oncopanel poster aacr 2008
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OncoPanel Cell Line Gene Mutation Chart*
ASSESSING CANCER THERAPEUTIC AGENTS ACROSS A FIFTEEN HUMAN TUMOR CELL LINE PANELOvechkina, Y.Y., Nguyen, P.T.B., Keyser, R.F., Shively, R.D., Marcoe, K.F. and O’Day, C.O.
MDS Pharma Services
INTRODUCTIONThe US National Cancer Institute (NCI) panel of 60 human tumor cell lines has become a widely used resource for in vitro anticancer drug discovery. The extensive profiling of these cell lines now includes information on cell line specific cancer gene mutations that are known to render cells carrying them more sensitive to chemotherapeutic agents. A powerful attribute of cell-based screening with multiple tumor cell lines is these cells demonstrate diverse sensitivities to anticancer agents. The patterns of relative drug sensitivity and resistance found with standard anticancer drugs across different tumor cell lines with defined onocogenic mutations have been shown to reflect mechanisms of drug action. We have assembled a 15 human tumor cell line panel to examine mechanisms of cytotoxicity. The cell line panel was assembled with 5 common human tumor types including breast, lung, colon, skin and leukemia. Cell lines were chosen based on available published mutation profiling data (Sanger Institute) and represent the major mutations occurring in cancer genes. Standard cancer therapeutic agents were tested for proliferative, apoptotic and cell cycle arrest responses using multiplexed high content screening (HCS) with automated fluorescence microscopy and image analysis based technology (GE Healthcare INCell Analyzer 1000). We generated cell line profiles to reveal drug sensitivity and resistance patterns using multiplexed cellular response phenotypes. The combination of mutation profiling data with cellular response phenotypes allowed investigation of mechanisms of enhanced susceptibility to anti-cancer agents. Time course determinations for growth inhibition, apoptosis and cell cycle arrest detection were evaluated for all fifteen tumor cell lines. To determine the feasibility of using cryo-preserved cells as ready-to-use reagents for routine screening, cellular responses from continuous culture and cryo-preserved cells in regards to growth inhibition, number of doublings, apoptosis and cell cycle arrest were compared for the 15 tumor cell lines included in this panel. This automated multiplexed cell image based technology coupled with genetic analysis offers a robust and sensitive approach to assessing mechanism of action and sensitivity to chemotherapeutics agents. Further the use of cryo-preserved cells enables this cost-effective multiplexed OncoPanel screening platform to be conducted with enhanced efficiency.
METHODSCell culture Breast cancer cell lines MCF7, MDA MB 468, T47D, colon cancer cell lines HT-29, HCT-116, SW48, lymphoid cancer cell lines K562, RPMI 8226, THP1, skin cancer cell lines SK-MEL-28, A375, A431 and lung cancer cell lines NCI-H23, NCI-H1299, and NCI-H82 were grown in RPMI1640, 10% FBS, 1% L-Alanyl-L-Glutamine and 1% sodium pyruvate in tissue culture flasks in a humidified atmosphere of 5% CO2 at 37oC. Working stocks of log-phase cells were cryo-preserved using a standard slow cooling in 10% DMSO protocol. For plating continuous culture cells the cells were thawed from working stocks and passed once prior to seeding into plates. For cryo-preserved studies the cells were thawed, washed in media without DMSO, and then seeded into 384-well plates.
OncoPanel Anticancer Profiling Assay Caspase 3 activation (a marker of apoptosis), phospho-histone-3 (a marker of mitosis), and relative cell count (an index of cell proliferation) were measured. Each cell line was seeded at an experimentally determined optimal density with complete growth media into 384-well plates and incubated in a humidified atmosphere of 5%CO2 at 37oC. Test compounds were serially diluted 3-fold over 10 concentrations and added 24 hours post cell seeding with a final assay concentration of 0.002% DMSO. Following an additional 24 or 72 hour incubation in the humidified atmosphere of 5%CO2 at 37oC, cells were fixed and immunolabeled with anti-active caspase-3 for detection of apoptosis, anti-phospho-histone-3 for detection of cell cycle and stained with nuclei dye for cell proliferation quantification. Twenty eight standard cancer therapeutic agents were tested. A non-contact dispensing automation system accommodated cell plating and reagent additions (Thermo Combidrops or Multidrops) and test compound additions (Labcyte ECHO-550). Automated fluorescence microscopy was carried out using a GE Healthcare INCell Analyzer 1000, and images were collected with a 4X objective.
Data Analysis For HCS 12 bit tiff images were acquired using the InCell Analyzer 1000 3.2 and analyzed with Developer Toolbox 1.6 software. EC50 and IC50 values were calculated using nonlinear regression to fit data to a sigmoidal 4 point, 4 parameter One-Site dose response model, where: y (fit) = A + [(B – A)/(1 + ((C/x) ^ D))]. Curve-fitting and EC50 / IC50 calculations were performed using XLFit™ software (IDBS) or MathIQ based software.
Measured Parameters Cell proliferation was measured by the signal intensity of the incorporated nuclear dye. The cell proliferation output was referred to as the relative cell count. To determine the cell proliferation end point, the cell proliferation data output was transformed to percent of control (POC) using the following formula:
Percent of Control = relative cell count (compound wells) x 100 relative cell count (vehicle wells)
Relative cell count IC50 is the test compound concentration that produces 50% of the cell proliferation inhibitory response or 50% cytotoxicity level. A relative cell count EC50 is the test compound concentration that produces 50% of the maximum effective response that occurs at the curve inflection point. The output of each biomarker is fold increase over vehicle background normalized to the relative cell count in each well. Concentrations of test compound that cause a 5 or more fold induction in the caspase-3 signal were determined to induce significant apoptotic induction. When the fold induction of the phospho-histone-3 signal over background is ~1, there was “no effect” on the cell cycle. Two or more fold increase in phospho-histone-3 signal over vehicle background indicated significant test compound induction of mitotic block. Two or more fold decrease in the phospho-histone-3 signal may indicate G1/S block only when cytotoxicity levels are below the measured relative cell count IC80. When 2 or more fold decrease in the phospho-histone-3 signal are observed at concentrations higher than the relative cell count IC80, the decrease in mitotic cell counts are most likely due to a more general cytotoxicity effect rather than a true G1/S phase block. Wells with concentrations higher than the relative cell count nuclear IC80 are eliminated from the phospho-histone-3 analysis.
OncoPanel Anticancer Profiling Assay Continuous Culture Cells vs Cryo-preserved Cells
The relative cell count EC50 (half maximal effective concentration) measures cell proliferation. Growth rate is reflected as the time required for the cell population to double once.
41263331233319372118Doubling time, hrs
0.160.052.450.060.460.270.720.430.281.55Etoposide EC50, microM
T47DSW48SKMEL28 RPMI 8226 NCI H82 NCI H23 NCI H1299 MCF7 K562 HT29Cell line, Cryo -preserved
46314135243919302019Doubling time, hrs
0.060.052.010.020.270.211.170.380.121.72Etoposide EC50, microM
T47DSW48SKMEL28 RPMI 8226 NCI H82NCI H23NCI H1299 MCF-7K562 HT29Cell line, Continuous Culture
ONCOPANEL ANTICANCER PROFILING ASSAY
Dat
a A
cqu
isit
ion
/ A
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ysis
Ass
ay P
roce
du
r e Seed Cells in 384-well plate
Tipless Compound Addition (ECHO-550)
24 hrs
24 – 72 hrs
Cell Fixation
Read plates on the InCell 1000 Analyzer
(30 min / plate,60 plates – 20 hrs)
InCell Developer Software Analysis
(60 plates – 10 hrs)
AIM curve-fitting analysis (MathIQbased software)
Automated Report Generation
Immuno-label and Stain Cells(60 plates – 4 hrs)
Multiplexed cell proliferation, apoptosis and cell cycle image data
Representative images are shown for T47D cell line. Labels: Nuclei - blue; Apoptotic cells - green; Mitotic cells - red
Vehicle Taxol Staurosporine
Lack of Correlation Between Cell Growth Rate and EC50
# Doublings in 72 hrs
1.6 3 4.4
Lo
g (
EC
50)
mic
roM
0.1
0.3
0.5
0.7
0.9
1.1
1.3
Growth rate vs EC50
0.44.46HCT116
0.34.07HT29
0.93.45A375
1.33.39K562
1.13.36A431
0.93.27NCIH1299
0.93.15NCIH82
0.62.74SW48
0.52.31RPMI 8226
1.02.19SKMEL28
0.42.16NCIH23
1.01.96MDA MB 468
0.81.93MCF7
1.01.74T47D
0.11.55THP1
Log (EC50)microM
# Doublings in 72 hrs
Cell Line
The relative cell count EC50 (cell proliferation parameter) value was measured in 5-FU treated OncoPanelcell lines. EC50 values did not correlate with cell growth rate (# Doublings/72hrs), correlation coefficient = 0.003.
Cell Cycle Changes Detected at 24 hrs in the Absence of Growth Inhibition Are
Predictive of 72 Hour Toxicity.
Cell CycleApoptosisCell Proliferation
24 h
rs
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hrs
OncoPanel Anticancer Profiling Assay, representative curves at 24 and 72 hrs for cell proliferation, apoptosis induction, and cell cycle block induced with staurosporine in the T47D cell line and Taxol in the NCI H82 cell line are shown.
A summary of OncoPanel Anticancer Profiling parameters at 24 and 72 hrs in
T47D and NCI H82 cell lines
Cell Cycle Changes Precede Apoptosis Induction in OncoPanel Cell Lines Treated
with Staurosporine or Taxol
CompoundTime, hrs
Relative cell count IC50
(microM)
Relative cell count EC50
(microM)
Apoptosis induction (microM)
Mitosis cell cycle block
(microM)
Inhibition of mitosis (G1/S cell cycle block) (microM)
Staurosporine 24 > 3 > 3 − − 0.03Staurosporine 72 0.05 0.04 0.31 − 0.02
Taxol 24 > 0.3 0.011 0.009 0.012 −Taxol 72 0.002 0.002 0.002 0.002 −
The relative cell count IC50 (half maximal inhibitory concentration) and EC50(half maximal effective concentration) values measure cell proliferation. Compound concentrations are indicative of a 5-fold apoptosis induction inactivated caspase-3 signal and a 2-fold change in phospho-histone3 signal (mitosis marker) over vehicle back-ground.
Staurosporine Taxol
Average concentration ratio for apoptosis induction to cell proliferation EC50
in treated cells at 24 and 72 hr time points across all 15 OncoPanel cell lines is shown in red: [Apoptosis] / [Cell proliferation EC
50]. Average concentration ratio for cell cycle change to cell
proliferation EC50
in treated cells at 24 and 72 hr time points across all 15 OncoPanel cell lines is shown in blue: [Mitosis] / [Cell prolif-eration EC
50].
0
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4
24 hrs 72 hrs
[mitosis] / [cell count EC50
] [apoptosis] / [cell count EC50
]
aver
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[mitosis] / [cell count EC50
] [apoptosis] / [cell count EC50
]
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U0126, MEK Inhibitor, Selectively Inhibits the Growth of BRAF Mutant Cell Lines
OncoPanel In Vitro Selectivity for EGFR and Bcr-Abl Inhibitors
*Sanger Institute, http://www.sanger.ac.uk/genetics/CGP/CellLines/
Cell Lines Organ BRC-ABL CDKN2A PDGFRA TP53 EGFR KRAS NRAS ER+ PIK3CA RB1 MADH4 PTEN STK II bRAF BRCA2 CTNNB1 APC
MDA MB 468 Breast XXXX
MCF-7 Breast XXX
T47D Breast XXX
HT-29 Colon XXXXX
SW48 Colon XX X
HCT-116 Colon XXXXX
RPMI 8226 Lymphoid X X X
K562 Lymphoid X X X X
THP1 Lymphoid XXX
NCI-H1299 Lung X
NCI-H23 Lung X X
NCI-H82 Lung X
SK-MEL-28 Skin X X X
A375 Skin X X
A431 Skin X
RAS*
RAF*
ERK1/2
MSK1 RSK
MEK1
MEK2
MNK2ELK-1 STAT3
MNK1
C-M
YC
C-M
YC
Transcription/Proliferation
MEK inhibition; U0126
GTP
GDPRAS*
RAF*
ERK1/2
MSK1 RSK
MEK1
MEK2
MNK2ELK-1 STAT3
MNK1
C-M
YC
C-M
YC
Transcription/Proliferation
MEK inhibition; U0126
GTP
GDP
Activated BRAF mutants increase sensitivity to U0126 induced MEK inhibition in SK-MEL-28, A375 and HT29 cell lines. BRAF mutation has been shown to predict sensitivity to MEK inhibition in melanoma cell lines (D. B. Solitet al.). Developmentof MEK inhibitors for treatment of melanoma has resulted from this in vitro selectivity translating to in vivo xenograph efficacy.
-1.0
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SW48A375HT29
SKMEL28THP1
HCT116NCIH1299
MCF7A431K562
MDA MB 468NCIH23
T47DNCIH82
RPMI 8226
? log (average EC50) µM
UO126, MEK inhibitorsensitive resistant
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HCT116HT29
MCF7T47D
SKMEL28MDA MB 468
NCIH82NCIH1299
THP1A375
NCIH23A431
SW48
∆ log (average EC50) µM
Bcr-Abl inhibitorsensitive resistant
a) A431 cell line, known to over express the EGF receptor, showed the greatest sensitivity to AG 1478. b) K562 cell line, known to have activated BCR-Abl mutation, demonstrated a ten-fold lower EC
50 for cell proliferation inhibition and apoptosis induction response.
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MDA MB 468T47D
NCIH23SKMEL28
THP1A375HT29
NCIH1299HCT116
K562MCF7
NCIH82RPMI 8226
∆ log (average EC50) µM
AG1478, EGFR inhibitorsensitive resistanta) b)
Methotrexate Treatment OncoPanel Breast Cancer Cell lines: T47D and MDA MB
468 Resistant and MCF-7 Sensitive
CONCLUSIONSThe integration of mutation profiling data (Sanger Institute) with the cellular response phenotypes generated with this multiparametric OncoPanel profiling assay allowed investigation of mechanisms of enhanced susceptibility to anti-cancer agents. Biomarkers for cell proliferation and cell death coupled with genomic information linking the sensitivity/resistance of cell lines to activating or inhibiting mutations in oncogenes permit screening for sensitivity and selectivity of a compound towards different mutations within similar cancers. Methotrexate treated breast cancer cell lines T47D and MDA MB 468 were resistant to treatment while MCF-7 demonstrated significant sensitivity. Highly selective inhibitors for specific oncogene mutations demonstrated greater cell proliferation inhibition and apoptosis induction in the tumor cell lines in the panel possessing the targeted oncogene. Development of melanoma treatments have been based on the known selective sensitivity to MEK inhibition in BRAF mutant cells. Melanoma cell lines SK-MEL-28 and A375 demonstrated an enhanced sensitivity to U0126, a selective inhibitor of MEK in BRAF mutant cells. The cell line A431, known to over express EGF receptors, demonstrated the greatest sensitivity to EGFR inhibitor treatment. K562 cells contain a Philadelphia chromosome which forms due to translocation between chromosomes 22 and 9. This translocation leads to fusion of the Bcr and Abl genes to generate a chimeric oncogene, bcr-abl, which causes chronic myelogenous leukemia. Bcr-Abl inhibitor treatment led to a ten fold higher cell proliferation inhibition and apoptosis induction in the K562 cell line than the other oncopanel cell lines. The high selectivity of the Bcr-Abl inhibitor for the K562 cancer cell line growth inhibition could be directly attributed to inhibition of the activated oncogene product found in this particular cell line. Time course determinations for the multiparametric OncoPanel profiling assay showed that cell cycle detection with phospho-histone-3 was an early indicator of cell arrest with similar 24 and 72 hour endpoints. Where cell proliferation was not inhibited during the 24 hours of compound exposure, detection of cell cycle block and/or apoptosis with activated caspase-3 were indicative of potential cell proliferation inhibition at the 72 hour assay endpoint. The doubling time varied among the oncopanel cell lines, however, no correlation was found between inherent growth rate and the cell proliferation measurement. For all 15 human tumor cell lines included in the OncoPanel profiling assay the EC50 values for cell proliferation and growth rate in cryo-preserved cells highly correlated with values generated in continuous culture cells. The use of cryo-preserved cells enhances the efficiency of this cost-effective automated multiplexed OncoPanel screening platform in assessing mechanism of action and sensitivity to chemotherapeutic agents. Drug combination analysis using this OncoPanel profiling assay to determine synergistic activity of compound cocktails could provide pharmacologically relevant data for improving cancer treatment efficacy.
REFERENCESSanger Institute, http:/www.sanger.ac.uk/genetics/CGP/CellLines/Rodrigues RA, Alfonso J, O,Day C, Ovechkina Y, Ward C. 297 Distinct Cell Lines: A High-Content Analysis Assay and a Full Automation Design Solely Using Noncontact Liquid Dispensing. Journal of the Association for Laboratory Automation. 2007;12(5):318-326.Shoemaker RH. The NCI60 human tumour cell line anticancer drug screen. Nature Reviews. 2006;6:813-823.Ikediobi ON, Davies H, Bignell G, et al. Mutation analysis of 24 known cancer genes in the NCI-60 cell line set. Mol Cancer Ther, 2006;5(11):2606-2612.Huang R, Wallqvist A, Covell DG. Assessment of in vitro and in vivo activities in the National Cancer Institute’s anticancer screen with respect to chemical structure, target specificity, and mechanism of action. J Med. Chem., 2006;49:1964-1979.Solit DB, et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature, 2006;439:358-362.
More Sensitive Apoptosis Detection at the 72 Hour Time Point
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SKMEL28
HCT116
NCIH23
RPMI 8226
MDA M
B 468
A431NCIH1299
HT29A375
NCIH82
MCF7
K562T47D
THP1SW
48
[Ap
op
tosi
s24
hrs
]-
[Ap
op
tosi
s72
hrs
],m
icro
M
Apoptosis inducing concentration at 24 hrs subtracted from apoptosis inducing concentration at 72 hrs for staurosporine treated OncoPanelcell lines are shown. Positive values indicate apoptosis induction is more sensitive at the 72 hour time point for all cell lines tested.
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140 T47D
EC50= 8.49 nMIC50> 30 µM
[Methotrexate], M
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140 MDA MB 468
EC50= 26.3 nMIC50> 30µM
[Methotrexate], M
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40
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140 MCF-7
EC50= 16.4 nMIC50= 19.8 nM
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SW48THP1
NCIH82MCF7HT29
NCIH1299MDA MB 468
A375NCIH23
A431RPMI 8226
T47DSKMEL28
Δ log (average EC50) µM
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HCT116SW48K562THP1
NCIH82MCF7HT29
NCIH1299A375
NCIH23A431
RPMI 8226SKMEL28
MDA MB 468T47D
Δ log (average IC50) µM
sensitive resistant sensitive resistant
The relative cell count IC50
(half maximal inhibitory concentration) and EC50
(half maximal effective concentration) values measure cell proliferation. Methotrexate, a folic acid analog and a potent inhibitor of the enzyme DHFR, is commonly used for treatment of breast cancer. Several mechanisms associated with increased resistance to methotrexate have been identified, such as, diminished drug uptake, expression of an altered DHFR enzyme with a reduced affinity for methotrexate, and increased levels of the target enzyme, DHFR, and conversion of methotrexate to polyglutamates.
Rapamycin Treatment OncoPanel Cell Lines: Breast Cancer (MCF-7) and Myeloma
Cancer (RPMI 8226) Most Sensitive
Inhibitors of the PI3K/Akt/mammalian target of the rapamycin (mTOR) pathway are promising therapeutics for
5-fluorouracil (5-FU) is an effective treatment for non-melanoma skin cancer. A431, derived from non-melanoma epidermoid carcinoma, showed the greatest sensitivity to 5-FU treatment.
breast and myeloma cancer.
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NCIH23K562
NCIH1299HT29
HCT116A431A375
NCIH82SKMEL28
THP1SW48
∆log (average EC 50) µM
sensitive resistant
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A375A431
HCT116HT29K562
MDA MB 468NCIH1299
NCIH23NCIH82
SKMEL28SW48T47DTHP1
∆log (average IC 50) µM
sensitive resistant
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A431RPMI 8226
THP1HT29
NCIH23HCT116
SW48MCF7A375
NCIH1299MDA MB 468
T47DSKMEL28
NCIH82K562
∆log (average EC 50) µM
sensitive resistant
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A431RPMI 8226
THP1HT29
HCT116SW48
NCIH23A375
NCIH1299MCF7
NCIH82SKMEL28
K562MDA MB 468
T47D
∆log (average IC 50) µM
sensitive resistant
5-FU Treatment OncoPanel Cell Lines: Non-Melanoma Skin Cancer (A431) Most
Sensitive