title: Evaluation of assays for drug efficacy in a three-dimensional model of the lung.
abstract: BACKGROUND
  The focus of the outlined work is the establishment of a three-dimensional lung model for various drug-screening applications.
  
  
  METHODS
  The non-small cell lung cancer (NSCLC) cell line Colo699 was cultivated as monolayer (2D) on plates for 5 days or as microtissues (3D) using a hanging-drop system for 5 and 10 days. Cells and microtissues were treated with afatinib (10-80 µM), cisplatin (100-800 µM) or vinorelbine (25-200 µM) for 24 or 48 hours (h). Cell proliferation and viability were analysed by intra-cellular adenosine triphosphate (ATP) and lactate dehydrogenase release (LDH) assays, annexin V/propidium iodide (PI) staining, and cell cycle determination. Microtissue morphology and size, as well as cell death were evaluated via phase contrast microscopy.
  
  
  RESULTS
  Our results demonstrate the valid determination of viability and cell death using established assays in the 3D system for drug testing. The comparison of ATP, LDH and cytometry data showed moderate (0.40) to very strong (0.99) correlations. Thereby, we observed partially significant differences in drug efficacy between microtissues and 2D cultures dependent from the applied treatment and read-out method. Altogether, microtissues developed resistance to cisplatin and vinorelbine; but remained more vulnerable to afatinib. These findings were confirmed with microscopy.
  
  
  CONCLUSIONS
  In summary, we established an NSCLC 3D test system with multiple assays compatible for drug-testing applications of substances with different mechanisms of action. In addition, our data support the usage of microtissues as more accurate tools for drug-efficacy testing with the possibility of long-term cultivation and treatment.
authors.affiliation: Tyrolean Cancer Research Institute, Innrain 66, 6020, Innsbruck, Austria.|Oncotyrol, Innrain 66, 6020, Innsbruck, Austria.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Huber
authors.affiliation: University Hospital for Internal Medicine V (Hematology and Oncology), Medical University of Innsbruck, Anichstr. 35, 6020, Innsbruck, Austria.
authors.forename: Arno
authors.initials: A
authors.lastname: Amann
authors.affiliation: University Hospital for Internal Medicine V (Hematology and Oncology), Medical University of Innsbruck, Anichstr. 35, 6020, Innsbruck, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Koeck
authors.affiliation: University Hospital for Internal Medicine V (Hematology and Oncology), Medical University of Innsbruck, Anichstr. 35, 6020, Innsbruck, Austria.|Oncotyrol, Innrain 66, 6020, Innsbruck, Austria.
authors.forename: Edith
authors.initials: E
authors.lastname: Lorenz
authors.affiliation: InSphero AG, Wagistr. 27, 8952, Schlieren, Switzerland.
authors.forename: Jens M
authors.initials: JM
authors.lastname: Kelm
authors.affiliation: University Hospital for Pediatrics II, Medical University of Innsbruck, Anichstraße 35, 6020, Innsbruck, Austria.|Tyrolean Cancer Research Institute, Innrain 66, 6020, Innsbruck, Austria.
authors.forename: Petra
authors.initials: P
authors.lastname: Obexer
authors.affiliation: University Hospital for Internal Medicine V (Hematology and Oncology), Medical University of Innsbruck, Anichstr. 35, 6020, Innsbruck, Austria.
authors.forename: Heinz
authors.initials: H
authors.lastname: Zwierzina
authors.affiliation: University Hospital for Internal Medicine V (Hematology and Oncology), Medical University of Innsbruck, Anichstr. 35, 6020, Innsbruck, Austria. gabriele.gamerith@i-med.ac.at.|Oncotyrol, Innrain 66, 6020, Innsbruck, Austria. gabriele.gamerith@i-med.ac.at.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Gamerith
mesh_terms: D000077716:Afatinib; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D006801:Humans; D008168:Lung / Q000166:cytology* / Q000473:pathology; D008175:Lung Neoplasms / Q000473:pathology*; D011799:Quinazolines / Q000494:pharmacology; D018874:Spheroids, Cellular / Q000166:cytology*; D046509:Tissue Culture Techniques / Q000379:methods*; D014407:Tumor Cells, Cultured; D014747:Vinblastine / Q000031:analogs & derivatives / Q000494:pharmacology; D000077235:Vinorelbine
chemical_list: D011799:Quinazolines; D000077716:Afatinib; D014747:Vinblastine; D002945:Cisplatin; D000077235:Vinorelbine
keywords: Drug testing; Hanging-drop; In vivo mimicking; Lung cancer; Three-dimensional model