Analysis of Drug Resistant Properties of A2780 Ovarian ...€¦ · - Confluence: Segmentation...
Transcript of Analysis of Drug Resistant Properties of A2780 Ovarian ...€¦ · - Confluence: Segmentation...
Analysis of Drug Resistant Properties of A2780 Ovarian Cancer Cell Lines
Using Label-Free Automated Microscopy (IncuCyte Zoom)
Authors Andrew Bashford and Jim Cooper
Scientific Development Group
Culture Collections
National Infection Service
Public Health England
Abstract
The A2780 ovarian cancer cell line and its reported cisplatin and Adriamycin (generic name:
doxorubicin) drug resistant derivatives (A2780cis and A2780ADR) have been available for
over 20 years and offer researchers the potential to study drug resistance in the field of
cancer therapeutic research. The cell lines were quantitatively characterised for their
resistant properties soon after derivation by various technically demanding and inconsistent
methods, where relative IC50 values were reported as a measure of resistance. We
hypothesised that despite several decades of cell banking and storage, the A2780cis and
A2780ADR cell lines had retained their drug resistant characteristics as compared to the
parental cell line and by utilising novel automated microscopy and image analysis software,
we could quantify these properties using a label free kinetic assay and end point nuclear
fluorescence staining. Our results qualitatively and quantitatively demonstrated drug
resistance in the A2780cis and A2780ADR cells and the IC50 values derived using both the
kinetic assay and nuclear staining were consistent with previously published data derived
using alternative methodologies. This study has independently verified that the cell lines
retain the drug resistance properties as reported decades previously. We believe this novel
approach using an automated, high throughput format allows the rapid and robust
quantification of the drug resistant properties of cell lines and will be of use to researchers in
the field working with these and other cancer derived cells.
Introduction
The ovarian cancer cell line A2780 (ECACC 93112519) and its drug resistant derivatives
(A2780cis, ECACC 93112517 and A2780ADR, ECACC 93112520) are widely used tools for
cancer research and have been in general use for over 20 years. The resistant cell lines
were generated by monolayer culture of A2780 cells in increasing concentrations of
Adriamycin (generic name: doxorubicin) (Behrens et al. 1984) or cis-diammineplatinum(II)
dichloride (cisplatin) (Behrens et al. 1987). Together these cell lines represent an excellent
means to study cancer biology, resistance to chemotherapeutic agents and enable the
search for new therapeutic agents.
Drug resistance in cell lines is best compared using IC50 values; the dose of a drug which
causes a 50% reduction in cell viability. A number of different methodologies have been
employed to determine this value. These include the use of agar-based clonogenic assays
(Puck and Marcus 1956), a modified version: the human tumour stem cell assay (HTSCA)
(Hamburger and Salmon 1977), the resazurin assay for cell viability (Tardito et al. 2009),
ATP assays for cell viability (Fan et al. 2004), HTSCA combined with thymidine incorporation
(Friedman and Glaubiger 1982), HTSCA combined with the ATP assay (Sevin et al. 1988),
MTT cell proliferation assay (Yamaue et al. 1996) and dye exclusion methods (Weisenthal et
al. 1983). These approaches have different durations of growth, are labour intensive and can
often yield different experimental results.
The European Collection of Authenticated Cell Cultures (ECACC) and its official distributors
are the exclusive suppliers of A2780, A2780cis and A2780ADR cell cultures. Here we report
a modern approach using the automated IncuCyte ZOOM system and a label-free
assessment of IC50 values.
Methods
Authenticated (Short Tandem Repeat (STR) profiled) cell lines were provided from ECACC
and grown in accordance with the recommended procedures. In short, cells were grown in
RPMI-1640 media with 2mM Glutamine (R8758, Sigma Aldrich) and 10% FBS (Sigma
Aldrich). Initial cell expansion was performed in T175 flasks (431466, Corning) seeding at
densities of 5x104 cell/cm2 (A2780) or 3x104 cell/cm2
(A2780cis, A2780ADR). At the first
passage cells were washed with PBS, treated with 0.05% Trypsin-EDTA (Gibco®) and 100μL
of cells seeded into black walled cell bind 96 well plates (3340, Corning) at densities of
3x104 cell/cm2 (A2780) and 1x104 cell/cm2 (A2780cis, A2780ADR). For each cell type thirty-
two wells were seeded per ninety-six well plate and two plates were seeded in total.
Drug dilutions were added to cells immediately after they were plated. Cisplatin
(P4394, Sigma) was dissolved in PBS to make a 5mM working stock. This was serially
diluted using culture media and 100μL added to cells to make final concentrations of:
Fig 1) A2780 cells and its drug resistant clones; A2780cis, A2780ADR. STR profiles demonstrating cell line identity for a) A2780, b) A2780cis and c) A2780ADR. Representative images showing 0 hr time point for untreated cells d) A2780, e) A2780cis and f) A2780ADR. 72 hr time point for untreated cells g) A2780, h) A2780cis and i) A2780ADR. Scale bar: 70μm
500μM, 100μM, 10μM, 1μM, 0.1μM, 0.01μM, 0.001μM. For each cell type a total of 8 wells
(4 per plate) were treated with each concentration. Adriamycin (44583, Sigma) was
dissolved in DMSO to make a 10mg/ml stock. This was serially diluted using culture media
and 100μL added to cells to make final concentrations of: 150μM, 100μM, 10μM, 1μM,
0.1μM, 0.01μM, 0.001μM. For each cell type a total of 6 wells (3 per plate) were treated with
each concentration of Adriamycin. DMSO was also serially diluted using culture media to act
as a vehicle control for Adriamycin treatments; 100μL was added to cells to make final
concentrations of: 0.85%, 0.57%, 0.057%, 0.0057%, 0.00057%, 0.000057%, 0.0000057%.
For each cell type a total of 2 wells (1 per plate) were treated with each concentration of
DMSO.
Plates were incubated (37°C, 5% CO2) in the IncuCyte ZOOM (Essen BioScience, Ann
Arbor, Michigan, US) and phase images taken every two hours using the 20x objective lens.
Nuclear stain Syto16 (S7578, Life Technologies) was diluted in PBS to make 5μM stock.
After 120 hours wells were washed with PBS and Syto16 added. Plates were returned to the
IncuCyte ZOOM, incubated for 20 minutes and re-imaged using both phase and green
fluorescence channels. Data were analysed using the IncuCyte ZOOM software and
processing definitions modified to quantify confluence and cell number. Processing definition
were set as follows:
- Confluence: Segmentation Adjustment of 0.1, hole fill 0.00μm2, adjust size -2 pixels.
Filters: Area min 100μm2.
- Cell count (main): Parameters: Top-Hat threshold adjustment with radius of 10μm
and threshold = 2.000, edge split on with sensitivity = 0. Filters: area min 60.00μm2,
mean intensity min 25.00.
- Cell count (low, high debris): Parameters: Adaptive threshold adjustment with
threshold = 5.000. Edge split on with sensitivity = 11, hole fill 0.00μm2 and adjust size
= -1 pixels. Filters: area min 90.00μm2, area max 900.00μm2, eccentricity max 0.850.
Accuracy of processing definitions was qualitatively assessed to determine whether main or
low cell count processing definitions where appropriate.
GraphPad Prism 7 was used to plot drug concentration against confluence or cell number
and fit non-linear regression to determine the IC50 values. To test the effect of DMSO, values
obtained from Adriamycin treated wells were normalised using values obtained from
corresponding DMSO concentrations. No difference was seen in IC50 values calculated
using normalized or raw values.
Fig 2) The IncuCyte ZOOM is able to accurately quantify confluence and nuclear count. Representative images from A2780 cells after 120 hr showing a) phase data, b) fluorescence of nuclear stain Syto16. The IncuCyte ZOOM trained processing definition to quantify c) percentage confluence and d) nuclear count as number per mm
2.
Scale bar: 70μm
Results
All human cells supplied by ECACC undergo Short Tandem Repeat (STR) profiling to
ensure their correct identity (Fig 1a-c). To independently verify the drug sensitivities of
A2780, A2780cis and A2780ADR, cells were cultured using the IncuCyte ZOOM, an
automated live-cell analysis system which accurately quantifies confluence and cell number
which prompted us to test its use as a novel, label-free method for determining IC50 values.
Cells were grown in serial dilutions of cisplatin or Adriamycin and imaged every two hours
(Fig 1d-f). Untreated cells reached mid log phase after approximately 72 hours (Fig 1g-i) and
they showed similar morphology between the different cell types. Nuclear dye Syto16 was
added to the cells at the end of the experiment (120 hours) and cells imaged (Fig 2a,b). The
IncuCyte ZOOM created processing definitions which were modified to quantify cell
confluence, based on phase images, or cell number, based on the fluorescent nuclear stain
(Fig 2c,d). The two drugs caused different cellular effects at high concentrations, with
Adriamycin producing a greater amount of cellular debris. This resulted in the needs for two
different processing definitions in order to obtain accurate count cells; one to accommodate
the low cell number with high debris (low, high debris) and one to accommodate high cell
number with low debris (main).
IC50 values were calculated using cell confluence (72 hours, Fig 3) and cell count (120
hours, Fig 4) representing the mid-log phase and end-point of the experiment respectively
(summarised in Table1). Both methods of IC50 calculation produced very similar values for
each cell type, demonstrating equivalence between the two techniques. When treated with
cisplatin, the A2780 cells had a range of IC50 values between 0.4-0.7μM. The A2780cis cells
showed a ten-fold resistance to cisplatin with an IC50 range between 7-8μM and the
A2780ADR showed a slight resistance with IC50 values of 1μM. When treated with
Adriamycin, the A2780 cells had an IC50 range between 0.004-0.007μM. In comparison the
A2780ADR cells had an IC50 range between 0.06-0.9μM, again showing over ten-fold
resistance and the A2780cis cells had an IC50 range between 0.01-0.02μM. These results
are strikingly similar with those published over 20 years ago and generated using different
experimental techniques (Table 1).
Fig 3) Confluence-based IC50 values show A2780cis and A2780ADR have higher drug resistance than parental A2780 cells. Graphs used to calculate IC50 values from cell confluence data at 72 hours. Cells treated with cisplatin (a-c) or Adriamycin (d-f). a, d) A2780; b, e) A2780cis and c, f) A2780ADR. GraphPad Prism was used to fit non-linear regression to the data and calculate IC50 values. Error bars are SEM generated from 3 (Adriamycin) or 4 (cisplatin) technical replicates at each drug concentration.
Fig 4) Cell number-based IC50 values show A2780cis and A2780ADR have higher drug resistance than parental A2780 cells. Graphs used to calculate IC50 values from cell number data at 120 hours. Cells treated with cisplatin (a-c) or Adriamycin (d-f). a, d) A2780; b, e) A2780cis and c, f) A2780ADR. GraphPad Prism was used to fit non-linear regression to the data and calculate IC50 values. Error bars are SEM generated from 6 (Adriamycin) or 8 (cisplatin) technical replicates at each drug concentration.
Discussion
In this report we have demonstrated that A2780cis and A2780ADR are ten-fold resistant to
their respective therapeutic agents. These results are consistent with the originally published
data describing these cell lines (Behrens et al. 1984; Behrens et al. 1987).
The use of the IncuCyte ZOOM enabled the automation of imaging and data processing.
The label-free approach relied on the assumption that the reduced cell division and growth
caused by a drug resulted in fewer cells at a given time point, either through reduced
proliferation or increased cell death, ultimately leading to a lower confluence. In these
analyses an important consideration is that changes in cell morphology can also affect
confluence measurements. Importantly this did not appear to be a significant factor in this
study as demonstrated by the similarity in data distribution generated using the two methods
(compare Fig3 & Fig4). The cell count is not without its limitations, however, due to the
addition of a nuclear label it was only completed at a single time point and the debris created
by cell death required two processing definitions to accurately map cell number. Qualitative
inspection of the fluorescence-based cell counting map, when overlaid on the phase images,
suggested a small number of miscounted cells. This was consistent throughout the dataset
and could not be improved through modification of the processing definitions. Despite the
caveats of confluence and cell count methodologies both produced strikingly similar IC50
values. This agreement between the two approaches strengthens the validity of the
experimental methods.
The initial publications describing the development of A2780cis and A2780ADR used agar
based clonogenic assays that took between 14 and 21 days (Behrens et al. 1987; Behrens
et al. 1984; Louie et al. 1985; Ozols et al. 1980). Similar studies looking at A2780 have
previously used agar based clonogenic assays grown for 7-10 days (Masuda et al. 1988) or
even fixed and stained cells after 96 hours (Sasaki et al. 1991). Despite the difference in
experimental duration, the results generated in this study are highly consistent with the
originally published literature (Table1).
This study has demonstrated that A2780 and its drug resistant counterparts, A2780cis and
A2780ADR, have the same character reported over 20 years ago. It shows the banking
procedures used in ECACC have maintained the cellular phenotype ensuring the cells are fit
for purpose. Furthermore, this study has demonstrated a label-free, fast and convenient way
to determine IC50 values using the IncuCyte ZOOM.
Table 1. IC50 values for A2780, A2780cis and A2780ADR from experimental data and published
studies
Drug Method Time (hr)
IC50 values
A2780 (µM) A2780cis (µM) A2780adr (µM)
Experimental IC50
Cisplatin
Cell number 120 0.736 7.044 1.009
Confluence 72 0.438 7.812 0.997
Range : 0.4 - 0.7 7 - 8 1
Published IC50
Cisplatin
(Behrens et al., 1987) 1.1 8 -
(Sasaki et al., 1990) 0.303 - 1.364
(Masuda et al., 1988) 1.02 38.5 -
Range: 0.3-1.1 8-39 1.4
Experimental IC50
Adriamycin
Cell number 120 0.007 0.016 0.070
Cell number (DMSO normalised) 120 0.005 0.012 0.060
Confluence 72 0.004 0.023 0.071
Confluence (DMSO normalised) 72 0.004 0.010 0.086
Range : 0.004 - 0.007 0.01 - 0.02 0.06 – 0.9
Published IC50
Adriamycin
(Louie et al., 1985) 0.01 - 1.6
(Sasaki et al., 1990) 0.11 - 710
(Hamilton et al., 1984) 0.015 - 2.25
Range: 0.01-0.1 - 1.6-710
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