Using Protein Markers to Study Cell Proliferation in 2D and 3D

Cell proliferation is a fundamental property of cell populations and is essential to assess in a variety of biological contexts, particularly in studies of development, cancer progression, and cellular responses to therapeutic compounds. For standard two-dimensional (2D) cultures, several well-established methods are available to measure proliferation.

Dye dilution assays incorporate fluorescent dye (such as CFSE) that can be incorporated into cells to trace multiple rounds of cell division. DNA synthesis methods incorporate nucleoside analogs (BrdU and EdU) that can be measured in proliferating cells actively undergoing new DNA synthesis. Measuring the expression of specific protein markers involved in proliferation, such as cell cycle proteins, provides another direct means of assessing proliferation. Metabolic assays provide an indirect measure of proliferation by quantifying the conversion of substrates such as resazurin or tetrazolium salts, or by measuring intracellular ATP levels. Live-cell imaging technologies have enabled label-free, real-time monitoring of cell proliferation using specialized imaging platforms with integrated environmental control systems.

In this article, we focus on the benefits of using protein markers for studying cell proliferation and explore how these can be applied in the context of 3D cell culture models.

Proteins for assessing cell proliferation

Detection of key protein markers provides a direct and quantitative window into cellular proliferation, complementing conventional proliferation assays and enabling multi-target profiling alongside other proteins of interest. Among these is Ki-67, a well-known proliferation marker that is always present in dividing cells. Other common markers include cell cycle proteins, such as PCNA, MCM family proteins, and p-Histone H3 (Ser10).

“Using an antibody to detect proliferation is a solid approach while multiplexing with other protein markers. This allows you to relate proliferation to another variable such as levels for your protein of interest or to identify specific cell types,” says Virginia Bain, Ph.D., Immunofluorescence Group Leader at Cell Signaling Technology.

“You might also turn to a protein marker to look at specific phases of the cell cycle, for instance Ki-67 detects proliferating cells in G1, S, G2, and mitosis, but not in the G0 resting phase, while PCNA levels are highest during S phase and phospho-Histone H3 (Ser10) levels are highest during mitosis. You might also include an antibody when working with a thymidine analog like BrdU. There is also some fascinating work done on measuring the mean duration of synthesis phase, which involves pulse labeling with multiple thymidine analogs like BrdU and EdU.”

An important advantage of protein markers is that they are directly related to the proliferative activity of cells. “Ki-67 protein expression is exquisitely tied to the proliferative state of cells and not to the number of cells in an assay well. In contrast, measures of viable cell number, using either metabolic surrogates or total DNA content, cannot effectively distinguish 10,000 proliferating cells from 10,000 nonproliferating, quiescent cells,” says Dan Lazar, Ph.D., Senior Research Scientist at Promega.

Cell cycle proteins such as Ki-67 can also exhibit relatively fast responses, which can be a useful property when conducting proliferation studies in culture. “For instance, while it can take three or more days after activation of quiescent PBMC or T cells to begin to see significant increases in cell number, dramatic increases in Ki-67 expression can already be observed within only 48 hours. Likewise, while treatment of cancer cells with a strong anti-proliferative agent might result in a modest 20% decrease in cell number (relative to untreated cells) within the first 24 hours, Ki-67 levels can decrease 70–80% within that same timeframe,” notes Dr. Lazar.

“The early and dynamic responsiveness of Ki-67 expression mitigates challenges faced by proliferation assays, which require longer treatment times, including the need for medium changes, drug redosing, and the confounding of antiproliferation studies by untreated control wells approaching confluence. Although the assay of new DNA synthesis offers some of the benefits of a Ki-67 assay, the commonly used BrdU and EdU assays require labor-intensive and nonhomogeneous methods, whereas the Lumit^® hKi-67 Immunoassay for Cell Proliferation is completely homogeneous, fast, and easy to execute.”

Protein markers are also particularly valuable in scenarios where cells may increase in size without undergoing division, as is often observed in cancer cells. For example, in response to anticancer drugs such as CDK inhibitors, cancer cells may arrest proliferation yet continue to grow substantially in size.

“This significant increase in cell volume, along with the corresponding increase in the levels of metabolic markers, can cause metabolic assays of cell viability to underreport or miss antiproliferative activity. In contrast, the assay of Ki-67 levels is refractory to this biological phenomenon and easily picks up the expected inhibition of cell proliferation. Given that cell growth has been reported to occur with other classes of antiproliferative agents, this provides a compelling argument for the assay of Ki-67 levels for the discovery of antiproliferative agents,” says Dr. Lazar.

Studying proliferation in 3D cultures

Transitioning from 2D to 3D cell culture models, such as spheroids and organoids, the use of protein markers such as Ki-67 continues to be a valid method for assessing proliferation, despite major differences in culture microenvironments. Small spheroids are known to display a homogeneous Ki-67 immunofluorescence staining pattern, while in larger spheroids the staining tends to be restricted to the outermost cell layers. In larger spheroids, a 3D imaging method that separates nuclear and cytoplasmic components makes it possible to quantify the distribution of Ki-67 and other protein markers even at the single-cell resolution. In multicellular tumor spheroids (MCTS), which typically feature three cell layers reflective of the heterogeneity in solid tumors, Ki-67 and PCNA tend to be found in the outermost proliferating layer.

It is important to remember that the 3D culture model introduces changes to the cellular microenvironment that can impact the performance and interpretation of cell-based assays traditionally developed for 2D cultures. The extracellular matrix or hydrogel components commonly used in 3D systems can interfere with optical measurements, such as fluorescence and absorbance. The low porosity of gels can also limit the permeability and diffusion of reagents and large molecules like antibodies or protein-binding drugs. Organoids, in particular, exhibit more advanced cellular differentiation and tissue-like architecture than spheroids, which can also affect the expression of certain markers. 

There are several considerations to ensure the proliferation assay appropriately accounts for the more complex 3D cultures. More preparative steps need to be taken prior to the assay, such as media exchange, hydrogel digestion, tissue clearing, or cell lysis, to ensure optimal readings. Reagents, such as assay kits and antibodies, should be validated, either by the manufacturer or independently, for use in 3D cell culture assays. In the case of organoids, it would be beneficial to use antibodies validated in immunofluorescence microscopy using methods such as whole-mount immunostaining. 

“The thickness of a 3D model, the nature of an ECM or hydrogel and the physical design of the plates or chips used to create the model can all influence the execution and performance of an assay. For the Lumit^® hKi-67 Immunoassay for Cell Proliferation, we have demonstrated the provided lytic reagent can effectively expose intracellular Ki-67 protein from cancer spheroids as large as 600 µm in diameter for subsequent detection by the luminescent assay chemistry. What’s more, with the Lumit^® hKi-67 assay, there’s no requirement to remove the culture medium before initiating lysis, an important convenience in the assay of scaffold-free, 3D models,” says Dr. Lazar. 

Reagent manufacturers are cognizant of the specialized needs for cells in 3D. As spheroids and organoids become more widespread, we can anticipate a growing availability of viability and proliferation assay technologies specifically optimized for the structural and biochemical complexities inherent to these advanced models. 

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