Journal: Science Advances
Article Title: Targeting uPAR with an antibody-drug conjugate suppresses tumor growth and reshapes the immune landscape in pancreatic cancer models
doi: 10.1126/sciadv.adq0513
Figure Lengend Snippet: ( A ) Left, representative flow cytometry histograms showing specific binding of A647-coupled FL1 to uPAR-expressing cells. Right, number of receptor binding sites per cell, estimated using QSC beads ( n = 3). Numbers below 1 × 10 3 reflect a lack of uPAR expression. ( B ) Confocal microscopy analysis showing uPAR-dependent FL1 uptake and lysosomal trafficking in murine receptor–positive KPC cells. Cells were incubated for 4 hours at 37°C with A647-labeled mAb in the absence or presence of E64d. No uptake is observed in uPAR KO cells. Cell membranes, in green, and nuclei, in blue, were stained with Alexa 488-labeled wheat germ agglutinin and DAPI, respectively. Scale bar, (pink) 20 μm and (white) 10 μm. ( C ) Real-time monitoring of FL1 uptake using the Incucyte S3 live-cell analysis system. The red fluorescent intensity shows a clear time-dependent increase in the uPAR-positive cells ( n = 3). ( D ) Scatter plot of total uptake at 20 hours normalized to the phase area (cell confluence) ( n = 3). ( E ) Flow cytometry analysis of A647-labeled FL1 internalization in the entire PDAC cell panel after 20 hours of incubation at 37°C with A647-FL1 and isotype control mAb, A647-IgG1. After incubation, surface-bound mAbs were removed by proteolytic treatment with a trypsin-EDTA, proteinase K solution ( n = 2). ( F ) Correlation between flow cytometry and Incucyte live-imaging data. The regression line (blue) and the R-squared value (red) are displayed. ( G ) Scatterplot illustrating the relationship between detected cellular uPAR receptor number ( y axis) and corresponding FL1 internalization ( x axis). ( H ) Histograms showing internalized 125 I labeled FL1 in selected human (left) and murine (right) uPAR-expressing cells in the presence and absence of 500 nM RAP ( n = 3). All data are representative of at least two independent experiments.
Article Snippet: Surface uPAR density was quantified using QSC calibration microspheres (Bangs Laboratories, #816), as per the manufacturer’s protocol.
Techniques: Flow Cytometry, Binding Assay, Expressing, Confocal Microscopy, Incubation, Labeling, Staining, Cell Analysis, Control, Imaging
