10th International Myeloma Workshop
(Sydney, april 2005)
Cross-talks between plasma cells and the
microenvironment: triggering and targeting
Federico Caligaris-Cappio
Università Vita-Salute San Raffaele
Milano, Italy

B cell clone
Precursors
IL-1b, TNF-b, M-CSF
Plasmablasts
Plasma cells
Adhesion molecules Angiogenetic factors
Adhesion molecules
Microenvironment Chemokines
Cytokines Extrallular matrix proteins
(IL-6)
Metalloproteinases
Macrophages
Fibroblasts
Myofibroblasts
Stromal cells
Osteoclasts
Endothelial cells

Cross-talks between plasma cells
and the microenvironment
Does Hypoxia play a role in shaping BM
microenvironment in MM ?

Vincent KA et al, TCM 2002
Under hypoxic conditions, HIF-1 is upregulated and mediates the
transcriptional activation of hypoxia-responsive genes including VEGF

Plasmacells from the BM of MM patient express HIF-1...
HIF-1
R1
HIF-1
R11
G1
..at levels significantly higher than plasmacells from MGUS

HIF-1/EPAS up-regulation by Stromal Cells
STROMAL CELLS
HIF-1
EPAS (HIF-2)
NT
ours
3h
Hypoxia
ON
EPAS, endothelial PER-ARNT-SIM homology domain
HIF-1, hypoxia-inducible factor-1 alpha

HUVEC CELLS: HYPOXIA TURNS ON THE
"ANGIOGENIC SWITCH"
A
Normoxia
Hypoxia
NT
Hypoxia
C
Normoxia
942
975
OD
30
Hypoxia
MMP-9
942
975
OD
er
MMP-9 MMP-2
20
821
1195
MMP-2
OD
basal (%)
821
1195
OD
lsnumb
10
Cel over
24 hrs
Normoxic CM
B
Hypoxic CM
30000
Normoxia
Hypoxia
25000
D
20000
HUVEC
15000
(number) 10000
5000
Migrated
3 hrs

UT
DFO
Sprouting
soid
Spher
Mean 0 ± 0.57 s.d.
Mean 4 ± 1 s.d.
Morphogenesis
Matrigel
HYPOXIA
50
Normoxia
EC
40
Hypoxia
30
HUV+ (%) 20
Matrigel
CD13
10
+VEGFR2
24 hrs

HYPOXIC HUVEC UP-REGULATE HIF-1, HIF-2, GLUT-1
A
DAPI
GLUT-1
HIF-1
B Hypoxia
Hypoxia
HIF-2
N
HIF-2a
HIF-1a
or
HIF-1a
moxia

Does Hypoxia play a role in shaping BM microenvironment in MM ?
Basement membrane
Hypoxic Plasma Cells
Pro angiogenic
factors
Migrating cell
iaoxm
nor
Hypoxic gradient

Cross-talks between plasma cells
and the microenvironment
· Does Hypoxia play a role in shaping BM
microenvironment in MM ?
· Is the regulation of endothelial
response to hypoxia affected by MM
therapy (PI) ?

Mg132-treated cells
Untreated cells
PI MG132 impairs
Annexin 5
mitochondrial activity of
HUVEC and plasma cells
Hoechst 33342
MitoTracker
E. Ferrero et al, submitted

MG 132 inhibits sprouting formation in vitro
NONE NONE
VEGF
MG132 VEGF+MG132
MG132 VEGF+MG132

MG132 inhibits angiogenesis in vivo
Matrigel plug assay
In vivo
Fischer 344 Rats

A
100
MG132 7.5 µM(normoxia)
MG132 7.5 µM(hypoxia)
80
ellsc ) 60
ing
(%
Liv
40
20
MG132 induces apoptosis
5 d
7 d
of HUVECs mainly when
B
they are hypoxic
NONE
VEGF
PI
2
4
2
3
79
15
72
23
MG132
VEGF+MG132
0.5
7.5
2
8
PI
52
40
29
61
Annexin V
Annexin V

Under hypoxia endothelial cells receive a proangiogenic switch that in turn
makes them more sensitive to MG132-induced apoptosis
Cells committed
to die
Cells committed
to die
Apoptotic bodies
Hypoxic gradient

Cross-talks between plasma cells
and the microenvironment
Are T cells (which T cells ?) acting in
MM BM microenvironment ?

Transduction of CD80 and CD40L into
MM cells generates autologous specific CTL
CD8
CD4
T cells only
T + Plasma cells
T + Stromal c.
T + PBMC
5x104
1.2x104
1.2x104
2.5x104
2.5x104
5x104
Cignetti et al, Human Gene Therapy, in press

Generation of anti-MM CTL correlates
with low stage/low tumor burden
Patient
CTL generation
Staging
% Malignant Plasma Cells
BM
PB
IE
YES
IA
25
0
VI
YES
IA
30
0
OB
YES
IA
34
0
FR
NO
IIB
25
0
MZ
NO
IIIA
92
2
CO
NO
IIIA
70
51
AL
NO
IIIA
56
6
MA
NO
IIIA
57
1
RA
NO
IIIB
58
15

Immunosurveillance Cell Players
Dendritic cells
NK
Treg
NKT
T cells
B cells
MM cells
T cells
GN/GE/Mf
Adapted From M Massaia

TWO MAIN SUBSETS OF T LYMPHOCYTES ARE KNOWN
Hematologic Tumors
Solid Tumors

MM plasmacells express MICA
U266
LP 1
A
anti - MICA/B
anti - MICA
anti - MICA/B
anti - MICA
18.7
194.7
28.7
34.7
B
MM 1
MM 2
MM 3
MM 4
64.2
14.8
14.6
69.7
MM 6
MM 7
MM 8
MM 9
M. Ferrarini et al, submitted

MICA expression by MM vs MGUS plasma cells*
Multiple M
MICA(Dmfi)
MGUS
MICA (Dmfi)
MM 1
18.7
MGUS 1
59.7
MM 2
194.7
MGUS 2
61.5
MM 3
28.7
MGUS 3
112.6
MM 4
34.7
MGUS 4
146.3
MM 5
64.2
MGUS 5
230.1
MM 6
14.6
MGUS 6
114.1
MM 7
14.8
MM 8
69.7
MM 9
10.8
MM 10
94.1
median
31,7
113,5
(p<0,05)
* As in indolent vs aggressive CLL (Zocchi et al, Cancer Res, 2004)

Pamidronate sensitizes MICA-positive
myeloma cell lines to cell lysis
A
U 266
LP 1
anti - MICA/B
anti - MICA
anti - MICA/B
anti - MICA
80
LP1 NT
LP1 Pam
U266 NT
60
U266 Pam
lysis
anti-TCR
40
anti-MICA
specific
anti-MICA/B
% 20
0
20
406080
% inhibition
0
2.5:1
5:1
10:1
E:T ratio

MICA enhances killing of MM plasma cells from patients by
pamidronate-activated V9/V2 T lymphocytes
60
50
MM3
lysis
40
MM3 Pam
MM9
30
MM9 Pam
60
specific
MM10
%
MM10 Pam
45
20
MM7 Pam
MM10
30
MM3
10
MM9
inhibition
15
%
0
0
1:1
3:1
10:1
1:1
3:1
10:1
E:T ratio

Possible involvement of lymphocytes
in the immune surveillance of multiple myeloma:
CD4
CD4
NKT
NKT
V
Pamidronate
V
U
U
U
U
PC
PC
CD8
PhosphoAg
V TCR
CD8
U MICA
U NKG2D
MHC
CD1d

Università Vita-Salute, IRCCS San Raffaele,
Milano
Department of Oncology
·Maurilio Ponzoni
· Elisabetta Ferrero
·Claudio Doglioni
· Marina Ferrarini
· Daniela Belloni
· Chiara Foglieni
· Simone Cenci
· Stefania Girlanda
· Roberto Sitia
· Silvia Scabini
· Paolo Ticozzi
·Aurelio Vicari
·Anna Borri
·Moreno Tresoldi

Document Outline