Cell viability assays are a bread-and-butter method for many researchers using cultured cells —everyday lab tools that are a part of many newsworthy papers, but rarely make news themselves.
Over time, cell viability assays have become easier to use and more “plug ‘n play”. Among modern assays, luminescent plate-reader based systems have been a favorite for several years because of their superior sensitivity, robustness, simple protocols and uncomplicated equipment requirements (all you need is a plate-reading luminometer). These qualities combine to allow easy scalability and adaptability from bench research to high throughput applications.
CellTiter-Glo® Luminescent Cell Viability Assay is an accepted go-to viability assay for many researchers. The assay measures ATP as an indicator of metabolically active cells. A quick search on Google Scholar returns 3,990 CellTiter-Glo results for 2017 and over 500 so far in January and February of 2018. A sampling of these recent publications gives a snapshot of some of the ways the CellTiter-Glo assay is used to support key areas of research today.
Does a treatment kill cells?
The obvious application of a cell viability assay is to understand whether cells are alive. In cancer research, the CellTiter-Glo assay is often used to confirm killing of tumor cells and to verify that normal cells survive. Therefore, these assays are a key part of the evaluation and screening of drug candidates and other therapies for cancer. Many papers reporting use of CellTiter-Glo are developing and evaluating the effectiveness of novel anti-cancer treatments.
Development of immunotherapies specifically targeting tumor cells has been one of the most exciting scientific advances in recent years. Several papers published in December and January used the CellTiter-Glo assay in studies evaluating effectiveness of new immunotherapies for various tumor types.
Can cancer cells be targeted with an antibody, then killed with light?
In a paper published in the International Journal of Cancer in January, authors Burley et al used the CellTiter-Glo assay to directly assess the effectiveness of a photo-activated approach to treatment of glioblastoma cells in 2D and 3D culture. Glioblastomas are aggressive brain tumors characterized by alterations in the epidermal growth factor receptor. The authors used an affibody directed against the EGF receptor to deliver a photo-reactive dye to glioblastoma cells in culture. When the dye is stimulated with near-infrared light, reactive oxygen is generated, killing the tumor cells. The CellTiter-Glo assay was used to assess viability in treated cells and spheroids after light exposure, and also to show that cells not exposed to light remained viable. The treatment successfully caused cytotoxicity in treated EGFR-positive cells, while limiting toxicity in normal cells.
Faster screening of lead antibodies
A paper by Bhatka et al, published online in Molecular Cancer Therapies in late December, illustrates use of the CellTiter-Glo assay to evaluate the effectiveness of antibody drug conjugates in small-scale screening experiments. In this paper, various anti-GDNF antibodies conjugated to candidate drug compounds were evaluated for their ability to kill breast cancer cells. Small-scale screening for functional antibodies was performed in 96-well plate format using 16 antibody drug conjugates and the CellTiter-Glo assay. The plate-based cell viability assay format allowed initial evaluation of lead antibodies in a way that preserved materials and saved time and labor, allowing efficient selection of the most promising candidates for further evaluation.
Screening and selection of the lead antibodies based on ADC-mediated cell killing would be far superior, but would be labor intensive and cost ineffective at typical 10-20mg scales. We therefore developed a 96-well format small-scale conjugation protocol (<1mg ADC) to conjugate all 16 murine anti-GFRA1 antibodies with a cleavable linker-payload. Bhatka et al 2017.
In addition to the basic question “Does my compound kill cells?” cell viability assays are also used to help determine how much of a test compound is required for toxicity to occur.
Evaluating Immunotoxin effectiveness: Evaluating dose:response and understanding competition
Published Dec 22 in Nature Scientific Reports, Kollmorgen et al., report that an immunotoxin composed of an anti-mesothelin antibody fragment and Pseudomonas exotoxin A shows promise for targeting and killing ovarian cancer cells. Immunotoxins are antibody fragments directed against specific target cell proteins and coupled to a toxic “payload” that is released into the cell upon antibody binding. The cell surface protein mesothelin has been pursued as a target for immunotherapy because it is overexpressed in certain cancers and is not expressed in vital organs.
These authors tested whether a circulating protein CA-125, present in ovarian cancers and known to bind mesothelin, could reduce the effectiveness of the immunotoxin by competing for mesothelin binding sites and blocking activity. To test this, they performed dose-response experiments using the CellTiter-Glo assay to determine that immunotoxin activity was not affected, even in the presence of excess CA-125, and so concluded that inhibition of effectiveness by CA125 was not an issue.
Cell viability assays are also used in antiviral research. In these studies, they are usually used to ensure that antiviral agents prevent viral replication, but do not kill host cells .
Searching for Zika and Ebola antiviral therapies: Ensuring killing of the right targets
Two papers published in January show how cell viability assays are used in the evaluation of antiviral drug compounds. In a study searching for compounds effective against Ebola virus, Luthra et al used CellTiter Glo in 384-well plate format to identify antiviral compounds with low toxicity to host cells. Having identified a candidate compound, they then synthesized and tested various concentrations in dose-response experiments to determine the 50% inhibitory concentration and the 50% cytotoxicity concentration—ideally there would be a large difference between the concentration that inhibited Ebola replication and the concentrations causing significant cytotoxicity.
In a January Virus Research paper evaluating various cell lines as hosts for Zika replication and antiviral testing, Vicenti et al took a similar approach. Having identified a Zika-friendly cell line, they tested the antiviral activity of two test compounds in that cell line, and again used CellTiter-Glo assay in companion viability assays to verify that the concentrations of drug that inhibited viral replication did not also kill the host cells.
500 2018 publications and counting
These papers show how CellTiter-Glo and other cell viability assays play a vital supporting role in areas of cell biology like biologics, immunotherapy, anti-viral and anti-cancer research, and more. In these publications, the cell viability assay may not be the most prominent experiment, but perhaps that is the point. A good cell viability assay will never get in your way, but will always make it easier to get the job done.
Here are the papers:
- Bhakta, S. et al., (2017) An anti-GDNF Family Receptor Alpha 1(GFRA1) Antibody-Drug Conjugate for the Treatment of Hormone Receptor-Positive Breast Cancer. Published Online December 27; DOI: 10.1158/1535-7163.MCT-17-0813
- Burley, T.A., et al., (2018) Near-infrared photoimmunotherapy targeting EGFR—Shedding new light on glioblastoma treatment. Int. J Cancer Jan 5. doi: 10.1002/ijc.31246. [Epub ahead of print]
- Kollmorgen et al., (2017) A re-engineered immunotoxin shows promising preclinical activity in ovarian cancer. Nature Scientific Reports Dec 22, 2017 DOI:10.1038/s41598-017-17329-7
- Luthra et al., (2018) A high throughput screen identifies benzoquinoline compounds as inhibitors of Ebola virus replication. Virus Research 244, 64–70.
- Vicenti, I., et al., (2018) Comparative analysis of different cell systems for Zika virus (ZIKV) propagation and evaluation of anti-ZIKV compounds in vitro. Antiviral Research 150, 193-201.
Learn more about the CellTiter-Glo® assay here.