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Characterization of Cell Spreading and Focal Adhesion
Formation in B16F10 Melanoma Cells
John Ganz1 and Karen H. Martin2
1Biology
Department, Class of 2009 Eckerd College, St. Petersburg, Florida
2 The Mary Babb Randolph Cancer Center and the Department of Neurobiology and Anatomy, West Virginia University, Morgantown, West Virginia
Principle of Fluorescent Immunostaining
Four Color Stain
Composite
Melanoma cancer cells were used in this study because of their
highly invasive characteristics and increased levels FAK. Indirect
immunofluoresence microscopy was used
to visualize
phosphorylated FAK and paxillin in focal adhesions. Melanoma
cancer cells were then fixed on plates at different time intervals
so the maturing of focal adhesions could be observed and then
quantitated.
Paxillin
1
Cas
GFRs
PI3K
FAK Kinase
PLC
Cell Growth
FAT
Grb2
FERM
Graf
Cell Survival
FAK overexpression is correlated with poor prognosis in
cancers. FAK regulates functions that contribute to formation of
tumors and cancer metastasis. FAK inhibitors are important
targets for therapeutic intervention.
Melanoma cells were plated on fibronectin-coated glass
coverslips for various time to monitor the process of cell
spreading. The cells were then stained with primary antibodies
for rabbit anti-pY397 FAK (the activated form of FAK) and
mouse anti-Paxillin (a marker for focal adhesions). Secondary
antibodies tagged with green AlexaFluor 488 (anti-rabbit) and
red AlexaFlour 546 (anti-mouse) were used to visualize the
primary antibodies.
Outlined areas represent Focal Adhesions
Secondary Antibody
Target
A fluorescent molecule is excited by a specific wavelength of
light. When a fluorescent molecule is excited, it will emit a
different, lower-energy wavelength of light. It is essential that
the excitation and emission spectra do not heavily overlap with
another fluorescent molecule’s excitation and emission spectra.
When overlapping occurs, one color will bleed through into
another color creating a false colocalization. The Fluorescent
Spectra Viewer graph (below) illustrates the four fluorescent
dyes used in these experiments.
½ hour- cells small and mostly symmetrical. Small immature
focal adhesions
1 Hour- Cells start to spread symmetrically. Numerous Focal
Adhesion form.
2 Hour
Melanoma Cell Lines
According to the American Cancer Society, an estimated 8,110
people will die from melanoma in 2007. The cells raised in
culture and subsequently photographed were B16 F10 murine
melanoma cells. The F10 indicates the cells were collected
from metastatic tumors and then serially passaged through ten
generations of mice. Growing and harvesting metastatic cells
from ten generations of mice selected for the most invasive
cancer cells.
3 hours- Cell in oblong shape. Focal adhesion thick and long
Time Course of Spreading In Melanoma Cells
Fluorescent Protein
ASAP1 Talin
Cell Migration
pY397
Quantitation of Focal Adhesions
A target protein is injected into an organism such as a mouse.
The mouse’s immune system will make antibodies to this
protein. The antibodies are the purified out of the mouse and
will become the primary antibody for the target protein. Mouse
antibodies can then be injected into another organism such as
a rabbit. The rabbit’s immune system will recognize the foreign
proteins and make antibodies to the mouse antibodies. The
rabbit anti-mouse antibodies can then be purified and will
become the secondary antibodies. The secondary antibodies
will then have a fluorescent dye attached. The secondary
protein that is rabbit anti-mouse will then work on any mouse
antibody. In this way the primary antibody is selective to
particular protein whereas the secondary antibody is selective
only to the organism that made the primary antibody.
Primary Antibody
Paxillin
1/2 Hour
pY397
Actin
1 Hour
Focal adhesions in a migrating cell
Nucleus
3 Hour
Focal Adhesion Kinase (FAK) is a member of a family of nonreceptor protein tyrosine kinases that regulate cell survival,
migration and proliferation. This kinase is often over expressed
in invasive cancer types such as breast, prostate and skin
cancers. FAK inhibitors are a target for therapeutic intervention
and are currently in phase I clinical trials.
Time Course of Spreading (Continued)
3 Hour
Abstract
Focal adhesions were outlined and then measured by the
program Image J in effort to quanitate the number and size
difference between the 1.5 and 3 hour time intervals. Three
sets of experiments with 3 images per time interval per
experiment were quantitated. The raw data were averaged for
each interval. These data show that focal adhesions become
larger and fewer in number between the 1.5 hour and 3 hour
interval.
Future Applications
2 hours- cell become polarized. Focal adhesions become
consolidated
The overexpression of FAK is a well known characteristic of
highly invasive/ metastatic cancers. The understanding and
inhibition of FAK could lead to powerful cancer treatments.
Pfizer currently is in Phase I trials with a series of FAK
inhibitors.