Tools for Measuring Cell Proliferation

Normal cell proliferation affects every aspect of cell-based life. Its measurement helps researchers to understand cells in context, whether during embryonic development, in the midst of disease, or during response to a test drug. “The investigation of cell proliferation arms researchers in various fields with valuable information about the growth, health, and behavior of cells within a given system,” says Afrida Rahman-Enyart, Scientific Liaison and Product Manager at Proteintech. “Since cell proliferation occurs as a natural biological process throughout the body or the outcome of various diseases, it is a relevant area of study for most of the life sciences from developmental biology to oncology.” This article looks at how cell proliferation measurements advance research across many fields, from basic research to disease biology and therapeutics.

Measuring cell proliferation

Measurements of cell proliferation are a crucial indicator of cell health, and “can be used as a readout when screening for activity of potential therapeutic compounds,” says Susan Keezer, Associate Director of Cell Biology at Cell Signaling Technology. But the best method for measuring cell proliferation depends upon the experiments and questions under study.

Generally, measurement is either direct or indirect. There are two types of direct methods: labeling new DNA in S phase cells that incorporate nucleoside analogs (BrdU, CldU, IdU and EdU); and using antibodies to detect specific proteins only expressed in proliferating cells (e.g., PCNA, Ki67 and MCM2). “Detection of these proteins with antibodies distinguishes proliferating from non-proliferating cells in a population, such as a tissue or a tumor,” says Keezer.

Some experiments may be more compatible with indirect methods, of which there are two main types: measuring cellular metabolism (e.g., MTT, XTT, or WST-1 assays); and observing effects of cell viability dyes such as Trypan Blue. Though these assays offer indirect measurements, they are useful for determining changes in the number of live cells—in contrast to a direct measurement using a BrdU assay, for example, which involves denaturing DNA.

Increasingly, cell proliferation assays are likely to be scaled up and moving faster. “The future of assays is high-throughput format and scale, particularly in drug discovery and other non-academic settings,” says Robin Clark, Global Product Manager, Cell Biology Reagents and Tools at MilliporeSigma (the life science business of Merck KGaA in the U.S. and Canada). Some proliferation detection methods are more amenable to higher samples and/or automation. For example, “the EdU assay does not require radioactive isotopes or immunodetection, unlike other thymidine analog approaches that are incorporated into DNA as cells replicate, making it a smart choice when throughput is critical,” says Clark.

Ultimately, the method [of cell proliferation assay] chosen will depend on the experiment and available lab resources, but should also take into account required sensitivity and throughput levels, according to Fang Tian, Director of Biological Content at ATCC. “For example, pathology labs use cell cycle marker Ki67 to study patient tumor samples, visualized directly by immunofluorescence,” says Tian. “But biopharmaceutical researchers doing drug screening might prefer an indirect metabolic-based cell proliferation assay, because it provides the high sensitivity and high throughput needed for plate-based screening.”

Cell proliferation encompasses many fields

The fundamental nature of cell proliferation means that it influences many different fields of research, including cancer, tissue repair, and immunology. “Direct proliferation measurement methods are important in cancer research, studies of immune cell function, and in specific treatments such as IVF, where proliferation and cell numbers have profound effects on embryonic development,” says Clark.

Immunology and stem cell research also benefit from a greater understanding of cell proliferation. “Immune system function relies on T-cell proliferation to maintain a repertoire of antigen specificity,” says Clark. [Additionally], “BrdU incorporation into limbal stem cells has been used in corneal stem cell research [to determine] proliferating properties of restricted progenitor cells.”

Any field that involves tissue growth or repair depends on increasing cell proliferation. “As cell propagation and growth are vital for repair following injury, scientists can also harness the power of cell proliferation for regenerative medicine, wound healing, and tissue engineering,” says Rahman-Enyart. Almost any cell-based field of medicine can benefit from greater insight into the mechanisms of cell proliferation. “Neurogenesis—proliferation and differentiation of neural stem cells—is studied extensively in neuroscience to further understand neurodevelopment and develop potential treatments for neurodegenerative diseases, such as Alzheimer’s,” says Rahman-Enyart.

Cell proliferation in cancer

A hallmark of cancer is cell proliferation that has gotten out of control. “Cancer cells typically have defective cell cycle checkpoint pathways and uncontrolled proliferation,” says Keezer. Because cancer cells exhibit sustained proliferation, understanding proliferative mechanisms is helping to advance cancer research and therapeutics. For example, the drug palbociclib, originally used as a CDK inhibitor, was shown to inhibit cell proliferation in an early breast cancer study. Sold as Ibrance by Pfizer, it is currently approved to treat patients with HR+, HER2- metastatic breast cancer. “In 2009, researchers at Pfizer and UCLA used cell proliferation as a readout to determine which human breast cancer cell lines, representing various molecular subtypes, were sensitive to palbociclib,” says Keezer. “This study was one of the first steps in getting palbociclib to the clinic, and it helped predict which patients would respond to the drug.”

Furthermore, cancer cells themselves may hold the keys to turning off their harmful growth characteristics. “Evaluating how cancer cells proliferate can assist in the development of therapies that target and inhibit this growth,” says Rahman-Enyart. And this may even extend to guiding therapies in the clinic, as “proliferation measurement is used increasingly to measure tumor aggressiveness,” says Clark.

Generating new research tools

Understanding the fundamentals of cell proliferation also provides opportunities to generate research tools. “People understand the value of primary cells, coming fresh from tissue and having more physiological relevance, but primary cells have limited lifespans and cannot proliferate forever,” says Tian. ATCC has used knowledge of cell proliferation to generate human telomerase reverse transcriptase (hTERT)–immortalized primary cells, which retain the physiology of primary cells, yet exhibit the extended growth and greater reproducibility of continuous cell lines. “With this unique program we keep primary cells proliferating so they continue to be physiologically relevant, and give the user more reproducibility,” she says.

Such tools accompanied by expertise are continuing to uncover important details about cell proliferation, further advancing our understanding of the mechanisms controlling cell growth and renewal.