The migratory/invasive activities of LuPanc-1 and LuPanc-2 were determined with xCELLigence® technology (ACEA Biosciences, San Diego, supplied by OLS, Bremen, Germany), as outlined in detail in previous publications [19 (link),20 (link)]. The lower side of the CIM plate-16 porous membrane was coated with a 1:1 mixture (v/v) of collagen I and collagen IV (30 μL) to facilitate the adherence of the cells and thus enhance the duration of the signal recording. Each well was loaded with 60,000 (LuPanc-1) or 80,000 (LuPanc-2) cells in standard growth medium (see above). Cells were allowed to settle in the laminar flow hood for 30 min at RT, after which the assay was started and run for 24 or 48 h. Some assays were performed in the absence or presence of recombinant human (rh) TGF-β1 (RELIATech GmbH, Wolfenbüttel, Germany) or the TGF-β type I receptor/ALK5 serine/threonine kinase inhibitor SB431542 (Merck, Darmstadt, Germany). Data acquisition (with signal recording every 15 min) and analysis were performed with the RTCA software (version 1.2, ACEA Biosciences).
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