1. ASSESSING CANCER THERAPEUTIC AGENTS ACROSS A FIFTEEN HUMAN TUMOR CELL LINE PANEL
Ovechkina, Y.Y., Nguyen, P.T.B., Keyser, R.F., Shively, R.D., Marcoe, K.F. and O’Day, C.O.
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INTRODUCTION Lack of Correlation Between Cell Growth Rate and EC50 Cell Cycle Changes Precede Apoptosis Induction in OncoPanel Cell Lines Treated OncoPanel In Vitro Selectivity for EGFR and Bcr-Abl Inhibitors OncoPanel Anticancer Profiling Assay Continuous Culture Cells vs Cryo-preserved Cells
with Staurosporine or Taxol AG1478, EGFR inhibitor Bcr-Abl inhibitor Cell line, Continuous Culture HT29 K562 MCF-7 NCI H1299 NCI H23 NCI H82 RPMI 8226 SKMEL28 SW48 T47D
Growth rate vs EC50
The US National Cancer Institute (NCI) panel of 60 human tumor cell lines has Data Analysis For HCS 12 bit tiff images were acquired using the InCell Analyzer Cell Line
# Doublings in
72 hrs
Log (EC50)
microM 4 5
a) sensitive resistant
b) sensitive resistant
become a widely used resource for in vitro anticancer drug discovery. The 1000 3.2 and analyzed with Developer Toolbox 1.6 software. EC50 and IC50 values THP1 1.55 0.1 1.3
3 4 RPMI 8226 SW48
Etoposide, EC50 microM 1.72 0.12 0.38 1.17 0.21 0.27 0.02 2.01 0.05 0.06
average ratio
NCIH82 A431
average ratio
T47D 1.74 1.0
extensive profiling of these cell lines now includes information on cell line specific were calculated using nonlinear regression to fit data to a sigmoidal 4 point, 4 MCF7 1.93 0.8 1.1 2
3
MCF7
K562
NCIH23
A375
THP1
Doubling time, hrs 19 20 30 19 39 24 35 41 31 46
HCT116
cancer gene mutations that are known to render cells carrying them more sensitive parameter One-Site dose response model, where: y (fit) = A + [(B – A)/(1 + ((C/x) MDA MB 468 1.96 1.0 2 NCIH1299 NCIH1299
Log (EC50) microM
1 HT29 NCIH82
^ D))]. Curve-fitting and EC50 / IC50 calculations were performed using XLFit™ 0.9 A375 MDA MB 468
to chemotherapeutic agents. A powerful attribute of cell-based screening NCIH23
SKMEL28
2.16
2.19
0.4
1.0 0
1
THP1
SKMEL28
SKMEL28
T47D
Cell line, Cryo -preserved HT29 K562 MCF7 NCI H1299 NCI H23 NCI H82 RPMI 8226 SKMEL28 SW48 T47D
0
with multiple tumor cell lines is these cells demonstrate diverse sensitivities to software (IDBS) or MathIQ based software. RPMI 8226 2.31 0.5 0.7
24 hrs 72 hrs 24 hrs 72 hrs
NCIH23
T47D
MDA MB 468
MCF7
HT29
HCT116 Etoposide, EC50 microM 1.55 0.28 0.43 0.72 0.27 0.46 0.06 2.45 0.05 0.16
anticancer agents. The patterns of relative drug sensitivity and resistance found NCIH82
SW48 2.74
3.15
0.6
0.9 0.5 [mitosis] / [cell count EC50] [apoptosis] / [cell count EC50] [mitosis] / [cell count EC50] [apoptosis] / [cell count EC50]
SW48
A431
RPMI 8226
K562
with standard anticancer drugs across different tumor cell lines with defined Measured Parameters Cell proliferation was measured by the signal intensity
Doubling time, hrs 18 21 37 19 33 23 31 33 26 41
-1.00
-0.75
-0.50
-0.25
0.00
0.50
1.00
-0.50
0.25
0.75
-1.00
-0.75
-0.25
0.00
0.50
1.00
0.25
0.75
NCIH1299 3.27 0.9
onocogenic mutations have been shown to reflect mechanisms of drug action. A431 3.36 1.1
0.3 Staurosporine Taxol
of the incorporated nuclear dye. The cell proliferation output was referred to K562 3.39 1.3 ∆ log (average EC50 ) µM ∆ log (average EC50 ) µM The relative cell count EC50 (half maximal effective concentration) measures cell proliferation. Growth rate is
We have assembled a 15 human tumor cell line panel to examine mechanisms as the relative cell count. To determine the cell proliferation end point, the cell A375 3.45 0.9
0.1
Average concentration ratio for apoptosis induction to cell proliferation EC 50 in treated cells at 24 and 72 hr time points across all 15 reflected as the time required for the cell population to double once.
of cytotoxicity. The cell line panel was assembled with 5 common human tumor proliferation data output was transformed to percent of control (POC) using the HT29 4.07 0.3 1.6 3 4.4 OncoPanel cell lines is shown in red: [Apoptosis] / [Cell proliferation EC50]. Average concentration ratio for cell cycle change to cell
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
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-
types including breast, lung, colon, skin and leukemia. Cell lines were chosen based following formula:
HCT116 4.46 0.4
# Doublings in 72 hrs
CONCLUSIONS
eration EC50]. activated BCR-Abl mutation, demonstrated a ten-fold lower EC50 for cell proliferation inhibition and apoptosis induction response.
on available published mutation profiling data (Sanger Institute) and represent
the major mutations occurring in cancer genes. Standard cancer therapeutic The relative cell count EC50 (cell proliferation parameter) value was measured in 5-FU treated OncoPanel
Percent of Control = relative cell count (compound wells) x 100 cell lines. EC50 values did not correlate with cell growth rate (# Doublings/72hrs), correlation coefficient =
agents were tested for proliferative, apoptotic and cell cycle arrest responses More Sensitive Apoptosis Detection at the 72 Hour Time Point Methotrexate Treatment OncoPanel Breast Cancer Cell lines: T47D and MDA MB
relative cell count (vehicle wells) 0.003. The integration of mutation profiling data (Sanger Institute) with the cellular
using multiplexed high content screening (HCS) with automated fluorescence
468 Resistant and MCF-7 Sensitive response phenotypes generated with this multiparametric OncoPanel profiling
microscopy and image analysis based technology (GE Healthcare INCell Analyzer
assay allowed investigation of mechanisms of enhanced susceptibility to anti-
Relative cell count IC50 is the test compound concentration that produces 50% of the
[Apoptosis 24hrs] - [Apoptosis 72hrs],
1000). We generated cell line profiles to reveal drug sensitivity and resistance 0.50
Cell Cycle Changes Detected at 24 hrs in the Absence of Growth Inhibition Are cancer agents. Biomarkers for cell proliferation and cell death coupled with
patterns using multiplexed cellular response phenotypes. The combination of cell proliferation inhibitory response or 50% cytotoxicity level. A relative cell count sensitive resistant sensitive resistant
Predictive of 72 Hour Toxicity. 0.25 genomic information linking the sensitivity/resistance of cell lines to activating or
mutation profiling data with cellular response phenotypes allowed investigation EC50 is the test compound concentration that produces 50% of the maximum effective
SKMEL28 T47D
T47D MDA MB 468
RPMI 8226 SKMEL28
inhibiting mutations in oncogenes permit screening for sensitivity and selectivity
of mechanisms of enhanced susceptibility to anti-cancer agents. Time course response that occurs at the curve inflection point. The output of each biomarker is A431
microM
RPMI 8226
0.00 NCIH23 A431
A375 NCIH23
of a compound towards different mutations within similar cancers. Methotrexate
determinations for growth inhibition, apoptosis and cell cycle arrest detection were fold increase over vehicle background normalized to the relative cell count in each Cell Proliferation Apoptosis Cell Cycle