1.0 mmol/L ethylenediamine tetra-acetic acid [EDTA]). The addi-
microliters DMEM with 0.1% BSA was added to each test well
tion of 5.0 mL DMEM neutralized the trypsin. Centrifugation for
and incubated for 1.5 hours at 37°C. Excess DMEM was aspi-
rated, and 2 ϫ 10 cells were incubated in each test well for 35
5
2
minutes at 500g (4°C) allowed separation of the cells.
Antibodies against integrin subunits used for the adhesion
minutes at 37°C.
assay included mouse monoclonal antibodies to alpha2 (P1E6,
Chemicon), alpha3 (P1B5, Chemicon), alpha5 (P1D6, Chemicon),
alpha6 (GoH3, Pharmingen), beta1 (6S6, Chemicon), and beta4
For the antibody inhibition assay, an equal volume of the
blocking antibody was added to the cell solution to achieve a final
antibody concentration of 10 g/mL and incubated on ice for 1
hour before adding the cell suspension to each microwell.
Five washes with 200 L PBS were followed by fixation with
100 L 1% formaldehyde for 15 minutes. The formaldehyde was
aspirated, and the adherent cells were stained for 20 minutes
with 60 L per well of 2% crystal violet in PBS. The plates were
(
ASC-3, Chemicon). Antibodies against integrin subunits used for
Western blot analysis (included alpha2 (sc-6586, Santa Cruz),
alpha3 (sc-6592, Santa Cruz), alpha5 (Transduction Laborato-
ries), alpha6 (A33, kindly provided by Arnoud Sonnenberg of the
Netherlands Cancer Institute), alphaV (VNR 139, Chemicon),
beta1 (HB1.1, Chemicon), and beta4 (439-9B, Pharmingen). An-
tibodies against integrin subunits used for fluorescence-activated
cell sorter (FACS) analysis included alpha2 (AK-7, Pharmingen),
alpha3 (C3 II.1, Pharmingen), alpha5 (IIA1, Pharmingen), alpha6
washed with distilled H O until all BSA wells were clear. The
2
wells remained inverted overnight. The cells were lysed with 100
L 2% SDS per well for 30 minutes, and the optical density (OD)
of each well was 562 nm. We applied Student t test to compare the
ability of JHU-022-SCC and UM-SCC-1 cells to adhere to the
substrate before and after incubation with blocking antibodies.
(
(
GoH3, Pharmingen), alphaV (VNR139, Chemicon), alphaVbeta6
E7P6, Chemicon), beta1 (6S6, Chemicon), beta3 (B3A, Chemi-
con), and beta4 (ASC-3, Chemicon).
Western Blot Analysis
Migration Assay
Seventy percent confluent cell cultures on 10-cm plates were
washed with calcium-free PBS. Two hundred microliters of lysis
buffer (0.5% Triton, 50 mmol/L Tris [pH 7.5], and 1.0 mmol/L
MgCl2 with fresh 1.0 mmol/L phenylmethylsulfonyl fluoride
We measured tumor cell migration along purified extracel-
lular matrix (ECM) components collagen IV and laminin 5 (pro-
vided by Kaoru Miyazaki, PhD, Yokohama City University), us-
ing a modified Boyden chamber (Neuroprobe, Bethesda, MD). An
[
PMSF] and 1.0 mM/L N-ethylmaleimide [NEM]) were added to
8-m filter was precoated with the study ligand at a concentra-
each plate and swirled gently for 30 minutes. A cell lifter was
used to scrape the plate, and the whole cell lysate was placed in
a Microfuge tube and centrifuged for 5 minutes at 14,000g. The
supernatant was aspirated, and aliquots of 30 g of protein were
obtained by standardizing the samples using the Pierce assay.
Thirty micrograms of whole cell lysate from each cell were
prepared by adding sample buffer and heating at 95°C for 3
minutes. The samples were cooled to room temperature, loaded
onto 7% resolving sodium dodecyl sulfate polyacrylamide gels
tion of 0.5 g/mL for 1 hour. The lower well of the Boyden
chamber was filled with serum-free medium containing 0.1%
bovine serum albumin (BSA). Fifty microliters of cell suspension
was added to the upper chamber and then incubated for the
indicated time at 37°C. The tumor cell suspensions were also
incubated with antibodies directed against different integrin sub-
units. Cells on the top of the filter were removed by wiping, and
the filter was fixed in 1% formaldehyde in phosphate-buffered
saline (PBS). Migrated cells were then stained with 1% crystal
violet, and nine randomly chosen fields from triplicate wells were
counted at original magnification ϫ400.
(
SDS-PAG), and run at 130 V for approximately 60 minutes.
Semidry mini protein transfer to membrane filters (Immobilon-P
membranes) was performed at 13 V for 31 minutes with a max-
imum current limit set at 5.5 mA/cm .
2
Enhanced chemiluminescence (ECL) detected proteins
along the Immobilon-P membranes. The membranes were first
blocked overnight using a 5% blocking solution (5% powdered
milk in Dulbecco’s-PBS with 0.1% Tween). Three 10-minute
washes (0.1% Tween in Dulbecco’s-PBS) were followed by incu-
bation for 1 hour with the primary antibody diluted with the
blocking solution. A second set of three washes was followed by an
hour-long incubation with the appropriate horseradish peroxi-
dase (HRP) conjugated secondary antibody. A third set of washes
was followed by incubation with the detection solution (Amer-
sham) for 1 minute. The membranes were then exposed to x-ray
film for signal detection.
Orthotopic Mouse Model
One hundred microliters of PBS containing 5 million tumor
cells were injected into the cervical subcutaneous tissues of the
4-week-old athymic nude female mice (Harlan Sprague-Dawley).
Eight mice were injected with UM-SCC-1 cells, and seven mice
were injected with the JHU-O22-SCC cells. The mice were killed
at 30 days.
All specimens were harvested in an identical fashion. Five
coronal sections of the lower jaw and cervical region of each of the
mice were harvested in an anterior to posterior direction. An
experienced, board-certified pathologist specializing in head and
neck tumors systematically evaluated representative slides pre-
pared from each of the specimens in a blinded fashion. The
presence or absence of perineural invasion was determined. Dur-
ing histological evaluation, a tally was kept from each specimen
for evidence of lymphatic and vascular invasion.
Fluorescence-Activated Cell Sorter Analysis
Flow cytometric analysis was performed on cells from each
of the two cell lines. Cultured cells of 70% to 90% confluence were
harvested by brief trypsinization (0.25% trypsin and 1 mM/L
EDTA) for approximately 2 minutes. One million cells were re-
suspended in 200 L phosphate-buffered saline–fetal calf serum
(PBS-FCS).
Adhesion Assay and Blocking Assay
The ECM components studied were collagen IV, fibronectin,
vitronectin, and laminin 1 (all from Becton Dickinson); laminin 5
(
conditioned medium); and laminin 10 and laminin 11 (Gibco).
Primary antibody was added to each cell solution to a final
antibody concentration of 2.5 g/mL. The cells were incubated on
ice for 1 hour followed by three 200-L PBS-FCS washes. The
secondary antibody was added, and the solution was incubated on
ice for 30 minutes. Four 200-L washes with PBS-FCS were
performed. Propidium iodide was added to a final concentration of
1 g/mL. The cells were then resuspended in 500 L PBS-FCS
and loaded into the FACS machine.
Subunits of interest included alpha2, alpha3, alpha5, alpha6,
alphaV, beta1, and beta4. Seventy-five microliters of each ligand
at a concentration of 10 g/mL was used to coat wells of
Immulon-4 (Dynatech Laboratories) enzyme-linked immunosor-
bent assay (ELISA) 96-well plates and incubated for 1 hour at
3
7°C. One percent BSA was the negative control. Excess ligand
was aspirated, and each well was washed with PBS. Two hundred
Laryngoscope 112: November 2002
Dyce et al.: Integrins in Head and Neck Carcinoma
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026