Transcript brightfield, 10X magnification dead cells

Quantitative Analysis of Tumor Cell Responses to Chemotherapy
1
Grubin ,
1
Hamed
1,
2
Roth
Jeremy
Salaheldin
and Charles M.
1Department of Biomedical Engineering, 2Department of Chemical & Biochemical Engineering,
Rutgers University, 98 Brett Road, Piscataway, NJ 08854, USA
Abstract
Our research is directed towards determining which treatments
for cancer are the most effective on a cellular level. To obtain
this information, quantitative techniques for monitoring cellular
phenotypes (growth, migration, and apoptosis) and molecular
expression (real-time PCR and DNA microarrays) are needed. A
useful cell growth assay has been utilized for current
experiments involving treatment of cancer cell arrays with
different cocktails. Cocktails that are most effective will be
studied at the cellular and molecular levels and possibly
optimized for efficacy and specificity of action. The results of
these experiments will hopefully lead to better cancer treatments
and a better understanding of how cancer cells behave.
Preliminary Results
DAY 0
Cells are seeded in 24 well plate
DAY 1
Cells are treated with varying
concentrations of etoposide.
DAY 3
Cells are washed with saline.
This is a preparatory step for the
staining procedure.
Introduction and Background
Despite decreases in incidence and mortality, cancer remains
the second leading cause of death in the United States. Current
chemotherapies are ineffective against many malignant forms of
tumors. Such tumor cells have mutations that increase both their
ability to proliferate and their resistance to apoptosis. Since
each tumor is unique, special treatments must be developed to
treat each patient.
The short-term goal of this project is to determine the response
of tumors to different cancer drug cocktails. Current experiments
study the effect of etoposide on tumor cells. Etoposide inhibits
DNA synthesis by interfering with the activity of topoisomerase,
an enzyme required for replication. It is being explored as a
possible treatment for various types of tumors.
We are studying the effect of etoposide on a human hepatoma
cell line (HepG2) and a human glioblastoma cell line (A172).
Cells are treated with varying concentrations of etoposide. 48
hours later, their viability is quantified using a live-dead assay.
The assay being used is a two-color fluorescence assay that
simultaneously determines the amount of live cell number and
dead cell number.
Live cells contain esterases that convert cell-permeable, nonfluorescent calcein acetoxymethyl to intensely fluorescent
calcein. Dead cells have damaged membranes, which allow
otherwise impermeable ethidium homodimer-1 to enter. Once
inside the dead cell, ethidium binds to nucleic acids, producing a
bright red fluorescence. These fluorescence values can be
quantified using the Cytofluor Multiwell Fluorescent Plate
Reader. The values can then be matched to known cell number
values to determine the amounts of live cells and dead cells.
The photographs to the right demonstrate the fluorescence
caused by ethidium and calcein.
Acknowledgements: We are grateful to the National Science
Foundation (CAREER Award to C.M.R.) for financing this
project. We are also thankful for the continual help and
guidance of our laboratory group members, especially Dr. Li
Kim Lee.
DAY 3
Ethidium stains are added to
cells. Calcein is not added since
all cells have been killed by
methanol. Plate is scanned by
Cytofluor. The fluorescence data
quantifies the total amount of
cells in each well.
DAY 3
Ethidium and calcein stains are
added to cells, causing them to
fluoresce. Plate is scanned by
Cytofluor. The fluorescence data
received from Cytofluor is used to
quantify cell viability.
DAY 3
Cells are killed with 75%
methanol and washed with
saline. This is done to normalize
for the effects of varying seeding
densities among wells, so the
fraction of dead cells to total cells
can be obtained.
The dose response of HepG2 cells to etoposide was quantified
using the calcein/ethidium staining assay. The amounts of dead
cells from the first fluorescence reading were divided by the
amounts of dead cells from the second reading to determine the
fraction of cells killed in each well.
Conclusions
A172 Cells
HepG2 Cells
The graph demonstrates a dose-dependent response between
etoposide concentration and fraction of cells killed. In order to
deliver the etoposide to the cells, minimal amounts of
dimethylsulfoxide (DMSO) were added to etoposide so it would
dissolve in saline. To account for the toxic effects of DMSO, it
was added to the control cells, which explains the 40 percent cell
death when no etoposide was added.
Future Work
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live cells
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live cells
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live cells
•More viability data will be obtained for HepG2 cells being
treated with etoposide.
•The etoposide experiments will be performed on A172 cells.
Results will be compared to those obtained from HepG2 cells.
•The calcein/ethidium assay being used currently only
distinguishes between live and dead cells. New complementary
cellular assays will be developed to determine viability in terms
of cell damage.
•We will measure changes in cell cycle and DNA repair gene
expression for these cells when they are treated with etoposide.
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dead cells
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•As mentioned above, DMSO is used to improve etoposide
solubility, but DMSO is toxic. Therefore, there is some
uncertainty as to whether it is etoposide or DMSO killing the
cells. To circumvent this problem, we will develop a new
polymeric system for drug delivery.