Research ArticleExperimental Studies

The AutoDiSC: Development and Validation of a Novel Chemotherapy Sensitivity and Resistance Assay

TIMO SCHINKÖTHE, DHAKSANAN THIRUCHITTAMPALAM, ANNETTE SCHMIDT, NILS BOMHOLT and PETER STAIB
Anticancer Research June 2013, 33 (6) 2491-2498;

Abstract

Backround: Measurement of cell survival as performed by the differential staining cytotoxicity (DiSC) assay allows for a comprehensive prediction of individual chemotherapy response. This assay has not reached routine clinical application because of its several limitations. In order to improve the DiSC assay, we developed a new type of chemotherapy sensitivity and resistance assay (CSRA) by combining a novel evaluation methodology with automated measurement of cell survival based on flow cytometry, termed AutoDiSC. Materials and Methods: Tumour cell lines were treated with cytostatic drugs and cultured in 96-well plates for four days at 37°C. Subsequently, propidium iodide was added and 96-well plates were analyzed using flow cytometry in order to determine the number of living cells in each well. Each component was evaluated for reproducibility and impact on assay read-out. Results: The evaluation process allowed for specification of all relevant parameters that determine assay performance. Conclusion: AutoDiSC is a new fully-automated CSRA which meets all criteria required for routine clinical use.

Pre-therapeutic, ex vivo, chemotherapy sensitivity and resistance assays (CSRAs) represent powerful tools to increase the efficacy of currently used chemotherapy regimens (1-4). In this respect, measurement of cell survival as performed by the differential staining cytotoxicity (DiSC) assay developed by Weisenthal et al., allows for a comprehensive prediction of individual treatment response. The method of this assay consists of incubating dissociated cells from biopsy specimens in the presence or absence of anticancer drugs for 4-6 days concluding in the discrimination of dead and viable cells by light microscopy. The discrimination is carried out by using a dye, e.g. fastgreen. The ratio of dead cells to total cells is the measure of cell kill (5, 6). The DiSC assay has not reached clinical routine use because of its limitations in terms of technique (labour-intensive), susceptibility to operator variation and interpretation of the result (end-point measurement, no simple informative value). Numerous publications have, however, demonstrated the clinical value of chemosensitivity testing with the DiSC assay. This CSRA requires few cells and can be performed within 4-6 days (7, 8). In order to improve the DiSC assay and to overcome its major pitfalls, we developed a new type of CSRA by combining a novel evaluation methodology described by Staib et al., (chemosensitivity index, Ci) (9) with automated measurement of cell survival based on flow cytometry, termed AutoDiSC. The evaluation process allows for specification of all relevant parameters that determine assay performance, such as type of 96-well plates, culture conditions, set up of controls, drug concentration range and staining reagents. AutoDiSC is a new fully-automated CSRA which meets all criteria required for routine clinical use.

Materials and Methods

Cell cultures and flow cytometry. For the validation process of AutoDiSC, established cell lines (obtained from the University of Cologne, Germany) were set-up at identical cell count and identical suspension volume. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum in a humidified atmosphere with 5% CO2. After incubation, cells were stained with propidium iodide (PI) (Sigma Chemicals, St. Louis, MO, USA) and cell counting or live/dead discrimination respectively was carried out by flow cytometry using a FACS® array flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). Thresholds were set on forward scatter (FSC) 200 and sideward scatter (SSC) 400. The flow cytometer data analysis program FlowJo® Software (TreeStar Inc., Ashland, OR, USA) was employed for colour gating analysis of interest. The results are given as a percentage of cell survival compared to that of the positive control, i.e. tumour cell survival (TCS%).

Development and design of the AutoDiSC assay. The important criterion concerning the design of AutoDiSC is the reproducibility of results. In this context, the single components of AutoDiSC were validated according to result reproducibility and stability. Altogether, 17 96-well microtiter plates from Corning (Corning Inc., Corning, NY, USA), BD Bioscience, Greiner (Frickenhausen, Germany) and Nunc GmbH (Wiesbaden, Germany) were tested concerning inter-well divergence in cell growth. Wells were loaded with 100 μl Jurkat cell suspension at 2.5×105 cells/ml and incubated for 96 h. After staining with PI, cell count was performed by flow cytometry. The influence on cell proliferation of the material and well shape [flat (F) and round (U) bottom] were validated. A dilution series (0.5-5 M) of 99.5% sodium chloride (Sigma chemicals, St. Louis, MO, USA) was prepared to determine a suitable concentration for the negative control. Again 100 μl of cell suspension (2.5×105 cells/ml) was placed into the wells. After incubation with 1 μl of each sodium chloride concentration for 96 h and staining, cell survival was measured by flow cytometry. By means of Biomek® FX-Workstation (Beckman Coulter, Krefeld, Germany), six concentrations on a logarithmic scale were produced for commonly used anticancer drugs (data only shown for bleomycin sulfate). For validation of the optimal culture volume, cells were set at 5×105 cells/ml, and either 50 μl or 100 μl of cell suspension were placed into the wells. After 96 h incubation and staining cell count was carried out by cytometry. To determine the optimal cell density, cells were diluted either to 2×105 or 2×106 cells/ml and 100 μl of both dilutions were placed in wells. After 15 min of incubation and staining, cell count was performed. Finally, the optimal dye concentration was determined by using PI over a wide concentration range (180-0.008 μM).

AutoDiSC design. AutoDiSC consists of a 96-well microtiter plate with cytotoxic drugs. Each drug section consists of two identically arranged strips (16 wells). The first row represents the positive control (no drug added) and the last row the negative control (sodium chloride). Each drug was prepared at six concentrations on a logarithmic scale. As soon as density gradient isolation is performed, hematopoietic cancer cells (with identical cell count) were pipetted into every single well. Further incubation for 96 h at 37°C with 5% CO2 is needed. After staining cells with propidium iodide, cell survival is measured through flow cytometry. The results are given as the percentage of cell survival compared to that of the positive control (TCS%). Dose-response curves were then generated as functions of the dose response relationship. Using a linear regression plot the area under the curve (AUC) was then calculated. The area under the curve is the total dose response relation. Finally, the chemosensitivity index, Ci, as described by Staib et al., is set with values of between 0 and 1. Ci values of 0-0.5 indicate resistance and those above 0.5 predict sensitivity to the drug being tested. In conclusion, an efficiency rank of potential anticancer drugs against certain hematopoietic neoplastic diseases can be created for the individual patient.

Results

Propidium iodide for vital/death cell discrimination. To ensure optimal discrimination of dead from vital cells we tested PI at a wide range of 180-0.008 μM. Using PI, a maximum median fluorescence ratio dead/live cells of 269.62 was reached with a dye concentration of 1.843 μM (Figure 1 A).

Validation of the optimal cell density and incubation volume. The absolute density of cells can influence the quality of data acquisition. Whereas few cells result in high fuzziness, too many cells lead to an event overflow. To find the optimal cell density a wide range of cell densities were tested. At a density of 500 cells/ml, the relationship between seeded and measured cell counts showed a linear correlation (R2=0.999). A cell count below 500 cells/ml resulted in high standard deviations (Figure 1 B). For defining the optimal incubation volume, 50 μl and 100 μl were tested with six different cell lines in suspension. The mean expansion rate ranged from 1.55 to 4.53 using 50 μl, whereas for 100 μl incubation volume, the different cell lines had a mean expansion rate of 2.51 to 5.31. In all tested cases, cells had a higher expansion rate when using 100 μl, which was significant for all cell lines except Jeko-1 (data not shown).

The appropriate 96-well microtiter plate for AutoDiSC. A further factor which might influence the quality of test results might be the microtiter plate used. Plates with different surfaces, different physical modifications, from different manufacturers were tested, as described in Materials and Methods. Seventeen different plates from four manufacturers were analyzed. For the different tested plates, a mean cell count deviation of 5.8% (Corning, high-binding U) to 25.4% (Greiner, medium-binding U) was observed. The following 96-well microtiter plates had standard deviations under 10%: 8.4%, BD Bioscience, Primaria F; 8.1%, BD Bioscience, TC-treated F; 5.8%, Corning, high binding U; 7.4%, Corning, TC-treated F; 8.5%, Corning, high-binding F; 7.6%, Greiner, TC-treated F; 6.5%, Nunc, Immobilizer F; and 6.8% Nunc, Multisorp F (Figure 2). The wells of microtiter plates are of different shapes. Therefore wells with flat (F) and round (U) bottoms were compared regarding the effect on cell growth. The results illustrated that, all cell lines, except Granta-519, proliferated more strongly in microtiter plates with flat-bottom wells than in those with round-bottom wells. The quotients (F-/U-bottom) of cell proliferation in flat- and round bottom wells were as follows: All cell lines, except Granta-519, had a quotient above 1, with 1.95, Jeko-1; 1.74, Jurkat; 1.63, JVM-3; 1.51, Mec-1; and 1.37, Mec-2; Granta-519 had a quotient of 0.59 (Figure 3 A).

Exploring the optimal six-log anticancer drug concentration. The main step of performing AutoDiSC is the exposure of hematopoietic cancer cells to certain anticancer drugs in an increasing concentration range and the subsequent drawing of the dose-response curve. As mentioned above, every AutoDiSC consists of drugs at six concentrations on a logarithmic scale. To determine the appropriate concentration ranges, titration of several anticancer drug dilution levels was performed (figures for doxorubicin hydrochloride, etoposide phosphate, vincristine sulfate, gemcitabine hydrochloride and fludarabine phosphate are shown in the supplementary section). The gradual killing of the tested cell lines with bleomycin sulfate was effective at concentrations from 0.25 up to 60 μg/ml. Regarding lethal concentration 90% (LC 90), the concentration of a cytotoxic drug at which 90% of the cells die, values for bleomycin of several cell lines were 5.572 μg/ml (Granta-519), 5.579 μg/ml (Jeko-1), 8.381 μg/ml (JVM3), 5.579 μg/ml (Jeko-1), 8.381 μg/ml (JVM-3), 3.825 μg/ml (Mec-1), 11.137 μg/ml (Mec-2) and 1.259 μg/ml (WSU-FSCLL), respectively. Cell line WSU-FSCLL had an AUC value of 49.04 μg/ml × TCS% with the corresponding Ci index of 0.99 and JVM-3 had an AUC of 486.59 μg/ml × TCS% with the corresponding Ci of 0.93. (Figure 3 B).

Sodium chloride as a negative control. The negative control of AutoDiSC should create a toxic condition for all cell lines except for the operator. Sodium chloride represents such a chemical, able to kill every cell at certain concentrations due to osmotic stress. Concerning AutoDiSC, the negative control should kill every cell after 96 h of incubation. In this sense, several sodium chloride concentrations were tested, which allow for a stable and reproducible cell kill of 100%. The cells were killed at 0.5 M (Jurkat, L-540) and 1 M (Mec-1, JVM-3). At 3 M, no cell survival (Jurkat, L540, Mec-1, JVM-3) was measured by flow cytometry (data not shown).

Discussion

We have developed a novel CSRA, named AutoDiSC, able to predict the individual treatment outcome in cases of neoplastic hematologic diseases. AutoDiSC represents the enhancement of the DiSC assay, based on light microscopy described by Weisenthal et al. (5, 6). Concerning ex vivo cellular drug resistance, the application of DiSC assay has been described and extensively employed (7, 10-13). Nevertheless, the DiSC assay has disadvantages, it is labor-intensive, is susceptible to operator variation and the results require interpretation (1). In summary, AutoDiSC was developed by improving the DiSC assay and overcoming its major pitfalls.

AutoDiSC consists of a 96-well microtiter plate. Microtiter plates feature different surface properties and well shapes, with effect on cell growth. For this reason, we compared sixteen plates from four different suppliers. With a cell count standard deviation of 5.8%, the high-binding U microtiter plate of Corning Inc. gave the best results in regard to the inter-well reproducibility of cell growth. The medium-binding U plate of Greiner had the worst performance, with a standard deviation of 25.4%. In summary, while we gathered the best results with standard deviations below 10% from the Corning Incorporated products, the Greiner products had the worst results, with standard deviations above 10%, except the TC-treated F plate.

A closer look at the effect of well shape revealed that all tested cell lines (F/U ratios above 1), except for Granta-519 (F/U ratio 0.59), grew more strongly in wells with flat bottoms than in round ones. Considering cell growth and inter-well reproducibility, the high-binding microtiter plate of Corning Inc. with a flat bottom appeared to be the most appropriate 96-well microtiter plate for AutoDiSC.

We incubated several cell lines with sodium chloride at different concentrations, so that a negative control could be established. Practically every cell line died at a concentration of 3 M NaCl. Based on this measurement, we considered that for AutoDiSC, the concentration of 5 M sodium chloride provides a secure negative control.

To determine the optimal drug concentration ranges, we performed logarithmic titration (six logs) of anticancer drugs (data only shown for bleomycin sulfate) and incubated different cell lines with them for 96 h at 37°C. We generated fair dose-response curves, which gives a function of the dose response relationship, over the range of 0.25 to 60 μg/ml bleomycin sulfate.

AutoDiSC is based on the concept of co-incubating hematopoeitic cancer cells with several anticancer drugs (at a logarithmic concentration range) for 96 h at 37°C and the subsequent discrimination of dead and vital cells by flow cytometry. Therefore, we evaluated whether cells proliferate more strongly in 50 μl or 100 μl of medium. In addition, we determined the minimal cell density for optimal plate read-out. The tests showed that for cell proliferation, 100 μl medium is preferable to 50 μl. Cell counts above 500 cells per well illustrated satisfactory reproducibility and repeatability.

Finally, the object of this work was to optimize the dead and vital cell discrimination. Therefore, a dilution series of PI (180-0.008 μM) was prepared and Jurkat cells were stained at different PI concentrations. We estimated that a PI concentration of 1.843 μM was optimal for dead/vital cell discrimination.

It is well-known that individual patients with identical tumour histologies do not always respond identically to the same anticancer drug (14). Therefore, since the late 1950s, many scientists have attempted to develop a CSRA, so that the response of therapy can be predicted before it begins (15-21). However, none of the CSRAs have reached clinical utility. Colony-forming assays are limited by long incubation times, their labor intensivity, prone to individual interpretation and represent an end-point measurement. Dye exclusion assays, for example the DiSC assay, have a limited dynamic range, are labor-intensive and are subject to observer error. Cell metabolic assays such as the ATP-based tumor chemosensitivity assay are also labor-intensive and represent end-point measurements.

Figure 1.
Figure 1.

A: Jurkat cells were fixed with formaldehyde and the proportion (1:1) of vital and dead Jurkat cells were assessed. A propidium iodide (PI) dilution series (180-0.008 μM) was prepared and the vital/dead cell proportions were determined by staining with different PI concentrations (n=3). B: Jurkat cells were set either to 2×105 cells/ml or 2×106 cells/ml and a dilution series was performed of both (n=6). After staining with PI, the cell count was performed by flow cytometry.

Figure 2.
Figure 2.

Jurkat cells were set-up at identical counts. Then 100 μl of cell suspension was pipetted into each well of a 96-well microtiter plate and cells were incubated for 96 h at 37°C. After staining with propidium iodide (PI), cell vitality was measured by flow cytometry. The ordinate is the measured cell count per 20 μl cell suspension (percentage of cell vitality in each single well relative to the mean cell vitality of the whole 96-well microtiter plate). The abscissa shows the tested 96-well microtiter plates with flat (F) and round (U) bottoms: A: BD Bioscience: I untreated-F, II Primaria-F, III TC-treated-F; B: Corning Inc.: IV high-binding-U, V TC-treated-F, VI high-binding-F; C: Greiner: VII high-binding-U #1, VIII high-binding-U #2, IX medium-binding-U, X TC-treated-U, XI TC-treated-F; D: Nunc: XII Immobilizer-F, XIII MediaSorp-F, XIV MaxiSorp-U, XV MultiSorp-F, XVI TC-treated-U. A TC-treated-U 96-well microplate acted as control C.

AutoDiSC is a validated CSRA using flow cytometry. Because of its automation, AutoDiSC is not labour-intensive, is not prone to individual interpretation, and the Ci, described by Staib et al., represents a simple informative value defining anticancer drug sensitivity or resistance which can be derived from AutoDiSC.

AutoDiSC meets all criteria for a tool of pre-therapeutic ex vivo efficacy testing of anticancer drugs against hematopoetic neoplasms. Moreover, AutoDiSC may also be useful in screening of new potentially antitumour agents.

Figure 3.
Figure 3.

A: Jurkat, JVM-3, Mec-1, Mec-2, Granta-519 and Jeko-1 cells were set at identical counts and 100 μl of cell suspension were pipetted into each well (16 wells each with flat bottom and round bottom per cell line). Cells were then incubated for 96 h at 37°C. After staining with propidium iodide (PI) cell vitality was measured by flow cytometry. The ordinate is the measured cell count per 20 μl suspension. The abscissa shows the tested cell lines. B: Jeko-1, JVM-3, Granta-519, Mec-1, Mec-2 and WSU-FSCLL cells were incubated with different logarithmic concentration ranges of bleomycin for 96 h at 37°C (n=2). After staining with propidium iodie (PI) cell survival (CS) was measured by flow cytometry. The ordinate shows the CS at each bleomycin concentration relative to that of the positive control (no drug added).

Figure 4.
Figure 4.

Jurkat, Jeko-1, JVM-3, Granta-519, Mec-1, Mec-2 and WSU-FSCLL cells were incubated with different logarithmic concentration ranges of different cytotoxic drugs for 96 h at 37°C (n=3). After staining with propidium iodie (PI) cell survival (CS) was measured by flow cytometry. The ordinate shows the CS at each cytotoxic drug concentration relative to that of the positive control (no drug added). A: doxorubicin hydrochloride. B: etoposide phosphate. C: vincristine sulphate. D: gemcitabine hydrochloride. E: fludarabine phosphate.

Acknowledgements

This work was supported by TumorTec GmbH.

Footnotes

  • ↵* These Authors contributed equally to this work.

  • Conflicts of Interest

    There are no relevant conflicts of interest to disclose.

  • Received February 20, 2013.
  • Revision received April 23, 2013.
  • Accepted April 24, 2013.

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The AutoDiSC: Development and Validation of a Novel Chemotherapy Sensitivity and Resistance Assay
TIMO SCHINKÖTHE, DHAKSANAN THIRUCHITTAMPALAM, ANNETTE SCHMIDT, NILS BOMHOLT, PETER STAIB
Anticancer Research Jun 2013, 33 (6) 2491-2498;
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