A rapid and sensitive fluorometric screening assay using YO-PRO-1 to quantify tumour cell invasion through Matrigel
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A new quantitative assay for the study of tumour cell invasionin vitro is described. Employing the novel fluorescent dye YO-PRO-1, cells that penetrate Matrigel-coated transwells are counted on the basis of dye-bound cellular nucleic acid content. Following transmigration, the cells in the lower compartments are lysed by freezing in water. After a brief incubation with YO-PRO-1, nucleic acid or DNA content is measured as fluorescence intensity in 96-well microplates and quantitated by a cell- or DNA-calibration curve. Using standard curves, a linear relationship between fluorescence intensity and cell number was found in the range tested (from 100 to 80 000 cells). The mean relative intra- and inter-assay variability of the cell quantitation in this range was 3.5 and 4.2%, respectively. When applied to Matrigel invasion studies, as few as 400 cells could be counted. The quantitation could be performed within 3 h. HCT 116, MDA MB 231 and HT 29 cells were investigated as examples of tumour cells with different invasive abilities in the 48-h Matrigel invasion assay. Using YO-PRO-1, 6.5 ± 0.6% invasive HCT 116 cells and 52.6 ± 4.5% MDA MB 231 cells (percentage of the inoculated cell population) were measured. HT 29 cells were practically non-invasive. These results were confirmed by visual scoring of DAPI-stained nuclei. In conclusion, the main advantages of the assay are its sensitive, reproducible and rapid quantitation of tumour cell invasionin vitro and the applicability to extended sample numbers by measuring in 96-well microplates.
- Liotta LA, 1984, Tumor invasion and metastases: role of the basement membrane.Am J Pathol,117, 339–48.
- Terranova VP, Liotta LA, Russo RG and Martin GR, 1982, Role of laminin in the attachment and metastasis of murine tumor cells.Cancer Res,42, 2265–9.
- Jones PA and DeClerck YA, 1980, Destruction of extracellular matrices containing glycoproteins, elastin and collagen by metastatic human tumor cells.Cancer Res,40, 3222–7.
- Kramer RH and Vogel KG, 1984, Selective degradation of basement membrane macromolecules by metastatic melanoma cells.J Natl Cancer Inst,72, 889–97.
- Turpeenniemi-Hujanen T, Thorgeirsson UP, Rao CN, and Liotta LA, 1986, Laminin increases the release of type IV collagenase from malignant cells.J Biol Chem,261, 1883–9.
- Mignatti P, Robbins E and Rifkin DB, 1986, Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade.Cell,47, 487–98.
- Hujanen ES and Terranova VP, 1985, Migration of tumor cells to organ-derived chemoattractants,Cancer Res,45, 3517–21.
- Poste G, Doll J, Hart IR and Fidler IJ, 1980,In vitro selection of murine B16 melanoma variants with enhanced tissue-invasive properties.Cancer Res,40, 1636–44.
- Liotta LA, Lee WC and Morakis DJ, 1980, New method for preparing large surfaces of intact basement membrane for tumor invasion studies.Cancer Lett,11, 141–7.
- Hart IR and Fidler IJ, 1978, Anin vitro quantitative assay for tumor cell invasion.Cancer Res, 38, 3218–24.
- Starkey JR, Hosick HL, Stanford DR and Liggitt HD, 1984, Interaction of metastatic tumor cells with bovine lens capsule basement membrane.Cancer Res,44, 1585–94.
- Terranova VP, Hujanene ES, Loeb DMet al. 1986, Use of a reconstituted basement membrane to measure cell invasiveness and select for highly invasive tumor cells.Proc Natl Acad sci USA, 83, 465–9.
- Albini A, Iwamoto Y, Kleinman HKet al, 1987, A rapidin vitro assay for quantitating the invasive potential of tumor cells.Cancer Res,47, 3239–45.
- Repesh LA, 1989, A newin vitro assay for quantitating tumor cell invasion.Invasion Metastasis,9, 192–208.
- Parish CR, Jacobsen KB, and Coombe DR, 1992, A basement-membrane permeability assay which correlates with the metastatic potential of tumour cells.Int J Cancer,52, 378–83.
- Mosmann T, 1983, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.J Immunol Meth,65, 55–63.
- Schlechte W, Brattain M and Boyd D, 1990, Invasion of extracellular matrix by cultured colon cancer cells: dependence on urokinase receptor display.Cancer Commun, 2, 173–9.
- Imamura H, Takao S and Aikou T, 1994, A modified invasion-3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay for quantitating tumor cell invasion. Cancer Res,54, 3620–4.
- Rago R, Mitchen J and Wilding G, 1990, DNA fluorometric assay in 96-well tissue culture plates using Hoechst 33258 after cell lysis by freezing in distilled water.Anal Biochem,191, 31–4.
- Adams CJ and Storrie B, 1981, A simple DNA-dependent fluorescence enhancement assay for cell number.J Histochem Cytochem,29, 326.
- Blaheta RA, Franz M, Auth MKH, Wenisch HJC and Markus BH, 1991, A rapid non-radioactive fluorescence assay for the measurement of both cell number and proliferation.J Immunol Meth,142, 199–206.
- Wan CP, Sigh RV and Lau BHS, 1994, A simple fluorometric assay for the determination of cell numbers.J Immunol Meth,173, 265–72.
- Haughland RP, 1992, Nucleic acid stains. In: Larison KD, ed. Handbook of Fuorescent Probes and Research Chemicals, 5th edition. Eugene, OR, Molecular Probes, pp. 221–9.
- Grotendorst GR, 1987, Spectrophotometric assay for the quantitation of cell migration in the Boyden chamber chemotaxis assay.Methods Enzymol,147, 144–52.
- Kinsella AR, Lepts GC, Hill CL and Jones M, 1994, Reduced E-cadherin expression correlates with increased invasiveness in colorectal carcinoma cell lines.Clin Exp Metastasis,12, 335–42.
- Hollas W, Blasi F and Boyd D, 1991, Role of the urokinase receptor in facilitating extracellular matrix invasion by cultured colon cancer.Cancer Res,51, 3690–5.
- Price JE, Polyzos A, Zhang RD and Daniels LM, 1990, Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice.Cancer Res,50, 717–21.
- Fraker LD, Halter SA and Forbes JT, 1984, Growth inhibition by retinol of a human breast carcinoma cell linein vitro and in athymic mice.Cancer Res,44, 5757–63.
- de Vries JE, Dinjens WNM, DeBruyne GKet al. 1995,In vivo andin vitro invasion in relation to phenotypic characteristics of human colorectal carcinoma cells.Br J Cancer,71, 271–7.
- A rapid and sensitive fluorometric screening assay using YO-PRO-1 to quantify tumour cell invasion through Matrigel
Clinical & Experimental Metastasis
Volume 14, Issue 5 , pp 451-458
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