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. Author manuscript; available in PMC: 2021 Aug 27.
Published in final edited form as: Methods Mol Biol. 2020;2078:329–340. doi: 10.1007/978-1-4939-9929-3_23

Determination of ADC Cytotoxicity in Immortalized Human Cell Lines

Shengjia Wu 1, Dhaval K Shah 1,*
PMCID: PMC8396111  NIHMSID: NIHMS1729112  PMID: 31643068

Abstract

Cytotoxicity assay is a necessary first step to triage ADC molecules before moving them forward to relatively time-consuming and expensive in vivo studies. When cells are exposed to ADC molecules, antigen expressing cells can effectively take up those molecules and eventually die as a result of the released payload. This cytotoxic property of ADCs can be evaluated by measuring the percentage of living cells at the end of the incubation period. Tetrazolium colorimetric assay (MTT) is a widely used method that can be used to measure cell viability. Here we have demonstrated how to use MTT assay to measure cytotoxic effect of ADC and calculate IC50. Besides the cytotoxic behavior on antigen expressing cells, ADCs can also demonstrate bystander killing effect on antigen negative cells in the vicinity of antigen expressing cells. Here, we have demonstrated how to use a co-culture experiment to evaluate the bystander effect of ADC with the help of fluorescent protein transfected antigen negative cells.

Keywords: Antibody-drug conjugate (ADC), cytotoxicity assay, MTT assay, bystander effect, co-culture system, cell viability assay

1. INTRODUCTION

Cytotoxicity assay is an essential experiment during the development of drug molecules like antibody-drug conjugate (ADC), which influence cell proliferation or demonstrate direct killing effect. It is a very useful tool to triage promising ADC candidates, predict in-vivo efficacy of ADCs, and evaluate the specificity of ADCs. The most important outcome of cytotoxicity assay is the information about remaining number of viable or dead cells at the end of the experiment. Several types of method can be used to measure this outcome, including tetrazolium reduction, resazurin reduction, protease markers, and ATP detection [1]. In this chapter, we have demonstrated the use of MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium] assay, which is one of the most commonly used assay developed by Mosmann [2] that is typically performed in 96-well plate. This assay relies on the ability of mitochondrial NADH to transfer electrons to MTT, and actively convert MTT into purple needle-like formazan crystals [3][4]. The purple color generated by these crystals is then used to assess the quantity of living cells in a well using two assumptions: (i) the reduction reaction happens only in viable cells, and (ii) the formazan production is directly proportional to the number of living cells. [5]

In order to conduct a thorough cytotoxicity assessment of ADCs, it is ideal to use both antigen positive (Ag+) and antigen negative (Ag−) cell lines. Since ADC molecules are large and relatively polar, they are not readily permeable through cell membrane, and reply on cell surface antigen to gain entry into the cell. Consequently, one would expect much more toxicity (i.e. lower IC50) of ADCs in Ag+ cells compared to Ag− cells. As such, ADCs are designed to provide advantage by targeting Ag+ tumor cells and sparing the damage to Ag-healthy cells. However, some ADCs can also kill Ag− cells that are in the vicinity of Ag+ cells by an effect known as the bystander effect. [6] This phenomenon stems from the diffusion of cytotoxic payload, which is generated by degradation of ADC in Ag+ cells, into the standing by Ag− cells. ADCs with cleavable linker and hydrophobic payload, like brentuxiamb vedotin (SGN-35) with val-cit linker and MMAE as the payload, are known to demonstrate the bystander effect. [7,8] In order to evaluate the therapeutic index of ADCs it is also important to assess their bystander effect in vitro.

There are two different type of assays that can be used to evaluated the bystander effect of ADCs in-vitro: (i) co-culture assay [8,9], and (ii) medium transfer assay [10]. Medium transfer method is performed by treating Ag+ cells with a certain amount of ADC first, and then transferring the conditioned medium to wells with Ag− cells after certain period of time. If the medium taken from Ag+ cells shows more killing than treating Ag− cells directly with the same concentration of ADC, then there is a conformation of the bystander effect. The co-culture method starts with culturing of Ag+ and Ag− cells together, and comparing the viability of Ag− cells in the co-culture system with the viability in Ag− monoculture system at the same ADC concentrations. If Ag− cells are killed to a greater extent in the co-culture system compared to the monoculture system at the same ADC concentrations, then there is a conformation of the bystander effect. The viability of Ag− cells in the co-culture system can be measured by either flow cytometer, where the Ag− cells are detected using fluorescently labelled antibody targeting an antigen specifically expressed on these cells, or fluorescent plate reader that can selectively quantify Ag− cells that are transfected with a fluorescent protein.

In order to provide methodological details of routinely preformed cytotoxicity experiments with ADCs, here we have demonstrated how to evaluate cytotoxicity of ADC in a monoculture system using MTT assay, and how to evaluate in vitro bystander effect of ADCs in a co-culture system using green fluorescence protein transfected Ag− cells.

2. MATERIAL

  1. Cell lines (MCF7 and N87 cells in this chapter)

  2. Cell culture medium (Roswell Park Memorial Institute (RPMI) 1640 Medium, supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% Fetal bovine Serum (FBS) in this chapter).

  3. Dulbecco’s Phosphate-buffered saline (DPBS), pH 7.4

  4. 0.25% Trypsin-EDTA (1X)

  5. Pipette or multichannel pipette

  6. Reagent reservoirs for multichannel pipette

  7. Hemocytometer or cell counting machine

  8. Tissue culture treated 96-well plate with clear wall & clear bottom (flat bottom)

  9. Tissue culture treated 96-well plate with black wall & clear bottom (flat bottom)

  10. Plate reader equipped with the filters for required wavelength

  11. 5 mg/ml MTT solution: Dissolve MTT (thiazolyl blue tetrazolium bromide) in sterile DPBS to 5 mg/ml; filter the prepared MTT solution through a 0.2 μM filter into a sterile, light protected container (see Note 1)

  12. Solubilization solution: 10% (w/v) SDS-solution with 0.01M HCL: Dissolve sodium dodecyl sulfate (SDS) in sterile DI water and make the final concentration as 10% (w/v) (See Note 2,3)

3. METHOD

All agents used in the assay should be sterile, and all the steps before reading the plate should be conducted in a biosafety cabinet.

3.1. Determining optimal cell counts and incubation time:

  1. Prepare cells for the assay. Harvest suspension cells by centrifugation. If cells are adherent, detach them by trypsinization. (see Note 4)

  2. Resuspend cells at a density of 1×106 per mL.

  3. Prepare serial dilution of cells from 1×106 per mL to 1×103 per mL.

  4. Add 100μL of the dilutions into wells in triplicate. Leave three wells of medium only as blank control (background control). (see Note 5)

  5. Incubate the plate at 37°C for 6–24h for cell adaptation. (see Note 6)

  6. Add 20uL of 5mg/mL MTT solution into each well.

  7. Incubate at 37 °C for 1–4 hours (see Note 7)

  8. Add 100uL 10% SDS-HCL solution and incubate the plate in dark at 37 °C overnight. (see Note 8)

  9. Read the absorbance at 570nm using a plate reader. (see Note 9)

  10. Get the average absorbance values from each cell number group and subtract the average value of the blank. Plot the absorbance vs. cell numbers, a suitable working range should lie within the linear portion of the curve. Determine the initial seeding number for the cytotoxicity assay based on doubling time and the desired duration of the assay.

3.2. Monoculture cytotoxicity study using MTT assay:

  1. Seed the cells in 96-well plate at density of 1,000–10,000 cells/well (actual seeding number depends on the optimal assay result) in 50uL media volume. Arrange the plate as shown in Fig.1 with 6 main groups: Blank medium for Ag+ and Ag− cells, Ag+ control, Ag− control, and ADC treated antigen positive and negative groups. Treat the ‘Blank’ wells with 50uL fresh medium instead. (see Note 10)

  2. Incubate the plate at 37°C with 5% CO2 overnight to let cell attach.

  3. Prepare the ADC with different concentrations, while keeping each dilution 2 times more concentrated than the desired concentration in the well. Add 50uL of prepared ADC solution into each drug treatment well. Add 50uL of fresh medium into blank and control wells. (see Note 11,12)

  4. Incubate the plate at 37°C for 48–144 hours. (see Note 13)

  5. Add 20uL of 5mg/mL MTT solution into each well.

  6. Incubate at 37 °C for 1–4 hours depending on the optimal assay result.

  7. Add 100uL 10% SDS-HCL solution and incubate at 37 °C overnight.

  8. Read the absorbance at 570nm.

  9. Calculate the cell viability at different ADC concentrations. Plot the data as % viability vs. ADC concentration, and fit the data to sigmoidal curve to get IC50 value. (fig.3 and fig.4) (see Note 14)

Fig 1.

Fig 1.

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Typical plate format for monoculture MTT assay. The groups are set in triplicate. For each cell line, there are three main groups: Blank (medium only); untreated cell control, and cells treated with ADC.

Fig 3.

Fig 3.

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N87(Ag+) viability curve following treatment with Trastuzumab-vc-MMAE.

Fig 4.

Fig 4.

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MCF7(Ag−) viability curve following treatment with Trastuzumab-vc-MMAE. Data digitized from Singh et al. [9]

3.3. Co-culture study to determine bystander effect of ADC.

From the results of cytotoxicity assay on monoculture cell lines, the IC50 values for both Ag+ and Ag− cells can be achieved. The concentration of ADC used for bystander effect should be larger than the IC90 in antigen positive cell line, and less than the IC50 in antigen negative cell line. In the method described here, the Ag− cell line needs to be transfected with fluorescence protein. Below, we have demonstrated a co-culture method using green fluorescence protein (GFP) transfected cell line, which was developed by Singh et al. [9] (see Note 15).

  1. Prepare both Ag+ and GFP-transfected Ag− cells. (see Note 16)

  2. Arrange the plate as shown in Fig.2 with 7 main groups: Blank (Background control), Ag− cell only group, and 5 co-culture groups with different antigen negative cell percentage: 90%, 75%, 50%, 25%, 10%. (see Note 17) Divide the Ag− only and co-culture groups further into two arms: ADC-non-treated (control) and ADC-treated.

  3. Seed cells at a density of total 10,000 cells/well at a final medium volume of 100uL, except for the blank group. (see Note 18) Add only 100uL of fresh medium into the blank group.

  4. Incubate the plate at 37°C with 5% CO2 overnight.

  5. Remove the old medium in each well. Add 100uL of ADC containing medium to each ADC-treatment well, and 100uL fresh medium to control wells. (see Note 19)

  6. Incubate the plate at 37°C with 5% CO2 for 48h.

  7. Read the plate at 485/535 nm (excitation/emission). (see Note 20)

  8. Repeat steps 6 and 7 at 96h and 144h after ADC treatment. (see Note 21)

  9. Normalize the fluorescence intensity value in each well by subtracting the reading from blank group. Divide fluorescence values of ADC treated wells with the value from non-treated wells to get % viability. (see Note 22) (fig.5)

Fig 2.

Fig 2.

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Typical plate format for co-culture bystander effect assay. It contains: Blank (medium only), untreated co-culture control, and ADC treated co-culture with different ratios of Ag+ and Ag− cells.

Fig 5.

Fig 5.

Fig 5.

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(a) Ag− cell viability over time in N87(Ag+) and MCF7(Ag−) co-culture system with different ratios of the cells. Data digitized from Singh et al. [9] With higher percentage of Ag+ cells, the bystander effect is more obvious. (b) Viability of Ag− cell at a single time point (144h). Each co-culture group shows statistically significant higher cytotoxicity than Ag− monoculture cells. A one-way ANOVA with multi-comparison was used in this case to determine the significance.

4. NOTE

1.

MTT dissolves in DPBS slowly and it takes about 5–10 min by vortex or stirring. The sterilized MTT solution can be stored away from light, at 4°C for short-term usage or at −20 °C for long-term storage up to one-month. The easiest way to make a light protected container is warping an aluminum foil outside a conical tube.

2.

Various agents can be used to solubilize the formazan product, including organic solvent like dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and isopropanol; and detergent like sodium dodecyl sulfate (SDS) and combinations of organic solvent and detergent. [1,11,12] Many media used for cell culture have phenol red (a pH indicator) in their ingredients, which can affect the absorbance. [11] HCL can help get rid of its influence, [12] and 0.01M is found to be the optimal concentration of HCL that can effectively reduce the influence of phenol red and provide maximum absorbance for MTT assay. [13] While dissolving formazan with DMSO provides a purple color, using 10% SDS (w/v) with 0.01M HCL gives a dark yellow color.

3.

Since SDS is a detergent, dissolving this chemical generates bubbles. To avoid these bubbles, avoid heavy vortexing or stirring. Heating can also help dissolve SDS. In general, HCL is added to 0.01M concentration after the 10% SDS is prepared. This solution can be stored at room temperature.

4.

Cells should be maintained at good condition (more than 95% live) before they are used for cytotoxicity assay. Do not use the cell immediately after thawing, and sub-culture them for at least one time before using for the assay.

5.

Make sure the cells are evenly seed inside the well and form single layer if using adherent cells. To have even seeding, put pipette tip at the wall of the well and form an angle around 30° before pipetting the cells slowly. If multichannel pipette is used to seed the cells, about 1–2 mL more cell suspension is needed in the reagent reservoir. Pay attention to the difference between cells/well and cells/ml notations.

6.

The recovery time can vary between cell types. For most of the cells 12–16h is sufficient.

7.

Evaluation of different MTT incubation times (e.g. 1h, 2h, 4h) during assay optimization can be helpful. Within a certain range, the longer the incubation time with MTT the higher the absorbance and resolution that can be obtained. However, when the cell density is too high, the MTT substrate can run out and the linear relationship between formazan production and cell viability can deviate. When conducting an MTT assay, an absorbance of 0.75–1.25 for the control group at the endpoint is optimal.

8.

Using 10% SDS-HCL solution as solubilization solution takes more time than DMSO to totally dissolve formazan crystal. A relatively longer incubation time provides higher and more stable absorbance. 12h-18h will be enough for most cases.

9.

The greatest absorbance for formazan is at 570nm. However, some plate reader with filter channels do not have the exact wavelength. In this case, choose the reading wavelength within 550–600nm.

10.

When doing cytotoxicity assay using 96-well plate, the plates are incubated at 37°C for several days. This leads to an evaporation problem, where the medium around the perimeter evaporates faster than the inner wells, which can cause unwanted noise and known edge effect. To get rid of it, one solution can be leaving the outermost wells unused and filling them with 200uL DPBS. If doing this, take into consideration that only 60 wells are available from a single 96-well plate when designing the experiment.

11.

The concentration range of ADC is designed based on ADC efficacy. The range should cover the concentrations with no killing effect on Ag+ and Ag− cells and have concentration which saturate killing capacity on at least Ag+ cell. When screening the ADC efficacy, a dilution factor of ten is a good start. After an understanding of the ADC working range, a smaller dilution factor can be applied to get a more precise estimation of IC50, since this will offer more points at log-linear phase of Hill equation.

12.

As an add-on for note 11, drug-to-antibody ratio (DAR) is also important to consider while deciding the concentration range of ADC in the experiment. An ADC with a DAR value of 4 shows more cytotoxicity in vitro than the one with a DAR of 2. Therefore, payload concentration (ADC concentration* DAR value) rather than ADC concentration is more relevant when using plain payload IC50 as a reference to set up the concentration range.

13.

The incubation time depends on the type of toxic payload the ADC has and the cytotoxic mechanism-of-action for the payload. If the payload is a tubulin inhibitor (e.g. MMAE or MMAF), 72h or 96h is better to evaluate cytotoxicity for these agents, since these payloads require cell-cycle arrest and cause delayed cell killing. DNA damaging payloads (e.g. PBD or calicheamycin) can induce the cell death relatively quicker.

14.
To process the data, the absorbance obtained from all the seeded wells should be subtracted by the average value of blank group. The percentage of live cells is then calculated by dividing the normalized absorbance of ADC treated groups with the normalized absorbance from control group. The equation is:
Viability%=ODTreatedODcontrol∗100%. (1)
Plot data with % viability on y-axis and concentration of ADC on x-axis in log-scale. Fit the data using a dose-response equation with four parameters:
Viability=Boundarylow+Boundaryhigh−Boundarylow/1+ConcentrationADCγIC5γ. (2)
Boundarylow is the lowest viability when treated with drug, while the Boundaryhigh represents the highest viability that one can get. γ is the hill slope. IC50 stands for the ADC concentration that can have 50% of maximum killing. Software, like GraphPad Prism can be used for the curve fitting by choosing the nonlinear regression option. Normally, IC50 value is used to evaluated ADC toxicity, sometimes IC90 value is also used instead. IC90 is the concentration that induced 90% of maximum killing. From equation 2, we can get IC90 by making the viability value equal to Boundarylow + 0.1 ∗ (Boundaryhigh − Boundarylow). Therefore, the IC90 equals to (9 ∗ 𝐼𝐶50𝛾)1/𝛾. IC10 can also be calculated by the same way, which equals to (1/9 ∗ 𝐼𝐶50𝛾)1/𝛾. Fig. 3 and Fig. 4 shows a viability curve for N87 and MCF7 cells after the treatment with Trastuzumab-vc-MMAE ADC. Of note, the %viability can sometimes be higher than 100% at low drug concentration. This may be due to increased metabolism of cells at low concentrations, as this treatment could create stress instead of killing the cells.
15.

Other fluorescence protein can also be used for this co-culture method. The cell line needs to be stably transfected, which means the fluorescence protein expression per cell needs to remain unchanged, and the fluorescence intensity needs correlated with cell number.

16.

Different cell lines sometimes have different recommended medium. Normally, when doing this co-culture method, mixing different medium does not affect the cell growth much. One concern is, if the transfection of Ag− cells is performed in-house, the medium may contain selection antibiotic. Before the co-culture assay, these cells need to be acclimated in normal medium without antibiotic.

17.

A tissue culture treated black plate with clear bottom is the most suitable plate type for this kind of assay. The black wall absorb light, which helps reduce background noise and interference from nearby wells. With a clear bottom, the fluorescence signal can be read from the bottom, which provide better resolution when reading fluorescence from adherent cells. The material of the bottom is not much crucial in this kind of assay, both plastic or glass bottom works.

18.

It requires time to observe the bystander effect compared to the direct cytotoxicity assay, and sometimes is can take up to three weeks. [9] The initial seeding density can be adjusted considering the incubation and doubling time. Bystander effect requires the payload to be cleaved from the ADC and penetrate into the nearby Ag− cells. Therefore, adding media during the assay will influence the bystander effect by diluting the free payload concentration, and thus it is not advised.

19.

The volume of medium added per well can go up to 200uL if the assay needs to last long.

20.

GFP has a maximum excitation wavelength at 488 nm and a maximum emission wavelength at 510nm. As mentioned in Note 15, reading from bottom could provide better signal-to-noise ratio.

21.

The reading time points and the duration of the assay are adjustable. As long as the initial seeding number is proper, the assay can go up to 20 days according to Singh et al. [9].

22.

To evaluate the bystander effect, compare the viability of co-culture system with Ag− cell only. If the difference is statistically significant and less viability is observed in co-culture system, the ADC molecule has bystander effect. For some ADC molecules, the bystander effect can only be observed when the ratio of Ag+ cell is larger than 50%.

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