Evolving research methods in cytology

With more than 200 cancer types identified,¹ oncology research is a vast and complex field providing constant challenges for research scientists. Intensive research into the mechanisms behind oncogenesis and the identification of biomarkers for cancer has led to the successful development of a number of anticancer therapies and, more recently, to therapies directed toward cancer-specific molecular targets. This article attempts to highlight the evolution of the methods and technologies used for the identification of biological targets and the evaluation of anti-cancer therapeutics, which has seen rapid and significant changes over the past 10 to 15 years. These changes have involved the progression from simple biochemical assays to in vitro cell-based assays and, more recently, the study of 3D models such as cell colonies, tissues, and whole organisms.

Early research identifying biologically relevant targets and studies of the effect of inhibitory compounds on them used traditional biochemical assays. Not only were these assays time-consuming, but the study of a single biochemical reaction did not provide sufficient information about physiological interactions or the membrane permeability of potentially therapeutic compounds. Along with the increasing knowledge of the complexity of biological pathways and the number of compounds being synthesised, it has become essential to minimize the number of potential drugs failing at later stages of the drug discovery process by obtaining as much information about potential new drug/compounds at an early stage.

Cell-based assays and High Content Screening (HCS)

The development of image-based microscopy for cell-based assays has proved invaluable for the study of both oncogenesis and potential anti-cancer compounds. With the introduction of fluorescent labelling technology and fluorescent imaging microscopy it became possible to study a number of cellular morphological and phenotypic parameters (Figure 1). Furthermore, the introduction of laser-scanning technology enabled faster data acquisition, and, along with improvements in automation and software design, laser-scanning imaging cytometry now provides rapid and efficient high-content screening (HCS) capabilities for cell-based assays. There are a number of cellular processes relevant to tumor cell biology that lend themselves to laser-scanning imaging cytometry. Such processes include cellular replication (cell cycle), apoptosis, cell morphology, migration, and the expression of proteins such as oncology-related kinases which are known to be dysfunctional in many cancers.

Figure 1.
HeLa Cells stained with 1.5uM Calcein-AM. 1a. whole well scan image taken using TTP Labtech’s Acumen® eX3. Image generated as a TIFF file and analyzed using Image Pro software (Media Cybergenetics); 1b. shows representative section enlarged 10x from the whole well scan.

Cell cycle analysis

Mutations in the genes causing deregulation of cell-signaling events which control the cell cycle have been identified to be the cause of a large number of cancers. As a result, cell cycle analysis is a major area of research, and the ability to study the effects of potential anti-cancer compounds on the cell cycle is important in the drug discovery process. The effect of compounds on the cell cycle can be easily studied using laser-scanning imaging cytometry with DNA stains such as Propidium Iodide (PI), the Hoechst stain, and, more recently, DRAQ5. These dyes allow identification of G1, S, and G2/M cell phases according to their fluorescence intensity. In addition, using laser-scanning imaging cytometry, it is possible to rapidly screen entire wells. Whole well cell-based screening enables cell number enumeration to be performed as part of the assay, thus ensuring normalization of response to cell numbers and robustness of data.

In a recent study, cell cycle analysis was employed using PI staining to study the therapeutic targeting of renal cell carcinoma cell lines expressing low concentrations of folliculin protein. Loss of function mutations in this protein have been attributed to Brit-Hogg-Dube syndrome (BHD), an autosomal dominant familial cancer associated with an increased risk of kidney cancer.² In this study it was shown that mithramycin arrested and blocked cells in the S and G2-M phases and that addition of a low dose of rapamycin potentiated mithramycin sensitivity, thus providing a rationale for the further evaluation of mithramycin as a potential drug for renal cell carcinoma associated with BHD.

Regulation of kinase activity

Kinases, which are responsible for protein phosphorylation, play a key role in the regulation of a number of cellular processes, including metabolism, replication, and angiogenesis, and defects in more than100 kinases have been implicated in the field of oncology. Studies which screen compounds that inhibit kinase activity have been successfully carried out using fluorescent antibodies against phosphorylated proteins.³ In a recent study, Phong et al. investigated the role of two kinases, p38 MAPK (a mitogen activated protein kinase) and Chk1 (a serine/threonine-protein kinase) on cell cycle arrest at the G2 DNA damage checkpoint.⁴

Using a scanning imaging cytometer with multiple lasers and a range of fluorescent reagents, it is possible to study more than one parameter simultaneously. By employing the multiplexing capabilities of this imaging system, cell enumeration was carried out by counterstaining with PI. Using a second fluorescently labelled antibody conjugate against the phosphorylated protein, phospho-histone H3, the effect of the inhibition of p38 and Chk 1 on G2 mediated checkpoint arrest was quantified. The use of multiplexing helped to provide robust data showing that although inhibition of Chk1 activity prevents G2 damage and checkpoint arrest by anti-cancer drugs, p38 is not involved in G2 mediated checkpoint arrest. However, p38 was shown to play an important pro-survival role through the regulation of apoptotic and survival pathways that allow cells to recover from DNA damage, suggesting that p38 MAPK activity may play a role in resistance to chemotherapy.

Cell migration and chemotaxis

The study of cell migration assays using laser scanning imaging cytometry enables direct visualisation of the cells at multiple time points during the assay. Using multiplexed staining with multiple laser imaging, it is possible to obtain reliable and quantitative high throughput imaging information on the phenotypic features of migrating cells alongside changes in various cellular processes such as cellular differentiation states. Recently, Gough et al. demonstrated the inhibition of endothelial colony forming cells (ECFCs) by a Src kinase inhibitor, dasatinib, highlighting the use of this technology to measure cellular migration to classify compounds by biological phenotype early in the drug discovery process.⁵

The future: 3D imaging analysis

Although 2D cell-based HCS is a well established technology, recent studies highlighting differences in drug sensitivities between cancer cell lines and 3D cultures suggest that the value of using cell-based assays in predicting clinical response is limited.^6,7 These differences are thought to be due to the fact that cells cultured in 3D often adopt a different morphology, gene expression profile, and growth rate compared to cells cultured on plates.^8,9 In vivo tumor cells are supported by an extracellular matrix microenvironment which plays an important role in resistance to penetration by anti-cancer drugs. With an increasing demand for obtaining maximum information early in the drug discovery process, 3D models such as soft agar assays have been employed to simulate the tumor microenvironment by providing additional knowledge on drug permeability, interactions, and toxicity.

Using microscope-based technology, the process of locating cell colonies in soft agar and obtaining colony profile images requires continual refocusing at different depths of field, making it unsuitable for high throughput screening assays. However, the use of nonconfocal cytometers with wide field objective lenses allows simultaneous and rapid scanning of entire wells to assess colony number and growth. A high depth of field eliminates the need to focus between wells, enabling high scan speeds of a plate and providing an automated high content readout.¹⁰ Laser scanning imaging cytometers with multiple laser facilities allow additional information to be obtained—for instance, the effect of compounds on proliferating cells within the colony. High-quality laser scanning imaging cytometers, capable of scanning larger organisms such as Drosophila larvae, C. elegans and even Zebra fish, can provide further information on multicellular drug interactions (Figure 2).

Conclusion

Laser scanning imaging cytometers are becoming a popular method of choice for cell-based HCS assays, with applications in both the clinical laboratory and throughout the entire drug discovery process. The ability to acquire data using multiple lasers, with instruments such as TTP LabTech’s Acumen ^eX3, enables researchers to analyze multiple events within a cell, thereby gaining information about potential mechanisms and efficacy of novel therapeutic compounds. Not only are laser scanning imaging cytometers able to provide information on the effect of compounds on colony formation and tissue structure, but it is also possible to study more than one cell phenotype in an assay. Analysis of cells within mixed cultures such as endothelial cells, bone cells, and macrophages can provide information, simulating as close as possible the in vivo multi-cellular tumor microenvironment.

Figure 2.
GFP labelled Zebrafish image acquired by TTP LabTech’s Acumen ^eX3 software.

The study of 3D models using multiplexing and laser-scanning imaging technology also may provide increased knowledge about tumor development and physiology, potential drug interactions, and toxicity at all stages of clinical analysis and drug discovery.

The rapid advances in laser-scanning imaging technologies over the past 10 years have provided researchers with tools to gain new insights into the mechanisms of disease and are helping to accelerate the development of more targeted therapeutics. With laser-scanning imaging cytometry and fluorophore technology continually evolving, the versatility and application repertoire of this technology will enhance research.

Wendy Gaisford, PhD, is the Medical Writer at TTP LabTech, a UK-based company specializing in the development and design of innovative instruments for pharmaceutical and biotech research. For further information visit www.ttplabtech.com

References

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