Figure 2
DSG2 KO in AsPC-1 cells leads to loss of cell–cell adhesion and reduced attachment in perfused livers. (a) Representative Western blot analysis on the left with corresponding densitometric analysis on the right of the desmosomal proteins desmoplakin (DP), desmoglein-2 (DSG2), desmocollin-2/3 (DSC2/3), plakophilin-2 (PKP2), plakoglobin (PG) and the adherens junctions molecules E-cadherin (E-Cad), and β-catenin (β-Cat) in AsPC-1 control (Ctrl) and DSG2 knockout (KO) cells. GAPDH served as loading control. n = respective mean of 8 independent biological replicates, two-tailed unpaired t test. (b) Dissociation assay of AsPC-1 control and DSG2 KO cells. Corresponding images of monolayer fragmentation after application of defined mechanical stress are depicted on the left side. n = respective mean of 4 independent biological replicates, two-tailed Mann–Whitney test. (c–e) Ex vivo liver perfusion of AsPC-1 control and DSG2 KO cells into wild-type (wt) mouse livers as depicted in the scheme on the left. The number of tumor cells per perfused liver cells was determined using flow cytometry in (c) as outlined in Fig. 1. n = 13 (control) and 9 (DSG2 KO) perfused livers, two-tailed Mann–Whitney test. (d) Representative immunostainings of perfused liver sections. Sections were stained with laminin to label vessel walls (yellow), DAPI to visualize cell nuclei (blue) and CellTrace Far Red (CTFR, red) marks perfused AsPC-1 tumor cells. The green background is a result of the autofluorescence of the liver tissue. Magnified areas show the local invasion of AsPC-1 tumor cells into the liver tissue. (e) Corresponding analysis of stained liver sections to determine the number of attached cells per total cell number determined via DAPI nuclear staining. n = 5 perfused livers, mean of 2 consecutive sections per transversal level (top, middle, bottom of the lobe), two-tailed unpaired t test.
