Clinical issues
parameter simultaneously. By employing the multiplexing capa- bilities of this imaging system, cell enumeration was carried out by counterstaining with PI. Using a second fl uorescently labelled anti- body conjugate against the phosphorylated protein, phospho-histone H3, the effect of the inhibition of p38 and Chk 1 on G2 mediated checkpoint arrest was quantifi ed. 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 mi- grating 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 profi le, and growth rate compared to cells cultured on plates.8,9 In vivo tumor cells are sup- ported 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 profi le images requires continual refocusing at different depths of fi eld, making it unsuitable for high throughput screening assays. However, the use of nonconfo- cal cytometers with wide fi eld objective lenses allows simultaneous and rapid scanning of entire wells to assess colony number and growth. A high depth of fi eld eliminates the need to focus between wells, enabling high scan speeds of a plate and providing an auto- mated 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 fi sh, 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 effi cacy 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 in- formation, simulating as close as possible the in vivo multi-cellular tumor microenvironment.
Figure 2. GFP labelled Zebrafi sh image acquired by TTP LabTech’s Acu- men 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 fl uorophore technology continually evolv- ing, 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
1. CancerHelp UK.How many different types of cancer are there? http://cancerhelp.
cancerresearchuk.org/about-cancer/cancer-questions/how-many-different-types- of-cancer-are-there. Accessed November 2, 2011.
2. Lu X, Wei W, Nahorski MS. et al. Therapeutic targeting the loss of the Birt-Hogg- Dube Suppressor gene. Mol. Cancer Ther. 2011;10:80-89.
3. Chresta CM, Davies BR, Hickson I. et al. AZD8055 is a potent, selective, and orally bioavailable ATP-competitive mammalian target of rapamycin kinase inhibitor with in vitro and in vivo antitumor activity. Cancer Res. 2010;70:288-98.
4. Phong MS, Van Horn RD, Li S. et al. p38 mitogen-activated protein kinase promotes cell survival in response to DNA damage but is not required for the G(2) DNA damage checkpoint in human cancer cells. Mol. Cell. Biol. 2010;30:3816-26.
5. Gough W, Hulkower KI, Lynch R. et al. A quantitative, facile, and high-throughput image-based cell migration method is a robust alternative to the scratch assay. J. Biomol. Screen. 2011;16:155-63.
6. Horning JL, Sahoo SK, Vijayaraghavalu S. et al. 3-D tumor model for in vitro evalu- ation of anticancer drugs. Mol. Pharm. 2008;5:849-62.
7. Prestwich GD. Evaluating drug effi cacy and toxicology in three dimensions: using synthetic extracellular matrices in drug discovery. Acc. Chem. Res. 2008;41:139- 48.
8. Fischbach C, Chen R, Matsumoto T. et al. Engineering tumours with 3d scaffolds. Nat. Methods. 2007;4:855-60.
9. Smalley KS, Lioni M, Herlyn M. Life isn’t fl at: taking cancer biology to the next dimension. In vitro Cell. Dev. Anim. 2006;42:242-7.
10. Wylie PG, Bowen WP. Detemination of Cell Colony Formation in a high-content screening assay. JALA. 2005;203-6.
32 December 2011 ■ MLO
www.mlo-online.com
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52