Cell-based assays are more complex than cell-free test systems but still reflect a highly artificial cellular environment. Incorporation of organotypic 3-dimensional (3-D) culture systems into mainstream drug development processes is increasingly discussed but severely limited by complex methodological requirements. The objective of this study was to explore a panel of standard assays to provide an easy-handling, standardized protocol for rapid routine analysis of cell survival in multicellular tumor spheroid-based antitumor drug testing. Spheroids of 2 colon carcinoma cell lines were characterized for evaluation. One of the assay systems tested could reliably be used to determine cell viability in spheroids. The authors verified that the acid phosphatase assay (APH) is applicable for single spheroids in 96-well plates, does not require spheroid dissociation, and is linear and highly sensitive for HT29 and HCT-116 spheroids up to diameters of 650 µm and 900 µm, consisting of 40,000 and 80,000 cells, respectively. Treatment of HT29 and HCT-116 cells with 5-fluorouracil, Irinotecan, and C-1311 revealed critically reduced drug efficacies in 3-D versus monolayer culture, which is discussed in light of literature data. The experimental protocol presented herein is a small but substantial contribution to the establishment of sophisticated 3-D in vitro systems in the antitumor drug screening scenario. (Journal of Biomolecular Screening 2007:925-937)
Schmeichel KL , Bissel MJ: Modeling tissue-specific signaling and organ function in three dimensions. J Cell Sci2003 ;116:2378-2388.
2.
Kunz-Schughart LA, Freyer JP, Hofstaedter F., Ebner R.: The use of 3-D cultures for high-throughput screening: the multicellular spheroid model. J Biomol Screen2004;9:273-285.
3.
Nelson CM, Bissel MJ: Modeling dynamic reciprocity: engineering three-dimensional culture models of breast architecture, function, and neoplastic transformation. Semin Cancer Biol2005;15:342-352.
4.
Zhang X., Wang W., Yu W., Xie Y., Zhang X., Zhang Y., et al: Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing. Biotechnol Prog2005; 21:1289-1296.
5.
Freyer JP: Tumor spheroids in radiobiology research. In Bjerkvig R (ed): Spheroid Culture in Cancer Research. Boca Raton, FL: CRC Press, 1992:217-275.
6.
Olive PL, Durand RE: Drug and radiation resistance in spheroids: cell contact and kinetics. Cancer Metastasis Rev1994;13:121-138.
7.
Dubessy C., Merlin JM, Marchal C., Guillemin F.: Spheroids in radiobiology and photodynamic therapy. Crit Rev Oncol Hematol2000;36:179-192.
8.
Mueller-Klieser W.: Multicellular spheroids: a review on cellular aggregates in cancer research. J Cancer Res Clin Oncol1987;113:101-122.
9.
Kunz-Schughart LA: Multicellular tumor spheroids: intermediates between monolayer culture and in vivo tumor. Cell Biol Int1999;23:157-161.
Kunz LA, Groebe K., Mueller-Klieser W.: Oncogene-associated growth behavior and oxygenation of multicellular spheroids from rat embryo fibroblasts. Adv Exp Med Biol1994;345:359-366.
14.
Yang TT, Sinai P., Kain SR: An acid phosphatase assay for quantifying the growth of adherent and nonadherent cells. Anal Biochem1996;241:103-108.
15.
Burger AM, Jenkins TC, Double JA, Bibby MC: Cellular uptake, cytotoxicity and DNA-binding studies of the novel imidazoacridinone antineoplastic agent C1311. Br J Cancer1999 ;81:367-375.
16.
Cholody WM, Martelli S., Konopa J.: 8-Substituted 5-[(aminoalkyl)amino]-6H-v-triazolo[4,5,1-de]acridin-6-ones as potential antineoplastic agents: synthesis and biological activity. J Med Chem1990;33:2852-2856.
17.
Cholody WM, Martelli S., Konopa J.: Chromophore-modified antineoplastic imidazoacridinones: synthesis and activity against murine leukemias. J Med Chem1992;35:378-382.
18.
Marusic M., Bajzer Z., Vuc-Pavlovic S., Freyer JP: Tumor growth in vivo and as multicellular spheroids compared by mathematical models. Bull Math Biol1994;56:617-631.
19.
Kunz-Schughart LA, Groebe K, Mueller-Klieser: Three-dimensional cell culture induces novel proliferative and metabolic alterations associated with oncogenetic transformation. Int J Cancer1996;66:578-586.
20.
Chignola R., Schenetti A., Andrighetto G., Chiesa E., Foroni R., Sartoris S., et al: Forecasting the growth of multicell tumor spheroids: implications for the dynamic growth of solid tumors. Cell Prolif2000;33:219-229.
21.
Hopfner M., Maaser K., Barthel B., von Lampe B., Hanski C., Riecken EO, et al: Growth inhibition and apoptosis induced by P2Y2 receptors in human colorectal carcinoma cells: involvement of intracellular calcium and cyclic adenosine monophosphate. Int J Colorectal Dis2006;16:154-166.
22.
Netzer P., Domek M., Pai R., Halter F., Tarnawski A.: Inhibition of human colon cancer cell growth by antisense oligodeoxynucleotides targeted at basic fibroblast growth factor. Aliment Pharmacol Ther2001;15:1673-1679.
23.
Chang YH, Chao Y., Hsieh SL, Lin WW: Mechanism of LIGHT/ interferon-gamma-induced cell death in HT-29 cells. J Cell Biochem2004;93:1188-1202.
24.
Cholody WM, Kosakowska-Cholody T., Hollingshead MG, Hariprakasha HK, Michejda CJ: A new synthetic agent with potent but selective cytotoxic activity against cancer. J Med Chem2005;48:4474-4481.
25.
Kunz-Schughart LA, Freyer JP: Adaptation of an automated selective dissociation procedure to two novel spheroid types. In Vitro Cell Dev Biol Anim1997;33:73-76.
26.
Connolly DT , Knight MB, Harakas NK, Wittwer AJ, Feder J.: Determination of the number of endothelial cells in culture using an acid phosphatase assay. Anal Biochem1986;152:136-140.
27.
Martin A., Clynes M.: Acid phosphatase: endpoint for in vitro toxicity tests. In Vitro Cell Dev Biol1991;27A:183-184.
28.
Martin A., Clynes M.: Comparison of 5 microplate colorimetric assays for in vitro cytotoxicity testing and cell proliferation assays. Cytotechnology1993;11:49-58.
29.
Si F., Shin SH, Biedermann A., Ross GM: Estimation of PC12 cell numbers with acid phosphatase assay and mitochondrial dehydrogenase assay: dopamine interferes with assay based on tetrazolium. Exp Brain Res1999;124:145-150.
30.
Nicholson KM , Bibby MC, Phillips RM: Influence of drug exposure parameters on the activity of paclitaxel in multicellular spheroids. Eur J Cancer1997;33:1291-1298.
31.
Mellor HR, Ferguson DJ, Callaghan R.: A model of quiescent tumour microregions for evaluating multicellular resistance to chemotherapeutic drugs. Br J Cancer2005 ;93:302-309.
32.
Sutton JE, Roos IA, Hillcoat BL: Combined actions of 5-fluorouracil and 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea on human colonic carcinoma cells in vitro. Cancer Res1982;42:5172-5175.
33.
Pizao PE, Winograd B., Peters GJ, Leyva A., Giaccone G., Pinedo HM: In vitro chemosensitivity testing of multilayered microcultures. Anticancer Res2006;12:1319-1322.
34.
Barone RM, Calabro-Jones P., Thomas TN, Sharp TR, Byfield JE: Surgical adjuvant therapy in colon carcinoma: a human tumor spheroid model for evaluating radiation sensitizing agents. Cancer1981;47:2349-2357.
35.
Harstrick A. , Gonzales A., Schleucher N., Vanhoefer U., Lu K., Formento JL, et al: Comparison between short or long exposure to 5-fluorouracil in human gastric and colon cancer cell lines: biochemical mechanism of resistance. Anticancer Drugs1998;9:625-634.
36.
Ciccolini J. , Peillard L., Evrard A., Cuq P., Aubert C., Pelegrin A., et al: Enhanced antitumor activity of 5-fluorouracil in combination with 2′-deoxyinosine in human colorectal cell lines and human colon tumor xenografts. Clin Cancer Res2000;6:1529-1535.
37.
Lowthers EL , Richard CL, Blay J.: Differential sensitivity to short-chain ceramide analogues of human intestinal carcinoma cells grown in tumor spheroids versus monolayer culture. In Vitro Cell Dev Biol Anim2003; 39:340-342.
38.
Hofmann C., Buttenschoen K., Straeter J., Henne-Bruns D., Kornmann M.: Pre-clinical evaluation of the activity of irinotecan as a basis for regional chemotherapy. Anticancer Res2005;25:795-804.
39.
Poindessous V., Koeppel F., Raymond E., Cvitkovic E., Waters SJ, Larsen AK: Enhanced antitumor activity of irofulven in combination with 5-fluorouracil and cisplatin in human colon and ovarian carcinoma cells. Int J Oncol2003;23:1347-1355.
40.
Guichard S. , Arnould S., Hennebelle I., Bugat R., Canal P.: Combination of oxaliplatin and irinotecan on human colon cancer cell lines: activity in vitro and in vivo. Anticancer Drugs2001;12:741-751.
41.
Kusnierczyk H., Cholody WM, Paradziej-Lukowicz J., Radzikowski C., Konopa J.: Experimental antitumor activity and toxicity of the selected triazolo- and imidazoacridinones. Arch Immunol Ther Exp1994;42: 415-423.
42.
Burger AM, Double JA, Konopa J., Bibby MC: Preclinical evaluation of novel imidazoacridinone derivatives with potent activity against experimental colorectal cancer. Br J Cancer1996;74:1369-1374.
43.
Hyzy M., Bozko P., Konopa J., Skladanowski A.: Antitumour imidazoacridone C-1311 induces cell death by mitotic catastrophe in human colon carcinoma cells. Biochem Pharmacol2006,69:801-809.
44.
Augustin E. , Wheatley DN, Lamb J., Konopa J.: Imidazoacridinones arrest cell-cycle progression in the G2 phase of L1210 cells. Cancer Chemother Pharmacol1996;38:39-44.
45.
Skladanowski A., Plisov SY, Konopa J., Larsen AK: Inhibition of DNA topoisomerase II by imidazoacridinones, new antineoplastic agents with strong activity against solid tumors. Mol Pharmacol1996;49:772-780.
46.
Mazerska Z. , Dziegielewski J., Konopa J.: Enzymatic activation of a new antitumour drug, 5-diethylaminoethylamino-8-hydroxyimidazoacridinone, C-1311, observed after its intercalation into DNA. Biochem Pharmacol2001; 61:685-694.
47.
Zaffaroni N. , De Marco C., Villa R., Riboldi S., Daidone MG, Double JA: Cell growth inhibition, G2M cell cycle arrest and apoptosis induced by the imidazoacridinone C1311 in human tumour cell lines. Eur J Cancer2001;37:1953-1962.
48.
Dziegielewski J., Slusarski B., Konitz A., Skladanowski A., Konopa J.: Intercalation of imidazoacridinones to DNA and its relevance to cytotoxic and antitumor activity. Biochem Pharmacol2002;63:1653-1662.
49.
Chau M., Otake Y., Christensen JL, Fernandes DJ, Ajami AM: The imidazoacridinone, C-1311(SymadexTM): the first of a potent new class of FLT3 inhibitors [abstract B35]. Paper presented at the 2006 American Association for Cancer Research (AACR) International Conference on Molecular Diagnostics in Cancer Therapeutic Development, Chicago, September 14, 2006.
50.
Douillard JY , Cunningham D., Roth AD, Navarro M., James RD, Karasek P., et al: Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet2000;355:1041-1047.
51.
Mans DR, Grivicich I., Peters GJ, Schwartsmann G.: Sequence-dependent growth inhibition and DNA damage formation by the irinotecan-5-fluorouracil combination in human colon carcinoma cell lines. Eur J Cancer1999;35:1851-1861.
52.
Grivicich I., Regner A., da Rocha AB, Grass LB, Alves PA, Kayser GB, et al: Irinotecan/5-fluorouracil combination induces alterations in mitochondrial membrane potential and caspases on colon cancer cell lines. Oncol Res2005;15:385-392.
53.
Grivicich I., Regner A., da Rocha AB, Kayser GB, Schunemann DP, Grass LB, et al: The irinotecan/5-fluorouracil combination induces apoptosis and enhances manganese superoxide dismutase activity in HT-29 human colon carcinoma cells. Chemotherapy2005;513:93-102.
