Aclonogenic assayis a cell biology technique for studying the effectiveness of specific agents on the survival and proliferation of cells. It is frequently used incancer researchlaboratories to determine the effect of drugs or radiation on proliferatingtumorcells[1]as well as for titration of Cell-killing Particles (CKPs) in virus stocks.[2]It was first developed byT.T. PuckandPhilip I. Marcusat the University of Colorado in 1955.[3]
Although this technique can provide accurate results, the assay is time-consuming to set up and analyze and can only provide data on tumor cells that can grow in culture. The word "clonogenic" refers to the fact that these cells areclonesof one another.
Procedure
editThe experiment involves three major steps:
- The treatment is applied to a sample of cells.
- The cells are "plated" in atissue culturevessel and allowed to grow.
- The colonies produced are fixed, stained, and counted.
At the conclusion of the experiment, thepercentageof cells that survived the treatment is measured. A graphical representation of survival versus drug concentration or dose ofionizing radiationis called acell survival curve.[4]
ForCell-killing Particle assays,the surviving fraction of cells is used to approximate the Poisson Distribution of virus particles amongst cells and therefore determine the number ofCKPsencountered by each cell.
Any type ofcellcould be used in an experiment, but since the goal of these experiments in oncological research is the discovery of more effective cancer treatments, human tumor cells are a typical choice. The cells either come from prepared "cell lines," which have been well-studied and whose general characteristics are known, or from abiopsyof a tumor in a patient.[5]The cells are put inpetri dishesor in plates which contain several circular "wells." Particular numbers of cells are plated depending on the experiment; for an experiment involving irradiation it is usual to plate larger numbers of cells with increasing dose of radiation. For example, at a dose of 0 or 1grayof radiation, 500 cells might be plated, but at 4 or 5 gray, 2500 might be plated, since very large numbers of cells are killed at this level of radiation and the effects of the specific treatment would be unobservable.
Counting the cell colonies is usually done under amicroscopeand is quite tedious. Recently, machines have been developed that usealgorithmsto analyse images.[6]These are either captured by animage scanneror an automated microscope that can completely automate the counting process.[7]One such automated machine works by accepting certain types of cell plates through a slot (not unlike a CD player), taking a photograph, and uploading it to a computer for immediate analysis. Reliable counts are available in seconds.
Variables
editThe treatment is usually adrug,ionizing radiation,or a combination of the two.[8]Some current research studies the potentiation of drug effects by concurrent irradiation—asynergisticeffect—and in this situation two groups are studied: a control group, which is not treated with the drug; and a treatment group, which is treated with the drug. Both groups are irradiated. If the slopes of their survival curves differ significantly, then a potentiating effect may be evident and could be studied further. Since many tumor cells won't grow colonies in culture, cell proliferation assay, which has a satisfactory accuracy reportedly in measuring synergistic effects between ionizing radiation and drugs, may be used as a surrogate[9]
A thorough discussion of the promising research being conducted with the aid of this technique is beyond the scope of this text, but some studies involve the effect of the expression of particulargenesorreceptorson the cell, the responses of different cell types, or synergistic effects of multiple drugs.
See also
editReferences
edit- ^Hoffman, Robert M. (1991). "In vitro sensitivity assays in cancer: A review, analysis, and prognosis".Journal of Clinical Laboratory Analysis.5(2):133–43.doi:10.1002/jcla.1860050211.PMID2023059.
- ^Ngunjiri, J. M.; Sekellick, M. J.; Marcus, P. I. (2008)."Clonogenic Assay of Type a Influenza Viruses Reveals Noninfectious Cell-Killing (Apoptosis-Inducing) Particles".Journal of Virology.82(6):2673–80.doi:10.1128/JVI.02221-07.PMC2258965.PMID18184709.
- ^Transcript of TWiV interview@http:// twiv.tv/TWiV197-082612.pdf
- ^Franken, Nicolaas A P; Rodermond, Hans M; Stap, Jan; Haveman, Jaap; Van Bree, Chris (2006). "Clonogenic assay of cells in vitro".Nature Protocols.1(5):2315–9.doi:10.1038/nprot.2006.339.PMID17406473.
- ^Hamburger, Anne W. (1987). "The Human Tumor Clonogenic Assay as a Model System in Cell Biology".The International Journal of Cell Cloning.5(2):89–107.doi:10.1002/stem.5530050202.
- ^Niyazi, Maximilian; Niyazi, Ismat; Belka, Claus (2007)."Counting colonies of clonogenic assays by using densitometric software".Radiation Oncology.2:4.doi:10.1186/1748-717X-2-4.PMC1770926.PMID17212832.
- ^Dahle, Jostein; Kakar, Manish; Steen, Harald B.; Kaalhus, Olav (2004). "Automated counting of mammalian cell colonies by means of a flat bed scanner and image processing".Cytometry.60A(2):182–8.doi:10.1002/cyto.a.20038.
- ^Carney, DN; Winkler, CF (1985). "In vitro assays of chemotherapeutic sensitivity".Important Advances in Oncology:78–103.PMID3916747.
- ^Liu, Q; Meng, W (2015)."Adapting a Drug Screening Platform to Discover Associations of Molecular Targeted Radiosensitizers with Genomic Biomarkers".Molecular Cancer Research.13(4):713–720.doi:10.1158/1541-7786.MCR-14-0570.PMC4410013.PMID25667133.