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Introduction

For many years, two-dimensional (2D) cell culture involving cells grown in a monolayer on a flat culture surface has served as a convenient system for investigating disease mechanisms and assessing the effects of potential new drugs. Cancer cell lines grown in 2D have long served as experimental surrogates for the cancers from which they were derived. In recent years, the culture of cancer cells, along with other cell types, in three-dimensional (3D) formats where they can form multi-layered structures is enabling new models for cancer research that are considered more biologically relevant.

While imaging and high-content analysis are often required to gain detailed information on the effects that drug candidates have on complex 3D cellular models, there is also a need for straightforward cell-based assays that can assess a single parameter, such as overall cell viability. An adenosine triphosphate (ATP) assay is a widely used method to assess a treatment’s effect on cell viability. Metabolically active cells use ATP as a source of energy, and when injured or depleted of nutrients, cells tend to show a rapid decline in cytoplasmic ATP. Assays used to measure ATP typically have a luminescent readout based on the firefly luciferase reaction, which requires ATP supplied by viable cells (Figure 1), and they are very sensitive thanks to the lack of background luminescence in cells and reagents. The rapid readout from an ATP assay can be used to quickly screen a large set of compounds and identify a subset that significantly affects cell viability. This reduced set may then be applied to more focused follow-up studies involving more labor-intensive experiments that provide more phenotypic detail.

Here we present a small-scale study of cell viability in 2D and 3D cell cultures, measured using the CellTiter-Glo® 3D Cell Viability assay (Promega). We assayed the viability of the following two cell types grown in 2D and 3D and challenged with several previously tested compounds1 : MCF-7, a commonly used breast cancer cell line that is estrogen receptor (ER)- and progesterone receptor (PR)-positive, and TU-BcX-4IC, cells from a patient-derived tumor explant that represents metaplastic breast cancer with a triple-negative (ER-, PR-, and HER2-negative) subtype2. Assay detection was performed using the SpectraMax iD5 Multi-Mode Microplate Reader, which has an ultra-cool PMT for low background luminescence and high sensitivity. All data were analyzed with SoftMax Pro Software, including curve fitting and IC₅₀ calculations.

Figure 1. Luciferase reaction used to measure cell viability. Cells are lysed and added to the luciferase reaction, where they provide the ATP needed to produce light. The more live cells are present, the more light is produced.

Materials

• CellTiter-Glo 3D Cell Viability Assay (Promega cat. #G9682)
• MCF7 breast cancer cell line (ATCC cat. #HTB-22)
• TU-BcX-4IC patient-derived primary triple-negative breast cancer cells (generously provided by the lab of M. Burow, Tulane University)
• Growth medium for MCF7: MEM (Corning cat. #10-010-CV) + 10% fetal bovine serum (FBS, Avantor Seradigm cat. #1500-500) + 1% Antibiotic-Antimycotic (Gibco 15240-062) + 0.01% mg/mL human recombinant insulin (Gibco cat. #12585-014)
• Growth medium for TU-BcX-4IC: DMEM (Corning cat. #15-017-CV) + 10% fetal bovine serum (FBS, Avantor Seradigm cat. #1500-500) + 1% Antibiotic-Antimycotic (Gibco cat. #15240-062) + 1% NEAA (Gibco cat. #11140-050) + 0.01% mg/mL insulin, human recombinant (Gibco cat. #12585-014)
• 384-well PrimeSurface 3D white culture plate, ultra-low attachment, U-bottom microplate (Sbio cat. #MS-9384WZ)
• 384-well white, tissue culture-treated, solid bottom microplate (Thermo Fisher Scientific cat. #164610))
• Test compounds:
  • Bortezomib (Tocris cat. #7282)
  • Ibrutinib (Tocris cat. #6813)
  • Idarubicin hydrochloride (Selleck Chemicals cat. #S1228)
  • Panobinostat (Selleck Chemicals cat. #S1030)
  • Romidepsin (R&D Systems cat. #3515)
  • Trametinib (Selleck Chemicals cat. #S2673)
• SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices)

Methods

Cell preparation

3D spheroids: MCF7 or TU-BcX-4IC cells were plated at 2500 cells per well into 384-well U-bottom microplates. Plates were centrifuged at 1000 rpm for 1 minute to aggregate the cells. Plates were then incubated at 37°C, 5% CO₂ for 72 hours.

2D monolayer culture: MCF7 cells were plated at 5000 cells per well into 384-well flat bottom microplates. The plates were then incubated at 37°C, 5% CO₂ for 24 hours. TU-BcX-4IC cells were plated at 2500 cells per well, then incubated for 48 hours.

(2) Oxaloacetate + L-Glutamate L-Aspartate + a-Ketoglutarate

Compound treatmen

TU-BcX-4IC and MCF7 cells cultured in 2D and 3D were treated in quadruplicate wells with a 1:5 dilution series of romidepsin ranging from 50 nM to 0.006 nM. Cells were incubated with compounds at 37°C, 5% CO₂ for 72 hours.

TU-Bcx-4IC cells in both culture formats were also treated with bortezomib, ibrutinib, idarubicin hydrochloride, panobinostat, and trametinib. Cells were incubated with compounds at 37°C, 5% CO₂ for 72 hours.

After incubation, CellTiter-Glo 3D reagent was added to the assay wells. For 3D spheroids, the plate was shaken for 5 minutes and then incubated for 30 minutes at room temperature to allow for cell lysis before reading. For 2D cultures, the plate was shaken for 2 minutes and then incubated for 10 minutes at room temperature before reading. Plates were read on the SpectraMax iD5 reader using the luminescence detection mode and settings shown in Table 1. All data analysis and graphing were done with SoftMax Pro Software.

Table 1. SpectraMax iD5 reader settings for the CellTiter-Glo 3D assay. Settings are specified in the plate section in SoftMax Pro Software. Read height is optimized by checking the box next to “Show Pre-Read Optimization Options” under More Settings and following the instructions that appear once the read is initiated.

Results

Results for each set of compound-treated cells were graphed using the 4-parameter logistic in SoftMax Pro Software, where the C parameter of each curve was taken as the IC50 value. In MCF7 cells treated with romidepsin, the IC₅₀ concentration was somewhat higher for 3D-cultured cells than for monolayer-cultured (Figure 2). TU-BcX-4IC treated with romidepsin exhibited a slightly lower IC₅₀ in 3D-cultured cells than cells in 2D (Figure 2E). For other compounds tested, IC₅₀ values were more often higher in 3D-cultured cells, with differences between 3D and 2D-cultured results often around two-to three-fold (Figure 3, Table 2).

A distinct difference between TU-BxC-4IC and MCF7 cells treated with romidepsin. In 2D culture, both cell types had similar IC₅₀ values, 1.13 nM vs. 1.88 nM. However, in 3D culture TU-BxC-4IC cells had a 12-fold lower IC₅₀ value of 0.443 nM, compared to 5.28 nM in MCF7, suggesting that the method of culture of the TU-BcX-4IC cells had a greater effect on the response of these cells than it did for the MCF7 cells.

Figure 2. Compound response in 3D vs. 2D cultured MCF7 cells. MCF7 cells cultured in 3D (red) vs. 2D (blue), were treated with romidepsin. IC⁵⁰ values were 5.28 nM and 1.88 nM in 3D and 2D, respectively.

Figure 3. Compound response in TU-BcX-4IC cells cultured in 3D vs. 2D. Red plots represent cells cultured in 3D, while blue plots are cells cultured in 2D. A, romidepsin-treated cells; B, bortezomib; C, ibrutinib; D, idarubicin; E, panobinostat; F, trametinib.

Table 3. . IC₅₀ values for compound-treated cells. Values are listed for MCF7 and TU-BcX-4IC cells cultured as spheroids (3D) or monolayer (2D). IC50 concentrations shown are nM.

Conclusion

Assays that use luciferase to generate a luminescent readout of relative ATP concentrations in cells under different experimental conditions provide a convenient way to rapidly assess cell viability. Cells can be placed in U-bottom plates, where they readily form spheroids and can be taken all the way through compound treatment and ATP assay without the need to wash wells or transfer spheroids. The resulting data on cell viability may be interesting on its own, and it may also bring to the researcher’s attention compounds or treatments that warrant further investigation by other methods, such as high-content imaging.

In this application note, we have demonstrated the feasibility of using the CellTiter-Glo 3D assay to compare the results of treatment with different compounds on the viability of two different cell types grown as either spheroids or monolayers. The cell culture methods and assay described are easy to set up and amenable to medium- and high-throughput screening. The SpectraMax iD5 reader provides the sensitivity needed for a robust assay readout, with data analysis and curve fitting that is automated by SoftMax Pro Software.

References

• Sirenko O, Brock CK, Lim A, Macha P, Nikolov E, Olsen C, McConnell EC, Wright M, Collins-Burow BM, Cromwell EF, and Burow ME. Evaluating Drug Response in 3D Triple Negative Breast Cancer Tumoroids with High Content Imaging and Analysis. 3 August 2022, PREPRINT (Version 1) available at Research Square https://doi.org/10.21203/rs.3.rs-1859525/v1.
• . Matossian MD, Chang T, Wright MK, Burks HE, Elliott S, Sabol RA, Wathieu H, Windsor GO, Alzoubi MS, King CT, Bursavich JB, Ham AM, Savoie JJ, Nguyen K, Baddoo M, Flemington E, Sirenko O, Cromwell EF, Hebert KL, … Burow ME. In-depth characterization of a new patient-derived xenograft model for metaplastic breast carcinoma to identify viable biologic targets and patterns of matrix evolution within rare tumor types. Clin Transl Oncol 24, 127–144 (2022). https://doi.org/10.1007/s12094-021-02677-8

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