Large-scale screens in mammalian cells demand for flexible high-throughput screening platforms that allow to analyze cellular traits on a genome-wide level or to identify small-molecule inhibitors (SMIs) from complex compound libraries. In this study we developed and tested high-density cell arrays made out of polydimethylsiloxane (PDMS) that support cell growth directly on standard glass microscope objective slides. We analyzed the effect of 3 reference inhibitors (MLN-8054, VX-680, and flavopiridol) and 4 exploratory, cell permeable small-molecule kinase inhibitors (two benzothiophene-based and two 4-amino-6-arylpyrimidine-based compounds) on different cell lines, using prototype 5 × 5 and 9 × 9 array carpets. We found that high-density PDMS cell arrays support growth of a broad range of cell types, are well suited for compound screens, and are compatible with high-content screening platforms. This novel array format is of particular advantage for compound screening to identify SMIs, when a combination of flexibility with respect to culture volume, well density, and high-resolution imaging is required. In addition, we demonstrated the suitability of this format for reverse transfection and siRNA experiments.
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References
1.
MalumbresM, BarbacidM. Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer, 2009; 9:153–166.
2.
BischoffJR, AndersonL, ZhuY, MossieK, NgL, SouzaB, SchryverB, FlanaganP, ClairvoyantF, GintherC, ChanCS, NovotnyM, SlamonDJ, PlowmanGD. A homologue of Drosophila aurora kinase is oncogenic and amplified in human colorectal cancers. EMBO J, 1998; 17:3052–3065.
3.
TanakaT, KimuraM, MatsunagaK, FukadaD, MoriH, OkanoY. Centrosomal kinase AIK1 is overexpressed in invasive ductal carcinoma of the breast. Cancer Res, 1999; 59:2041–2044.
4.
ManfrediMG, EcsedyJA, MeetzeKA, BalaniSK, BurenkovaO, ChenW, GalvinKM, HoarKM, HuckJJ, LeRoyPJ, RayET, SellsTB, StringerB, StroudSG, VosTJ, WeatherheadGS, WysongDR, ZhangM, BolenJB, ClaiborneCF. Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase. Proc Natl Acad Sci U S A, 2007; 104:4106–4111.
5.
HoarK, ChakravartyA, RabinoC, WysongD, BowmanD, RoyN, EcsedyJA. MLN8054, a small-molecule inhibitor of Aurora A, causes spindle pole and chromosome congression defects leading to aneuploidy. Mol Cell Biol, 2007; 27:4513–4525.
6.
HarringtonEA, BebbingtonD, MooreJ, RasmussenRK, Ajose-AdeogunAO, NakayamaT, GrahamJA, DemurC, HercendT, Diu-HercendA, SuM, GolecJM, MillerKM. VX-680, a potent and selective small-molecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med, 2004; 10:262–267.
7.
TylerRK, ShpiroN, MarquezR, EyersPA. VX-680 inhibits Aurora A and Aurora B kinase activity in human cells. Cell Cycle, 2007; 6:2846–2854.
8.
CarterTA, WodickaLM, ShahNP, VelascoAM, FabianMA, TreiberDK, MilanovZV, AtteridgeCE, BiggsWH3rd, EdeenPT, FloydM, FordJM, GrotzfeldRM, HerrgardS, InskoDE, MehtaSA, PatelHK, PaoW, SawyersCL, VarmusH, ZarrinkarPP, LockhartDJ. Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases. Proc Natl Acad Sci U S A, 2005; 102:11011–11016.
9.
SedlacekHH, CzechJ, NaikR, KaurG, WorlandP, LosiewiczM, ParkerB, CarlsonB, SmithA, SenderowiczA, SausvilleE. Flavopiridol (L86-8275, NSC-649890), a new kinase inhibitor for tumor therapy. Int J Oncol, 1996; 9:1143–1168.
10.
ByrdJC, LinTS, DaltonJT, WuD, PhelpsMA, FischerB, MoranM, BlumKA, RovinB, Brooker-McEldowneyM, BroeringS, SchaafLJ, JohnsonAJ, LucasDM, HeeremaNA, LozanskiG, YoungDC, SuarezJR, ColevasAD, GreverMR. Flavopiridol administered using a pharmacologically derived schedule is associated with marked clinical efficacy in refractory, genetically high-risk chronic lymphocytic leukemia. Blood, 2007; 109:399–404.
ChaoSH, PriceDH. Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo. J Biol Chem, 2001; 276:31793–31799.
13.
LangeBM, GullK. A molecular marker for centriole maturation in the mammalian cell cycle. J Cell Biol, 1995; 130:919–927.
14.
SedlacekHH. Mechanisms of action of flavopiridol. Crit Rev Oncol Hematol, 2001; 38:139–170.
15.
YangH, BurkeT, DempseyJ, DiazB, CollinsE, TothJ, BeckmannR, YeX. Mitotic requirement for aurora A kinase is bypassed in the absence of aurora B kinase. FEBS Lett, 2005; 579:3385–3391.
16.
KaurG, Stetler-StevensonM, SebersS, WorlandP, SedlacekH, MyersC, CzechJ, NaikR, SausvilleE. Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275. J Natl Cancer Inst, 1992; 84:1736–1740.
CatalanoSM, McLaughlinJ, HitoshiY, PayanD. Discovery and development of an aurora kinase inhibitor clinical candidate using an image-based assay for measuring proliferation, apoptosis, and DNA content. Assay Drug Dev Technol, 2009; 7:180–190.
22.
DeMoeJH, SantaguidaS, DaumJR, MusacchioA, GorbskyGJ. A high throughput, whole cell screen for small molecule inhibitors of the mitotic spindle checkpoint identifies OM137, a novel Aurora kinase inhibitor. Cancer Res, 2009; 69:1509–1516.
23.
KimJY, BaekJY, LeeKA, LeeSH. Automatic aligning and bonding system of PDMS layer for the fabrication of 3D microfluidic channels. Sens Actuators A Phys, 2005; 119:593–598.
