and Myeloma
Pre Rx
Final
500
80
M e dia n Le v e ls
Untreated
400
)
)
2
60
m
ml/
PC
/m
µg 300
n
(
(
tic
Ig
40
s
to
Zoledronate
n
st
200
op
al
ma
Ap
oc
Hu
20
e
100
%
st
O
0
0
Hu ma n Ig
A p opto tic P C
O steoc la sts
Con tro l P am .
Co n tro l Zo l.
Pa m id ron ate
Zo le d ro n ic Ac id
Myeloma Requires Osteoclasts
Inhibition of osteoclast formation has a
profound anti myeloma effect
Start
RANK-Fc
200
7000
Control
6000
160
)
)
l
l 5000
m
m
120
g/ 4000
µg/
µ
(
(
Ig
Ig 3000
80
n
an
2000
ma
umH
RANK-Fc
40
Hu 1000
0
0
0
1
2
3
456
7
8
9 10 11
0
1
234
56
7
8
9
10
Weeks
Weeks
Extramedullary myeloma is biologically
different from medullary disease
Extramedullary myeloma
Pre Rx
Final
does not require osteoclasts
for survival and proliferation
5000
Myeloma
Control
4000
Zoledronate
)
ml
µg/ 3000
(
Ig
n 2000
Myeloma
umaH
ta
S rt
+Zoled.
1000
Zoledronate
0
0
2
4
6
8
10
1214
1618
20
Week s fr om introduction of myelo ma cells
Myeloma Cell Osteoclast Co Culture
Osteoclasts + Myeloma Cells
Osteoclast
precursors
Myeloma Cells Alone
Osteoclasts
Osteoclast
Bone
Resorption
Resorption
Pits
Osteoclasts Sustain Myeloma Cell Survival
and Proliferation
100
90
80
70
Osteoclasts+Myeloma cells
(%) 60
Myeloma cells alone
50
40
Viability 30
ell 20
C
10
BUdR
0
0
7
14
21
28
35
42
49
Days in Culture
Osteoclast support of myeloma cells
requires contact.
Condition (N)
Viable cells Significance
Co-culture
308±31 x 103
--
Non-contact (12)
197 ±35 x 103
p<0.003
OC sup. (9)
213 ±45 x 103
p<0.01
Co-culture sup. (9)
229 ±25 x 103
p=0.05
IL-6 in myeloma
Parameter (N)
+Anti IL-6&R Coculture Significance
Total Cells x10-3 (16)
218 ± 36
314 ± 35
p<0.0001
% Viability (16)
84 ± 5
94 ± 1
p=0.05
Live Cells x10-3 (16)
200 ± 35
297 ± 34
p<0.0001
% Annexin V (14)
33 ± 4
18 ± 2
p<0.003
BrdU LI (12)
1.0 ± 0.3
1.1 ± 0.2
NS
Co-culture differentially affects gene expression of
myeloma plasma cells
Gene expression profiling of MM PC co-cultured with
osteoclasts reveal two disease classes
Osteoclast
Osteoclast
independent?
dependant?
1
2
Hierarchical clustering
of cocultured MM PC
by 134 commonly
altered genes
Cluster with
HMCL
NL PC
Genes commonly
5 in 8 of 11
234 in 6 of 8
changed
14 in 6 of 11
CA in
7 of 11
2 of 8
Myeloma cell osteoclast interactions
Myeloma cells affect osteoclast
progenitor's differentiation to mature
osteoclasts
Osteoclasts support survival and
sustained proliferation of myeloma cells.
Interactions with osteoclasts identify two
classes of myeloma.
Myeloma Depletes Osteoblasts
Progression:
Early
Apoptosis
Advanced
Co-culture system.
MM PC
OC
OB
Osteoblasts can support or kill myeloma
plasma cells in co culture
·Purified myeloma PC were cultured alone or co-cultured with Osteoblasts
·Viable MM PC recovered from cultures counted.
Live cells
MM+OB
MM PC from 12
MM PC from 6 patients
MM
OB from 12 different patients
OB from 3 different patients
2.5
2.0
1.5
OB
Neutral
1.0
0.5
0.0
12
3
45
6
MM PC
Osteoblasts inhibit
Osteoblasts support
OB
A
B
C
myeloma cells
myeloma cells
Varied Myeloma cell osteoblast interactions also in
the presence of osteoclasts
Viable Myeloma Cell Ratios
Co-culture/myeloma cells alone
3.0
PC + Osteoblasts / PC
PC + Osteoclasts
2.5
+ Osteoblasts / PC
2.0
1.5
1.0
0.5
0.0
Osteoblast restoration affects
myeloma growth and bone remodeling
MSC affect myeloma growth
and bone remodeling
500
hIg
BMD
400
300
change
200
ent
25
rceP 0
-25
-50
Control
Response
No Response
Myeloma osteoblast interactions
In vitro, osteoblasts support survival of
myeloma cells from some patients while
inhibiting others.
The interactions seems to be determined by the
myeloma cells.
In vivo, introduction of new MSC/osteoblasts
inhibits myeloma from some patients while
having no effect on others.
Summary and Conclusion
Myeloma interactions with the bone marrow
microenvironment
Support myeloma cells survival
Protect myeloma cells from therapy
Are responsible for manifestations
The bone marrow microenvironment is
a major factor in myeloma progression
and hence a critical target for therapy.
Thanks You!
X International Myeloma Workshop
Sydney, 2005