A High-Throughput Soft Agar Assay for Identification of Anticancer Compound
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A High-Throughput Soft Agar Assay for Identification
of Anticancer Compound
STEVEN N. ANDERSON, DANLI L. TOWNE, DAVID J. BURNS, and USHA WARRIOR
A 384-well soft agar assay was developed to identify potential novel anticancer compounds. Normally used to detect cell trans-
formation, the assay is used here to quantitate cell proliferation in a 3-dimensional (3-D) anchorage-independent format. HCC827
cells, which are highly sensitive to epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors, were used to develop the
method and a set of 9600 compounds used to validate the assay. Results were compared to a monolayer assay using the same
compound set. The assay provides a robust method to discover compounds that could be missed using traditional monolayer for-
mats. (Journal of Biomolecular Screening 2007:938-945)
Key words: soft agar, HTS, 3-D, anoikis, HCC827
INTRODUCTION found in a traditional screen using monolayers. Thus, a high-
throughput screen (HTS) developed to detect compounds with
G ROWTH OF CELLS IN SOFT AGAR is one of the hallmark char-
acteristics of cellular transformation and uncontrolled cell
growth, with normal cells typically not capable of growth in semi-
antiproliferative activity in soft agar would be predicted to be
superior to screening compounds in monolayers.
Using traditional soft agar methods to manually count colonies
solid matrices. Used to detect cancer cell transformation and val- would be difficult and cumbersome for an HTS assay. Several dif-
idate cancer targets, soft agar drug sensitivity assays have been ferent endpoints have been employed to quantitate colony forma-
used to test anticancer compounds since Hamburger and Salmon tion, including manual counting of colonies in 6- or 24-well
developed the human tumor clonogenic assay in 1977.1,2 In addi- plates,1,16,17 and in a limited scale by 3-H thymidine incorporation
tion, it has been proposed that testing drugs in a 3-dimensional and high-content analysis.18,19 Many of these assay methods
(3-D) format, like soft agar, is superior to using monolayer cul- require 3 to 4 weeks of growth before the colonies can be enu-
tures as cell growth in 3-D is more similar to the in vivo cellular merated, and the counting of each well is labor intensive. Recently,
environment.3-8 Normal epithelial cells are supported by basement some investigators have used metabolic tetrazolium dyes such
membranes providing survival and proliferative signals and undergo as MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
apoptosis when placed in suspension culture.9,10 Alternatively, bromide])11,20,21 and alamarBlue,22 a redox indicator, to provide an
cancer cells are able to evade attachment-regulated apoptosis indirect measure of viable cell number to quantify cell growth in
(anoikis), leading to uncontrolled proliferation. Therefore, assays soft agar. Although these methods do not measure colony size,
using anchorage-independent conditions have been developed to these assay plates can be read in a standard plate reader, which
discover compounds that cause reversion of transformed cells to eliminates manual counting errors and greatly increases through-
normal and inhibit cell growth under anchorage-independent con- put required for drug screening studies.
ditions.7,8,11-15 Anchorage-independent assays would be expected Here, we describe a 384-well HTS assay designed to discover
to discover not only compounds targeting cancer cell proliferation compounds inhibiting the growth of HCC827 human lung cancer
but also unique compounds inducing anoikis that would not be cells in soft agar. Optimal conditions for running a robust HTS
assay, including cell density, time of culture, Z′ factor, and DMSO
sensitivity, are determined for this assay format. Results are also
presented for a comparison study of 9600 compounds in a soft
Abbott Laboratories, Global Pharmaceutical Research and Development, agar assay screen versus a monolayer screen to evaluate and com-
Department of Biological Screening, Abbott Park, IL.
pare the robustness of the soft agar format. Results of several
Received Mar 13, 2007, and in revised form Jun 25, 2007. Accepted for publi- known kinase inhibitors, including staurosporine, Tarceva
cation Jun 27, 2007. (erlotinib), Zactima (vandetanib), Iressa (gefitinib), and Gleevec
Journal of Biomolecular Screening 12(7); 2007 (imatinib), are included in this study to demonstrate the feasibil-
DOI: 10.1177/1087057107306130 ity of HTS soft agar assays for identifying active compounds.
938 www.sbsonline.org © 2007 Society for Biomolecular Sciences
Downloaded from jbx.sagepub.com by guest on October 12, 2015384-Well Soft Agar HTS Assay
MATERIALS AND METHODS Add 10 µL of 0.6% agar/medium to
384-well nontreated plates to form base layer
Cells ↓
Add 50 µL cells in 0.4% agar
HCC827 cells (ATCC, Manassas, VA) are derived from human ↓
non–small cell lung carcinoma23,24 and maintained as monolayers Within 24 h, add 10 µL of compound in 1x medium
in 162-cm2 Costar flasks (Corning Costar Corporation, Corning, ↓
NY) using RPMI 1640 Medium supplemented with 10% fetal Incubate for 7 days
bovine serum (FBS), 10 mM HEPES, 1 mm sodium pyruvate, ↓
Utilize alamarBlue (7 µL) to quantitatenumbers of cells in each well
4.5 g/L glucose, and 100 U/mL penicillin-streptomycin (all from
Read using plate reader at ex 531: em 615 nm
Invitrogen, Carlsbad, CA). Cultures were maintained at 37 °C in a
humidified atmosphere of 5% CO2 in a Sanyo model MCO-20AIC FIG. 1. Flow diagram of 384-well soft agar assay.
incubator (Sanyo, Bensenville, IL).
Soft agar assay Compound preparation
The method presented here is modified from previously IC50 determination. Compounds for IC50 determinations were
published reports1,16,22 and designed for testing anticancer prepared as 5-mM stock solutions in DMSO. Compounds were
agents in 384-well plate HTS assays (Fig. 1). Noble agar serially diluted to their required concentrations in DMSO and 1 µL
(Sigma, St. Louis, MO) was prepared as a 5% solution of water transferred to a Greiner (Monroe, NC) polypropylene 384-well
and autoclaved in order to melt it into solution and for steril- plate. Using a Flexdrop automated dispenser (PerkinElmer),
ization. The agar was allowed to solidify and remelted for each which was previously rinsed with ethanol to help maintain steril-
assay using a microwave oven. A 0.6% agar/medium base layer ity, 70 µL of medium was then added to the drug plate. Next, 10
added to each well prevented cells from attaching and forming a µL of compound was transferred from the drug dilution plate to
monolayer on the plastic substrate. Then, 12 mL of 5% agar at plates containing cells in soft agar using a Beckman FX robotic
65 °C was mixed with 88 mL of supplemented medium as system (Fullerton, CA). The plates were placed back into the incu-
described above at 45 °C. Then, 10 µL of the 0.6% agar was bator, and the cells were allowed to grow a minimum of 7 days for
quickly pipetted into Costar nontreated plates (Corning Costar the soft agar format and 3 days for the monolayer format.
Corporation) that were previously warmed to 42 °C to prevent
the agar from solidifying to the sides of the plate. Each plate Primary screen. Compounds tested in the primary screen were
was tapped on a solid surface to ensure that the agar was on the processed in a similar fashion using mixtures of 10 compounds
bottom of the wells. The plates were left at room temperature per well as previously described.25 Then, 2.5 µL of the 500-µM
to allow the agar to solidify before placing them into a 42 °C compound stock mixtures, solubilized in DSMO, were placed in
incubator to warm before dispensing of the cells. a Greiner 384-well polypropylene plate. Next, 100 µL of medium
Cells to be seeded within agar were dissociated with trypsin- was added to each well as described above and 10 µL of the com-
EDTA (Invitrogen) and suspended in medium. Cells were pound solution transferred to the soft agar assay plate to produce
counted, centrifuged, and the resulting cell pellet triturated in a final screening concentration of 2 µM. Hits from the mixed drug
medium and kept at 40 °C. Then, 8 mL of 5% agar, cooled to plates were confirmed at a 10-µM concentration as single com-
45 °C, was added to each 92 mL of cells/medium, producing a pounds before determination of an IC50.
0.4% agar suspension. In addition, 50 µL of cell/agar suspension
was quickly added to each well before the solution solidified Monolayer assay format
using a Matrix 12-channel pipettor (Hudson, NH). The plates
were again tapped on a solid surface to ensure that all the con- To determine the quality of the soft agar assay, results were
tents of the well settled to the bottom. The plates were kept at compared to a monolayer format that was previously performed
room temperature for at least 1 to 2 h to ensure the contents com- using CyQUANT NF (Invitrogen) as an endpoint. CyQUANT NF
pletely gelled. The plates were transferred into a Sanyo humidi- was not used in the soft agar format, for a direct comparison to the
fied incubator overnight before the addition of compounds. Cells monolayer format, because the cells could not be easily separated
were incubated with compounds typically for 7 days before from the agar. The monolayer assays were performed in Greiner
alamarBlue (Invitrogen) was used to quantitate cell viability in black-walled/clear-bottom 384-well Poly-D-Lysine-coated plates
each well. Then, 7 µL of alamarBlue was added to each well and and were performed similarly to the soft agar assay with the
allowed to incubate at 37 °C. Plates were read on an Envision exception of the culture time reduced to 3 from 7 days. The assay
plate reader (PerkinElmer, Shelton, CT) using excitation/emission was terminated by discarding the medium on the cells and adding
wavelengths of 531:615 nm. 25 µL of CyQUANT NF Cell Proliferation Assay reagent to each
Journal of Biomolecular Screening 12(7); 2007 www.sbsonline.org 939
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well for 1 h. The fluorescent intensity was measured using a A
ViewLux reader with an excitation wavelength of 480 nm and an
emission wavelength of 540 nm. 8.E+06
Fluorescent Intensity
7.E+06
6.E+06
5.E+06
Data analysis 4.E+06
3.E+06
IC50s were determined using GraphPad Prism software 2.E+06
1.E+06 Untreated
employing a nonlinear regression analysis. 0.E+00
Detergent-treated
100 300 1000 3000 5000 background
RESULTS AND DISCUSSION Number of Cells Seeded
Assay optimization
B
To establish a robust HTS assay for testing compounds in soft
agar assays, we determined the optimal screening conditions for 7.E+06
Fluorescent Intensity
6.E+06
HCC827 cells. Five different cell concentrations ranging from 100 5.E+06
to 5000 cells per well were seeded as described above and allowed 4.E+06
to grow for 7, 10, and 18 days in soft agar. Then, 10 µL of BRIJ35 3.E+06
2.E+06
solution (0.3% final concentration) was added to control wells 1.E+06 Untreated
within 24 h after seeding to disrupt all cells and served as a nega- 0.E+00
Detergent-treated
100 300 1000 3000 5000
tive control for cell growth. AlamarBlue was added to wells, and background
Number of Cells Seeded
the plates were read after 6 h of exposure. Figure 2 illustrates the
results demonstrating that 5000 cells produced the best signal. At
day 7, the signal after 6 h of alamarBlue exposure was 6.3 million C
fluorescent units (FUs) compared to 1.6 million FU in the control
detergent-treated wells. A signal-to-noise window of 4 and a Z′ 5.E+06
Fluorescent Intensity
factor of 0.5 were observed for these conditions. Ten days of incu- 4.E+06
bation resulted in a less robust response with a window of 3.7 and 3.E+06
Z′ factor of 0.2. Results on day 18 showed an increased window 2.E+06
after 6 h of alamarBlue exposure using 3000 cells per well but an 1.E+06
Untreated
unacceptable Z′ factor of less than 0. From these results, it was 0.E+00
100 300 1000 3000 Detergent-treated
determined that future experiments would incorporate 5000 cells background
Number of Cells Seeded
per well in the testing of compounds.
In the same experiment, the optimum time for incubation with FIG. 2. Determination of optimal culture time in soft agar. (A)
alamarBlue was also determined to optimize the signal. Figure 3 Seven-day culture, (B) 10-day culture, and (C) 18-day culture. n = 288
illustrates that on day 7, 6 h of exposure to alamarBlue gave nontreated wells and n = 48 detergent-treated wells for each cell
optimal results with a Z′ of 0.5. Lower incubation times with number indicated. Error bars represent standard deviation.
alamarBlue produced less than optimal results. Plates read on day
10, after cell seeding, were also exposed for longer time periods
with almarBlue to determine if the signal and Z′ factor could be cell soft agar assay. Figure 5 shows the concentration response
further increased. Longer times of exposure increased the window of HCC827 cells in soft agar to the control compounds both at
and the Z′, but the results indicated a depletion of alamarBlue by 6 and 24 h of exposure to alamarBlue. Similar IC50s were observed
24 h (results not shown), which may make the assay less sensitive. for both time points. As expected, staurosporine, Tarceva,
Thus, it was established from the above optimization assays that Zactima, and Iressa demonstrated nanomolar IC50 potencies
compounds would be tested by seeding 5000 cells per well and against HCC827 cells in soft agar, whereas lapatinib, a dual
incubated an additional 7 days after compound addition. inhibitor of the receptors ErbB1 and ErbB2,26 was less potent and
Sensitivity of the assay format to DMSO was also determined Gleevec, an abl kinase inhibitor,27 was inactive up to 10 µM.
by adding different concentrations of DMSO 24 h after seeding Tarceva, Zactima, and Iressa (inhibitors of epidermal growth factor
the cells. Figure 4 illustrates that DMSO concentrations below receptor [EGFR] tyrosine kinase) are known to be very potent
0.3% have no effect on the assay at either 7 or 10 days. inhibitors of cell proliferation,28-31 with Tarceva and Iressa having
reported nanomolar potency against HCC827 cell proliferation.32,33
Test compounds Staurosporine is a well-known broad-spectrum kinase inhibitor,
and it is known to be highly potent in cell proliferation assays.34
Five kinase inhibitors—lapatinib, Tarceva, Zactima, Iressa, Because the control compounds in this 384-well soft agar assay
Gleevec, and staurosporine—were chosen to validate the HCC827 produced results similar to literature values, a soft agar screen
940 www.sbsonline.org Journal of Biomolecular Screening 12(7); 2007
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A Time of Incubation w/ AlamarBlue Day 7 (5,000 cells)
125 A EC50
Staurosporine 4e-009
8.E+06 Z' = 0.5 100 Lapatinib
2e-007
Fluorescent Intensity
7.E+06
% Inhibition
75 Tarceva
6.E+06 Zactima 4e-009
Z' = 0.3 FU Totals
5.E+06 50 Iressa 5e-009
4.E+06
Z' = 0 FU Background
25 Gleevec 4e-010
3.E+06
(detergent) 0
2.E+06 -9 -8 -7 -6 -5
1.E+06 -25 [compound], LogM
0.E+00
0 2 4 6
Hours alamarBlue
EC50
125
B Staurosporine 8e-009
100 Lapatinib
Time of Incubation w/ AlamarBlue Day 10 (5,000 cells) 3e-007
B
% Inhibition
Tarceva
75 5e-009
Zactima
50 Iressa 1e-009
1.E+07
Z' = 0.64 25 Gleevec 1e-010
Fluorescent Intensity
1.E+07
Z' = 0.38 FU Totals 0
8.E+06 -9 -8 -7 -6 -5
Z' = 0.2 -25
6.E+06 Z' = 0 [compound], LogM
FU Background
4.E+06 (detergent)
FIG. 5. Dose response of control compounds in soft agar at 6 and 24 h
2.E+06
of exposure to alamarBlue. (A) Concentration response of control com-
0.E+00
0 2 4 6 8 24
pounds read after 6 h of alamarBlue exposure. (B) Concentration
Hours alamarBlue
response of control compounds read after 24 h of alamarBlue exposure.
Each compound was tested in duplicate on separate 384-well plates.
FIG. 3. Determination of optimal incubation time with alamarBlue at Error bars represent standard deviation.
(A) day 7 and (B) day 10. Calculation of Z′ based on n = 225 for non-
treated control and 42 wells of detergent-treated wells. Error bars repre-
sent standard deviation. FU, fluorescent units. against a random collection of 9600 compounds was performed,
and the results were compared to a standard screen using HCC827
cells in a monolayer format.
A Soft Agar vs. DMSO (5,000 cells) Day 7
120 Soft agar screening assay
100
Percent Inhibition
80 Approximately 9600 compounds were screened in a 10
60 mixed-drug format with each drug present in the well at a con-
40
centration of 2 µM. The scatterplot from the soft agar 6-h
20
alamarBlue time point (Fig. 6A) demonstrated a very high scat-
0
-20 10 5 3 1.250 0.625 0.300 ter, whereas the 24-h alamarBlue time point (Fig. 6B) demon-
Percent DMSO Concentration strated a more reasonable scatter, where potential hits could be
readily separated from the scatter. Therefore, the 24-h time point
was used to select hits for the primary screen because we previ-
B Soft Agar vs. DMSO (5,000 cells) Day 10 ously demonstrated no differences in the IC50 of control com-
pounds at the 2 alamarBlue exposure times. The monolayer assay
120 results shown in Figure 6C demonstrate a similar scatterplot
100
Percent Inhibition
80 compared to the soft agar results. Assay wells that contained hits
60 were picked as described in Figure 6, and single compounds were
40
20
retested at a 10-µM concentration, in duplicate. Figure 7 demon-
0 strates the reproducibility of the soft agar HTS assay. As expected,
-20 single compounds tested at 10 µM with potent IC50s demonstrate
-40 10 5 3 1.250 0.625 0.300
the best activity and best correlation.
Percent DMSO Concentration
Compounds demonstrating activity in either the soft agar or
FIG. 4. Determination of soft agar assay sensitivity to DMSO at
monolayer format, at 10 µM, were tested in a 6-point dose-response
(A) day 7 and (B) day 10 of culture. n = 288 nontreated wells, n = 48 assay using a 1:10 dilution scheme. Compounds were obtained
detergent-treated wells, and n = 4 for each DMSO concentration. Error from 5-mM HTS liquid library stocks, and dose responses were
bars represent standard deviation. FU, fluorescent units. started at either 10 or 30 µM. From the 9600-compound soft agar
Journal of Biomolecular Screening 12(7); 2007 www.sbsonline.org 941
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A 120
80
Percent Inhibition
40
0
-40
-80
-120
0 384 768 1152 1536 1920
Well Location
120
B
80
Percent Inhibition
40 FIG. 7. Correlation plot of 10-µM single-point retests on differ-
0 ent test plates demonstrating reproducibility of the soft agar
method. The x- and y-axes demonstrate the percent inhibition values
-40
from each plate, whereas the color represents the IC50 values. As
-80 expected, the most potent compounds, in the upper right quadrant,
-120
0 384 768 1152 1536 1920 have the best reproducibility.
Well Location
Table 1. Unique Hits from Monolayer Primary Screen
120
C Retested from Powders
80
Percent Inhibition
40 Monolayer Monolayer Soft Agar
0
Compound Original IC50 Powder IC50 Powder IC50
-40 1a 0.3 0.5 5
-80 2 0.3 0.5 0.5
-120 3 0.3 0.4 0.4
0 384 768 1152 1536 1920
4 0.5 2 2
5 0.5 0.8 0.8
Well Location
6a 0.6 0.8 8
7 0.7 0.8 0.5
FIG. 6. Scatterplot of primary soft agar assay at (A) 6 h and (B) 24
8 0.7 0.03 0.03
h of exposure to alamarBlue. Scatterplot of monolayer assay (C) using
9 0.8 5 10
CyQUANT endpoint. , 384-well plate scatter; , 384 wells selected
10 0.8 2 0.8
for retesting. 11 0.9 2 3
12 0.9 2 2
13a 1 1 4
screen, 64 compounds with IC50 activities of at least 10 µM 14 1 3 2
were discovered. Similarly, 75 compounds were discovered 15 1 1 1
16 1 10 10
with activity of at least 10 µM in the monolayer assay. Although 17 1 Inactive Inactive
there was a high hit rate, the majority of the hits (47 compounds) 18 1 10 10
were found in both assay formats. Due to the high hit rate in the 19 1 6 6
primary screen mixed-drug format, several compounds were 20 1 1 1
identified as active compounds in either the soft agar or mono- 21 1 0.6 2
22 1 1 3
layer format that were not selected as hits in the alternate format.
Therefore, to determine the validity of the soft agar and mono- a. Denotes compound at least 4 times more active in monolayer versus soft agar format.
layer formats, 35 compounds with IC50s of at least 1 µM were
ordered as powders from the Abbott Drug Repository that were
not selected as hits, in either the original soft agar (Table 1) or both formats with few exceptions. Graphs of the compounds
monolayer primary screens (Table 2) and retested as single com- that produced IC50s with greater than a 4-fold difference are pre-
pounds at a 10-µM concentration. As shown in Tables 1 and 2, all sented in Figure 8. Compounds 1, 6, and 13 were more active
of the compounds that were retested produced similar IC50s in in the monolayer format than the soft agar format, whereas
942 www.sbsonline.org Journal of Biomolecular Screening 12(7); 2007
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Table 2. Unique Hits from Soft Agar Primary Screen compound 32 was more active in the soft agar format. These
Retested as Powders results confirm the robustness of the soft agar HCC827 cell
assay, demonstrating similar if not almost equivalent results to an
Soft Agar Monolayer Soft Agar
Compound Original IC50 Powder IC50 Powder IC50
established monolayer screening format.
23 0.1 10 10 CONCLUSIONS
24 0.2 1 1
25 0.3 1 2 The soft agar assay method presented here can be used to
26 0.5 4 4 screen large numbers of compounds in a simple and robust
27 0.5 2 2 fashion. The assay had a good signal-to-noise ratio and a posi-
28 0.6 1 2 tive Z′ factor. The soft agar assay has an advantage over mono-
29 0.6 1 2
layer techniques measuring cell proliferation because it
30 0.7 0.3 0.9
31 0.8 3 1 employs cells cultured in a 3-D matrix. Thus, this format
32a 1 8 2 should be able to discover compounds that induce anoikis that
33 1 10 10 would be missed in a monolayer screening assay. Although no
34 1 0.4 0.4 compounds were discovered in this screening set that induced
35 1 6 6 anoikis and possessed significant activity in soft agar versus
a. Denotes compound at least 4 times more active in soft agar versus monolayer format. monolayer cultures, compounds inducing anoikis have been
Compound 1 Compound 6
125 Soft Agar IC50 = 10 µM 125 Soft Agar IC50 = 0.7
Monolayer IC50 = 0.5 µM Monolayer IC50 = 0.1
100 100
Percent Inhibition
Percent Inhibition
75 75
50 50
25 25
0 0
-25 -25
-1 0 1 -1 0 1
Compound Concentration (M)
Compound Concentration (log µM)
Compound 13 Compound 32
125 Soft Agar IC50 = 10 µM 125 Soft Agar IC50 = 3 µM
100 Monolayer IC50 = 1.4 µM Monolayer IC50 = 11 µM
Percent Inhibition
100
Percent Inhibition
75 75
50 50
25 25
0 0
-25 -25
-1 0 1 -1 0 1
Compound Concentration (M) Compound Concentration (M)
FIG. 8. IC50 determinations of 4 compounds demonstrating differences between monolayer and soft agar results. Each compound was tested in
duplicate on separate 384-well plates. Error bars represent standard deviation.
Journal of Biomolecular Screening 12(7); 2007 www.sbsonline.org 943
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