l of thisented with EGF and B27 was prepared. 100 l of this cell suspension were plated on each well of poly HEMAcoated 96 well plates. The plates were centrifugated at 200 g during 6 min and then incubated in a humidified atmosphere of 5 CO2 at Cyclopamine 37. By using this technique we obtained single spheroids in each well, the variation of size between spheroids is less than 10 . In order to generate quiescent spheroids, after a first 4 days growth phase in defined medium, spheroids were washed twice with media containing 10 FCS, and then incubated with this media during 1 6 days. Spheroid viability quantification Spheroid viability was quantified by ATP monitoring with the Perkin Elmer ATPlite??assay system. This system is based on the production of light caused by the reaction of ATP, a cell viability marker present in cell lysate, with added luciferase and D luciferin.
We adapted ATPlite assay procedure Luteolin for spheroid application, especially concerning spheroid dissociation and cell lysis. Then 100 l of mammalian cell lysis solution were added to each well containing one spheroid in 100 l of culture medium. The plate was shaken for 20 min. In order to read luminescent signal, 75 l of the cell lysate was transferred to a black 96 well plate. Then 37 l of DMEM F12 medium containing 10 FCS and 37 l of ATPlite kit substrate solution were added. After 15 min of shaking, the luminescence signal was read on an Envision? plate reader. Immunofluorescence on frozen sections Capan 2 spheroids were rinsed with PBS and fixed in 4 neutral buffered formalin for 2 h. After fixation, spheroids were processed for 5 m frozen sections.
Sections were incubated overnight at 4 with antibodies directed against cleaved form of PARP, or gH2AX phosphorylated and Ki67 . After washing in PBS Triton 0.1 v v, the secondary antibody was applied. To determine cell cycle repartition, sections of Capan 2 spheroids expressing the green FUCCI probe were directly analyzed by fluorescence imaging. The observations were based on the examination of 3 sections from at least 5 spheroids. Each experiment has been repeated a minimum of 3 times. Cytotoxicity assays Spheroids were generated using 1000 cells in 100 l per well as indicated in spheroid generation section. After 4 days of culture, chemotherapeutic agents or combinations were added. Spheroid viability was evaluated by ATP quantification after 72 h compound treatment.
Tests were performed in triplicate and the data presented are from at least three separate experiments. ATP content percentage was calculated with regard to non treated spheroid and showed cell growth inhibition and or toxicity. The 50 effective concentration of a compound is the concentration which provokes 50 of the maximal effect of this drug. Curve fittings were performed with GraphPad Prism version 4.0 software using the sigmoidal dose response to determine EC50 values. Results Generation of Capan 2 spheroids in microplate In order to obtain a model of pancreatic multicellular spheroid, we tested several pancreatic cancer cell lines including BxPC3, MiaPaCa, Panc 1, AsPC 1, Capan 2. A pancreatic cancer spheroid model was obtained only with Capan 2 cell line. Seeding of 103 Capan 2 pancreatic cancer cells in DMEM F2 medium supplemented with 10 serum allowed cell association