Current Studies
One of the principal requirements for
cancer metastasis (cancer
spread) is tumor cell adhesion
and motility. Metastases from a primary tumor
to secondary locations throughout the body are a major cause of cancer
related deaths. In order to metastasize, tumor cells must undergo a
series of steps. First they are shed into the blood stream (intravasation), survive while traveling through the blood vessels, and
finally migrate again out of the vessels (extravasation)
into a new location in the body. Finally they must
proliferate.
The interactions between
cancer cells and the host immune system are of particular interest to our
group. Innate immune system processes can potentially promote tumor progression
through inflammation dependant mechanisms.
White blood cells,
neutrophils (PMN) in particular, are being studied to better understand
how the host immune system affects cancer cell adhesion and subsequent
migration in metastasis.
 This project
studies the adhesion and extravasation stages of the
tumor cell extravasation and metastatic process.
Primarily, we are studying factors
that promote a tumor cells' ability to extravasate
such as cellular dynamics, tumor-host interactions and
relevant hemodynamic forces. Regulation of adhesion molecules (ICAM-1, integrins, etc.), cytokine/chemokine interactions and responses are a few studies
currently being done. In addition, a migration assay has been developed to characterize tumor cell extravasation under
shear flow
conditions.
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Computational Fluid Dynamic (CFD) Modeling of Leukocyte
Facilitated Tumor Cell Adhesion in a Shear Flow
Previous
work done in our lab has demonstrated that PMNs can facilitate tumor cell
adhesion to the endothelium. Parallel plate flow chamber experiments have
shown that under different flow conditions, melanoma cells and PMNs adhere
to each other and to the endothelium with different efficiencies. These
results have suggested a two-step adhesion mechanism in which first PMNs
adhere to the endothelium, after which tumor cells adhere to those PMNs.
The primary focus of this work is the development of a computational fluid
dynamics, or CFD, model to explore what affect the fluid dynamics of the
system have on the interactions between PMNs and melanoma cells in a shear
flow.
MMP Studies
This study
aims to discover the mechanisms of tumor-endothelial interactions that aid
the melanoma cell in metastasis. Matrix Metalloproteinase (MMPs), in
particular, have been implicated in cancer angiogenesis, invasion,
migration and metastasis. In this study, we focus on the gelatinases
(MMP2, 9) and collagenase (MMP1).
Endothelial cells were co-cultured with melanoma cells in a 1:1 cell
ratio over 24 hours. The supernatant was collected and probed for MMP
activity using both zymographies and Western Blots. Upon detection of
upregulation in the secretion of a particular MMP, experiments were
designed to identify the cytokine that caused this upregulation. To study
the role that MMPs play in melanoma metastasis, melanoma invasion was
studied using a novel in-vitro chemotaxis invasion chamber. Endothelial
cells were grown on a layer of Matrigel that was coated on a filter in an
invasion assay to simulate the in vivo tumor extravasation and invasion
process through the blood vessel wall. Pharmacological inhibitors,
neutralizing antibodies, and/or siRNA knockouts from melanoma cells were
applied, respectively, to examine effects of inflammatory
cytokine-mediated MMP on melanoma invasion through the endothelium and
extracellular matrix
Human
umbilical endothelial cells (HUVEC) and 3 highly metastatic human melanoma
cell lines (Lu1205, A2058 and WM9) were co-cultured for 24 hours. It was
found that MMP-2 was upregulated and secreted into the supernatant from
the co-cultures of all 3 melanoma cell lines, with the highest expression
using Lu1205. Blocking IL-8 in the co-culture resulted in reduced
expression of MMP2, confirming that IL-8 is the target cytokine. The
secretion of tissue inhibitor of MMP 2 (TIMP2) is also found to be
upregulated from the melanoma-HUVEC co-culture. From literature, we
suspect the source of the MMP2 and TIMP2 is primarily from the
endothelials cells, however, we intend to confirm this. Invasion assays
using endothelial cells grown on Matrigel are currently being
investigated.
There
is strong evidence to suggest that IL-8 is responsible for increased
expression of MMP2 into the supernatant. MMP2, a gelatinase, has been
implicated in both metastasis and angiogenesis in cancer and this allows
us to understand more of the tumor microenvironment in order to
effectively apply therapy.
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 This study focuses on elucidating
melanoma-induced signaling events leading to dissociation of endothelial
cell junctional integrity. We have demonstrated
that contact of melanoma cells to human umbilical vein endothelial cells (HUVEC)
triggered rapid endothelial [Ca2+]i response.
In addition, alternation of endothelial adherens junctions following
contact of melanoma cell was also evidenced by the changes in tyrosine
phosphorylation of vascular endothelial (VE)-cadherin.
Melanoma contact induced a time-dependent increase in tyrosine
phosphorylation. Tyrosine phosphorylation reached a maximum at 45
min following the introduction of melanoma cells and diminished
thereafter. The immunological staining patterns of VE-cadherin are
strongly peripheral and junctional structures are intact prior to melanoma
contact (t = 0). Upon melanoma contact with endothelium, highly
localized dissociation of these junctions occurs (t = 45 min). In
addition, signaling molecules besides Ca2+ and receptors
involved in controlling the downstream junctional regulation following
contacts with melanoma cells are being studied.
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Melanoma Cell Migration to Type IV Collagen Requires Activation of NF-kB
Chemotaxis is the consequence of
environmental factors engaging their receptors to initiate signaling
cascades. However, the biochemical mechanisms controlling this phenomenon
are not clear. We employed an in vitro modified Boyden 48-well chemotaxis
migration system to characterize the role of signal transducers in type IV
collagen-induced A2058 human melanoma cell migration. Using specific
pharmacological inhibitors and a series of dominant-negative and
constitutively active signaling proteins, we show that Ras and Rac GTPases,
PI3-K, and PKC participate in cell migration mediated by
b1 integrins. Collagen also
induces a time-dependent degradation of IkB-a
and an increase in nuclear translocation of NF-kB
which is dependent on PKC pathway. More importantly, collagen-stimulated
melanoma cell migration directly correlated with an increase in NF-kB
transactivation. Furthermore, CIV induced an increase in
b1 integrin mRNA levels.
Specific NF-kB inhibitors
Helenalin and SN-50 inhibited melanoma cell migration to collagen,
indicating a novel requirement for NF-kB
transactivation in cell chemotaxis mediated by
b1 integrin signals. These
results describe signal transduction events that are initiated by type IV
collagen through b1 integrins and demonstrate an important role for NF-kB
in regulating melanoma chemotaxis.
This project is
in collaboration
with
Dr. Andrew Henderson in
Department of Veterinary Science at Penn State.
 Observing microscopic specimens is often useful in studies
of cellular interaction with a vascular wall. We have developed an in
vitro side-view flow chamber that permits observations from the side of
the cell's contact with various adhesive surfaces under dynamic flow
conditions. This side-view flow chamber consists of two precision
rectangular glass tubes called microslides. A smaller microslide is
inserted into a larger one to create a flow channel with a flat surface on
which either cultured vascular endothelium can be grown or purified
adhesion molecules can be coated. Two optical prisms with a 45 degree
angle chromium-coated surface are used along the flow channel to generate
light illumination and observation pathways. The side-view images of
cell-substrate contact can be obtained using a light microscope. This
design allows us not only to measure the effects of flow on cell-surface
adhesion strength, but also to have close observation of cell deformation
and adhesive contact to various surfaces in shear flow. Using this
side-view flow assay, the mechanics of white blood cell deformation and
adhesion to various endothelia cell adhesion molecules in dynamic shear
flow has been investigated.
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Previous Studies
 This study focuses on effects of
extracellular lipid groups on tumor cell pseudopodial protrusion and cell migration in
vitro. We are interested in understanding the interaction
between extracellular ligands (extracellular matrix protein molecules) with cell
receptors, and how extracellular bioactive lipids contribute to such
signaling mechanisms. Together with the initial signaling mechanism, we
are characterizing the subsequent cell activation via Ca2+
secondary messenger mediated pathway and how that affects the cellular
cytoskeleton reorganization. Glass micropipette assay system
(Figure A~C)
enables us to focally apply the chemotactic stimulant solution onto a specific region of
a suspended cell; a cell protruding pseudopods can be observed and
characterized in real-time using brightfield or fluorescence microscopy.
Adhered cells spreading and migrating toward chemotactic stimulation can
also be assayed in real-time using our system (Figure D).
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 We have developed a dual-micropipet technique to measure
cell locomotion. the objective of this work was to characterize tumor cell
locomotion in response to chemotactic stimulation. The assay involves two
micropipets. An individual tumor cell (A2058 human melanoma cell is shown)
was retained, without pressure gradient, in a pipette of approximately 14
microns i.d. A solution of type IV collagen (one of the important
extracellular matrix proteins), chosen as the chemotactic source, was
placed in another pipette (~10 microns o.d.) with zero pressure at the
pipette tip. The smaller pipette was then inserted into the larger one
containing the melanoma cell. The initial chemoattractant concentration
and the distance between the tip of the small pipette and the cell surface
provided a gradient for tumor cell locomotion toward stimulation. This
novel assay provides a direct measure of cell movement: cyclic pseudopod
protrusion and subsequent cell locomotion. The influences of different
adhesion substrates on cell locomotion were also studied.
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