Three CM cell lines, all derived from locally recurrent tumors, were used. The cell lines CRMM-1 and CRMM-2 were created by Gordon Nareyeck (Essen, Germany),
8 and kindly provided by Michele Madigan (Sydney, Australia). The cell line CM2005.1 was created in our laboratory by Sander Keijser (Leiden University Medical Center, Leiden, The Netherlands).
25 In Boston, CRMM-1 and CRMM-2 cells were grown in F-12K Nutrient Mixture, Kaighn's Modification (1×) supplemented with 10% fetal bovine serum (FBS) (both from Invitrogen, Grand Island, NY, USA), 1% 200 mM L-glutamine and 1% penicillin/streptomycin/amphotericin-B mixture (both from Lonza, Walkersville, MD, USA) at a 5% CO
2 atmosphere. The CM2005.1 cells were grown in RPMI-1640 (Lonza) supplemented with 10% FBS, 1% 200 mM L-glutamine, with 20 mM Hepes and 1 g/L NaHCO
3 (Gibco, Grand Island, NY, USA), 1% penicillin/streptomycin/amphotericin-B mixture, in 5% CO
2 atmosphere. In Leiden, the F-12K Nutrient Mixture, Kaighn's Modification (1×) was provided by Gibco (Life Technologies, Paisly, UK), supplemented with 10% FBS (Greiner Bio-one, Alphen aan den Rijn, The Netherlands), and amphotericin-B was not used. Photographs of the cultured cells were taken with a microscope (Zeiss AX10; Carl Zeiss Meditec, Jena, Germany) equipped with a camera (Axiocam MRm).