Oxygen levels in typical cell culture conditions do not accurately reflect the oxygen levels cells are exposed to within the body. Furthermore, oxygen levels can vary within the tumor microenvironment. These variances can affect how cells respond to a variety of drugs and small molecules. To further understand how oxygen levels affect drug sensitivity, the response of hormone-dependent MCF7 cells were compared to hormone-independent MDA-MB231 cells, cultured under low and high oxygen.

1. Michelle Yan, Marcy Wickett, Daniel Beacham, Leticia Montoya, Scott Clarke, and Michael O’Grady, Thermo Fisher Scientific/Molecular Probes, 29851 Willow Creek Rd, Eugene, OR, USA,
97402
RESULTS
Figure 1. High-throughput screening of Killer Collection using PrestoBlue
Figure 1: MDA-MB231 (A) and MCF7 (B) were treated with 10 µM of drug from the Killer
Collection. After addition of drug, cells were cultured under low (1%) or normal (19%) oxygen
for 24, 48 and 72 hours. At the end of each time point, cell viability was assessed using
PrestoBlue.
ABSTRACT
Oxygen levels in typical cell culture conditions do not accurately reflect the oxygen levels
cells are exposed to within the body. Furthermore, oxygen levels can vary within the tumor
microenvironment. These variances can affect how cells respond to a variety of drugs and small
molecules. To further understand how oxygen levels affect drug sensitivity, the response of hormone-
dependent MCF7 cells were compared to hormone-independent MDA-MB231 cells, cultured under
low and high oxygen.
The goal of this study was to examine the differences in the sensitivity of MCF7 and MDA-
MB231 cells to drugs from the Killer Collection (MicroSource Discovery Systems) when cultured
under low and high oxygen levels and for different drug exposure time periods. Cells were grown
using standard cell culture conditions (19% oxygen) or low oxygen (1%). Using high-content imaging
and high-throughput analysis methods, viability was assessed using two different mechanistic
readouts; cellular metabolic activity and membrane permeability. Post-screening analysis was
performed to confirm positive hits by performing dose-responses and determining IC50 and EC50
concentrations using the same reagents as in the screen, and assess oxidative stress and cellular
proliferation.
Results showed that viability differed depending on mechanistic readout and platform
method used to determine hits as well as duration of exposure to compound. In addition, the
response of MDA-MB231 cells was not always identical to that of the MCF7 cells. Post-screening
analysis of “hits” indicated different potencies of compounds tested depending on oxygen level and
cell type. These data suggest that some drugs may affect MCF7 and MDA-MB231 cells differently
depending on environmental oxygen levels and mechanistic readout used to determine cellular
health.
INTRODUCTION
 Assessment of cell viability is a foundational assay in drug discovery
and biological research
 Cell viability of hormone dependent (MCF-7) and hormone
independent (MDA-MB231) human breast cells were examined
 Cells were cultured under normal (19%) and low (1%) oxygen after
drug exposure
 Two different methods of measuring cellular viability were compared:
 PrestoBlue™ (HTS) a cell viability indicator that measures the
reducing power of living cells
 Live/Dead™ Cell Imaging Kit (HCS) is a two color cell viability
assay consisting of:
 Live Green: measures intracellular esterase activity
 Dead Red: enters only cells that have lost their membrane
integrity
MATERIALS AND METHODS
Cells & culture conditions: MCF7 and MDA-MB231 cells were cultured
in DMEM + 10% FBS at 5% CO, 19% O2 and 37⁰C until ready to use.
Cells were plated in 96-well plate and allowed to attach overnight. Cells
were then treated with drugs (as indicated) and transferred to incubator
to be cultured at 1% or 19% O2 for remaining treatment time.
PrestoBlue: Cells were loaded with 1x PrestoBlue for 60 min. under
culture conditions than read on a Varioskan™ Flash Multimode Reader.
Live/Dead Cell Imaging Kit: Cells were loaded with Live Green and
Dead Red plus Hoechst 33342 for 30 min. at room temperature. Cells
were then imaged and analyzed using the Cellomics Arrayscan™ VTI.
Data analysis: Data plots and dose-response curves were generated
using GraphPad Prism.
CONCLUSIONS
 Both PrestoBlue and Live/Dead Cell Imaging Kit were useful for the first
step in this drug screening experiment. Follow up experiments confirmed
hits determined by the initial screen.
 MCF7 and MDA-MB231 cells showed varied sensitivity to several drugs,
with MCF7 cells being less sensitive to Gambogic Acid and Celastrol.
 MCF7 cells treated with Tanshionone IIA were more sensitive to this drug
when cells were cultured at 19% O2 versus 1% O2.
 For cells treated with Dactinomycin, determination of dead cells by Dead
Red was the most sensitive assay to evaluate viability. This assay showed
a dose-response, while PrestoBlue and Live Green curves were more
linear.
 Further experiments are planned to confirm additional hits, assess cellular
proliferation and examine oxidative stress.
High content screening in MCF7 and MDA-MB231 cells show differential
responses depending on oxygen levels and mechanistic readout for viability
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Figure 2. High-content screening of Killer Collection using Live/Dead Cell
Imaging Kit
Figure 2: Results for high-content imaging screen with MDA-MB231 cells (A-B) and MCF-7
cells (C-D) using Live/Dead Cell Imaging Kit. Cells were treated with 10 µM drug from the
Killer Collection then incubated at 1% or 19% oxygen levels for 24 and 48 hours. At the end of
each time point, cell viability was assessed using Live/Dead Cell Imaging Kit.
A. B.
A. B.
C. D.
Figure 3. Dose-response curves for all three readouts by specific drug type
Figure 3: MDA-MB231 and MCF7 cells were treated with 0 – 30 µM of drug, then incubated
at 19% or 1% O2 for 24 hours. Differences in dose-response curves were observed
depending on assay type, and between cell types. MCF7 cells treated with Tanshinone IIA
showed the greatest difference in drug sensitivity depending on oxygen levels. MCF7 cells
grown at 19% O2 were more sensitive to the drug compared to cells grown at 1%. MDA-
MB231 and MCF7 cells showed differential responses when treated with Diactinomycin as
assessed by Dead Red, and with Pristimerin as assessed by PrestoBlue.
A.
B.
C.
Figure 4. Dose-response curves for PrestoBlue by cell type
Figure 4: MDA-MB231 and MCF7 cells were treated with 0 – 30 µM of drug, then incubated
at 19% or 1% O2 for 24 hours. Differences in dose-response curves (IC50) was observed
between cell types. MCF7 were less sensitive (greater IC50) to Gambogic Acid and Celastrol
as assessed by PrestoBlue, compared to MDA-MB231 cells.
Figure 5. Dose-response curves for Live/Dead Cell Imaging Kit
PrestoBlue, IC50 (µM)
Gambogic Acid,
19%
Gambogic Acid,
1% Celastrol, 19% Celastrol, 1%
MCF7 5.90 7.25 9.92 9.77
MDA-MB231 2.05 1.38 2.64 2.31
Live Green, IC50 (µM)
Gambogic Acid,
19%
Gambogic Acid,
1% Celastrol, 19% Celastrol, 1%
MCF7 8.23 7.39 6.45 6.36
MDA-MB231 3.00 3.36 6.73 5.02
Dead Red, EC50 (µM)
Gambogic Acid,
19%
Gambogic Acid,
1% Celastrol, 19% Celastrol, 1%
MCF7 4.68 4.71 4.06 4.32
MDA-MB231 2.71 3.64 3.14 3.35
#2169
Figure 5: MDA-MB231 and MCF7 cells were treated with 0 – 30 µM of drug, then incubated
at 19% or 1% O2 for 24 hours. When cellular viability was assessed with Live Green and
Dead Red, MCF7 cells were slightly less sensitive to Gambogic Acid and Celastrol. However,
differences were small. No differences due to oxygen levels were observed.
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