Microscopic images were taken every R788 60 s for up to 3 h (Zeiss Axiovert 200M; Zeiss, Göttingen, Germany). The images were analyzed with Visitron Metamorph 6.2 Software. COLO-357, MiaPaCa-2, Su8686, or T3M4 (1 × 106 in 2 mL) were cultivated in six-well plates (Nunc, Roskilde, Denmark) for 24 h when they reached confluence. Then, isolated PMNs (3 × 106), unfixed or fixed with 2% PFA for 10 min, was added and culturing was continued (37°C in a 5% CO2 humidified atmosphere).
Dyshesion was determined after various time intervals by quantifying the cell-depleted areas (see below). Alternatively, neutrophil elastase (Calbiochem, Darmstadt, Germany) (3 μg/mL) (≥ 20 U/mg) was added in serum-free medium. Furthermore, up to 1 × 107 PMNs with 15 μg/mL α-1-antitrypsin (Sigma, München, Germany), 50 nmol/mL of the neutrophil elastase inhibitor IV (Calbiochem), or 50 μmol/mL of the elastase substrate (N-(Methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethyl-ketone) (Sigma) were added in serum-free medium. Porcine elastase that was used for comparison was purchased from Calbiochem.
To exclude potential cytotoxic effects of PMNs on tumor cells, the tumor cells were preloaded for 30 min with 5 nM calcein (Sigma), and then Atezolizumab in vivo Adenylyl cyclase incubated with PMNs for different time points up
to 24 h. For comparison, porcine pancreas elastase (Calbiochem) was used. After various times, the cells were fixed in 100% ice-cold methanol for 1 min, then digital photographs of five representative areas were taken (Leica, Heerbrugg, Switzerland) at the magnification of tenfold of five independent experimental subsets. The cell-free areas were quantified using ImageJ software (open source). The “free” areas were digitally marked and quantified, following the calculation of the ratio: free area/area of the whole tumor cell layer. T3M4 (5 × 104 /mL) were cultivated in 24-well culture plates for 24 h. After washing with PBS, the cells were fixed in 4% PFA, prior to blocking with normal goat serum (KPL, Gaithersburg, MD, USA). Then, mouse mAb to E-cadherin (DAKO, 1:40) was incubated at room temperature for 1 h. After washing, the cells were incubated with a FITC-labeled secondary antimouse Ab, diluted 1:400 for 1 h. The cells were examined by digital immunofluorescence microscopy (Biozero; Keyence, Neu Isenburg, Germany). Isotypic IgG was used as “negative” controls. The tumor cells were harvested using ice-cold saline and a cell scraper. For intracellular staining, the membrane was permeabilized with methanol/acetone (75/25 v/v).