Haematologica 2003 - Supplements

141
Human myeloma cells adhere to fibronectin and
migrate in response to HGF, IGF-1 and SDF-1α
Randi Utne Holt, Vadim Baykov, Torstein Baade Ro,
Sigmund Brabrand, Anders Sundan, Anders Waage and
Magne Borset.
Norwegian University of Science and Technology, Dept. of Cancer
Research and Molecular Medicine.
Migration and adhesion are important events in the interaction
between myeloma cells and their surroundings. This cell behavior
is dependent on adhesion molecules and is regulated by stimuli
from the microenvironment in which the cell is located.
We examined adhesion of myeloma cell lines to several matrix
components (fibronectin (FN), VCAM-1, collagen I and IV,
laminin and vitronectin), after stimulation by various cytokines as
well as by Mn2+. The cytokines used were BMP-7, EGF, FGF2,
VEGF, HGF, IFN-α, IGF-1, IL-1β, IL-6, IL-15, IL-21, TNF,
SDF-1α, and MIP-1α. We found that INA-6 and ANBL-6 cells
adhered to FN and VCAM-1 but not to collagen, laminin or
vitronectin. Adhesion to FN increased upon stimulation of cells
by HGF (INA-6 only), IGF-1 or SDF-1α (7–9 fold), whereas
other cytokines gave little or no increase in adhesion.
Migration of myeloma cells was studied by use of transwell
chambers. HGF and SDF-1α increased migration of INA-6 and
ANBL-6 cells 3 – 4 fold compared to control, while IGF-1
increased migration 2 fold.
Both adhesion and migration may involve integrin activation. We
screened eight myeloma cell lines for surface integrins (β1, β2,
β7, α4, α5, αv, αL, αx) by flow cytometry, and found that α4β1
(VLA-4) was by far the most abundant integrin. Cytokinestimulated
adhesion was inhibited completely by use of blocking
monoclonal antibodies towards α4 or towards β1, whereas
migration decreased 50% by the same mAbs. The amount of
α4β1 varied widely between cell lines (more than 10 fold
variation in mean fluorescence intensity). Adhesion properties did
not only reflect surface level of α4β1, as INA-6 cells, with less
than half the amount of α4β1 integrin on their surface compared
to IH-1 cells, adhered much better to FN and VCAM-1 than IH-1
cells.
By use of blocking agents, we studied the intracellular pathways
for HGF, IGF-1 and SDF-1α signaling leading to activation of
α4β1 integrin. Cytokine-stimulated adhesion was blocked by PI-3
kinase-inhibitors (LY-294002 and Wortmannin), but not by MAP
kinase inhibitors (U-0126 and PD-98059). A tyrosine kinase
inhibitor specific for the HGF receptor c-Met inhibited HGFmediated
adhesion only, whereas pertussis toxin (an inhibitor of
Gi-protein-linked receptors including chemokine receptors)
inhibited only SDF-1α -mediated adhesion. This shows that IGF-
1- and HGF-induced α4β1 integrin activation does not act via the
receptor for SDF-1α or via other Gi-protein-linked receptors.
Similarly, IGF-1 or SDF-1α are not upstream of the signal from
the HGF receptor.
In conclusion, of many tested cytokines HGF, IGF-1 and SDF-1α
were unique in their ability to strongly activate α4β1-integrindependent
adhesion and migration of myeloma cells.
6.2 Angiogenesis and invasion
142
Bone marrow endothelial cells increase the
invasiveness of human multiple myeloma cells through
upregulation of MMP-9 : evidence for a role of
hepatocyte growth factor
Isabelle Vande Broek, Valerie Allegaert, Kewal Asosingh,
Liesbeth Hellebaut, Xavier Leleu*, Thierry Facon*, Karin
Vanderkerken, Ben Van Camp, Ivan Van Riet.
Department of Hematology and Immunology, Vrije Universiteit
Brussel (VUB), Brussels, Belgium; *Service des Maladies du Sang,
CHU, Lille, France.
The mechanisms underlying the homing of multiple myeloma
cells (MM) to the bone marrow (BM) are not fully understood. In
analogy to lymphocyte homing and trafficking in which matrix
metalloproteases (MMP) participate in the degradation of the
subendothelial basement membrane, one can hypothesize that
MMPs are implicated in the extravasation and homing of MM
cells to the BM. In this study, we investigated the role of MMP-9
on the in vitro invasiveness of human MM cells. MMP-9
expression and secretion was demonstrated by reverse
transcription polymerase chain reaction (RT-PCR) and/or
zymography in the human MM cell line RPMI 8226 and primary
MM cells isolated from the BM of 6 MM patients. CD138-
positive MM cells were isolated from patient BM samples by
MACS separation. The in vitro invasiveness of human MM cells
was investigated through the ability to invade Matrigel, a
basement membrane extract. Matrigel invasion assays revealed
that human MM cells indeed were invasive (range of invasive
cells between 3-29%) and that the number of invasive cells
considerably increased (from a mean value of 12 to 29%) when
invasion was assessed on Matrigel filters coated with BM
endothelial cells (EC) (transendothelial invasion). Neutralising
antibodies against MMP-9 inhibited transendothelial invasion by
56,5% (range 32-75%), indicating that MMP-9 is directly
involved in the invasion process. In parallel we could also
demonstrate by zymography that BM-EC induce an upregulation
of MMP-9 in primary MM cells, indicating that EC enhance MM
cell invasion through upregulation of MMP-9. Moreover, we
found that recombinant hepatocyte growth factor (HGF)
produced by BM EC upregulates MMP-9 production by the MM
cells, whereas anti-HGF neutralising antibodies inhibited MM
cell invasion by 59%.
In summary, our findings provide evidence that HGF produced
by BM EC enhances the invasion of human MM cells through
upregulation of MMP-9. We conclude that MMP-9 and HGF
potentially contribute to extravasation and homing of human MM
cells towards the BM compartment.
S150