title: Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation.
abstract: BACKGROUND
  There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed.
  
  
  RESULTS
  To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)γ inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease.
  
  
  CONCLUSIONS
  We have established and validated a suite of highly reproducible tumor microplate three-dimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds.
authors.affiliation: Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, Sutton, UK.
authors.forename: Maria
authors.initials: M
authors.lastname: Vinci
authors.forename: Sharon
authors.initials: S
authors.lastname: Gowan
authors.forename: Frances
authors.initials: F
authors.lastname: Boxall
authors.forename: Lisa
authors.initials: L
authors.lastname: Patterson
authors.forename: Miriam
authors.initials: M
authors.lastname: Zimmermann
authors.forename: William
authors.initials: W
authors.lastname: Court
authors.forename: Cara
authors.initials: C
authors.lastname: Lomas
authors.forename: Marta
authors.initials: M
authors.lastname: Mendiola
authors.forename: David
authors.initials: D
authors.lastname: Hardisson
authors.forename: Suzanne A
authors.initials: SA
authors.lastname: Eccles
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D016227:Benzoquinones / Q000494:pharmacology; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D004341:Drug Evaluation / Q000379:methods*; D053595:Embryonic Stem Cells / Q000187:drug effects*; D005663:Furans / Q000494:pharmacology; D018841:HSP90 Heat-Shock Proteins / Q000494:pharmacology; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D015398:Signal Transduction; D059016:Tumor Microenvironment / Q000187:drug effects
chemical_list: D000970:Antineoplastic Agents; D016227:Benzoquinones; D005663:Furans; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; C522973:PI103; D011725:Pyridines; D011743:Pyrimidines; C112765:tanespimycin