Fig 1: Evaluation of apparent collagen fiber density and distribution in CAM xenografts in 3D using MPM. (A) Freshly prepared tumor—including surrounding CAM—tissue imaged using bright-field microscopy and a digitally stitched MPM mosaic (scale bar = 500 µm). The color code shows the targeted molecules for the respective imaging channel. (B) Collagen network at the tumor interface under different tested conditions: Native CAM with an unstructured, Matrigel and DAPK1 ko clone with perpendicular, and HCT116 WT with parallel fiber orientation (scale bar = 50 µm). (C) Relative fraction of images with unstructured, parallel, or perpendicular collagen fibers’ orientation relative to the invasion front or the Matrigel interface (nsamples = 6; 2-way ANOVA: ** p < 0.01). (D) Collagen density at the Matrigel interface and the invasion front, respectively, expressed by normalized median pixel intensity of the SHG signal within the manually annotated ECM of one representative slice from each 3D stack (nsamples = 6; 1-way ANOVA: * p < 0.05; ** p < 0.01). (E) Western blot of DAPK1 ko cells after siRNA and scrRNA transfection, demonstrating the successful knockdown of uPAR without inducing cell death (original Western blot images presented in Figure S5A). (F) Representative MPM images of siRNA-tumors, resembling HCT116 WT tumors, while scrRNA-derived tumors resemble DAPK1 ko xenografts (scale bar = 50 µm). (G) Relative collagen fiber orientation of uPAR siRNA- and scrRNA-treated tumor cells (scr: nsamples = 6; si: nsamples = 9; 2-way ANOVA: ** p < 0.01; *** p < 0.001). (H) Normalized median pixel intensity of the SHG signal of uPAR siRNA- and scrRNA-treated xenografts (scr: nsamples = 6; si: nsamples = 9; Mann–Whitney test: n.s.). Data represented as mean ± SD.
Fig 2: uPAR-dependent tumor cell invasion in vitro. (A) Western blot analysis of DAPK1 (lower band at 160 kDa) and uPAR expression in HCT116 WT, DAPK1 ko, and SW480 cells. In contrast to HCT116 WT cells, DAPK1 ko and SW480 cells show the same expression pattern (DAPK1 low, uPAR high) (original Western blot images presented in Figure S5B). (B) 3D-tumor spheroid-based invasion assay with uPAR siRNA/scrRNA-treated HCT116 DAPK1 ko cells. (C) Area quantification of uPAR siRNA/scrRNA-treated HCT116 DAPK1 ko 3D-tumor spheroids after embedding in Matrigel (0 h) and after 72 h in mm2 (nsamples = 16; 2-way ANOVA: *** p < 0.001). (D) The 3D-tumor spheroid invasion of uPAR siRNA/scrRNA-treated HCT116 DAPK1 ko cells after 72 h relative to time point 0 h (nsamples = 16; Whitney test: *** p < 0.001). (E) Western blot of SW480 cells after siRNA and scrRNA transfection, demonstrating the successful knockdown of uPAR without inducing cell death (original Western blot images presented in Figure S5C). (F) A 3D-tumor spheroid-based invasion assay with uPAR siRNA/scrRNA-treated SW480 cells. (G) Area quantification of uPAR siRNA/scrRNA-treated HCT116 DAPK1 ko 3D-tumor spheroids after embedding in Matrigel (0 h) and after 72 h in mm2 (scr: nsamples = 18; si: nsamples = 17; 2-way ANOVA: *** p < 0.001). (H) The 3D-tumor spheroid invasion of uPAR siRNA/scrRNA-treated SW480 cells after 72 h relative to time point 0h (scr: nsamples = 18; si: nsamples = 17; Whitney test: *** p < 0.001). Black dots in graphs represent the first biological replicate and blue dots represent the second biological replicate. Data represented as means ± SD.
Fig 3: Evaluation of qualitative collagen fiber density and distribution, as well as uPAR expression in histological slices of CAM xenografts in 2D. (A) H&E staining of CAM xenografts derived from HCT116 WT cells (above) and DAPK1 ko tumor cells (below). Arrowheads indicate intra- and peri-tumoral chicken vessels. (B) Collagen staining of histological sections with Sirius red for the analysis of collagen network structure and density. (C) Label-free visualization of fibrillary collagen (blue) and natural auto-fluorescence from NADH (green) of unstained sections using MPM. (D) Conventional IHC stainings for the uPAR of CAM xenografts. (E) Cytoplasmic and membranous uPAR IHC score of HCT116 WT cells and DAPK1 ko CAM tumors at the invasion front and (F) at the tumor center (WT: nsamples = 7; DAPK1 ko: nsamples = 6; 2-way ANOVA: * p < 0.05; *** p < 0.001). (Scale bar main image = 400 µm; scale bar zoomed inlet 50 µm). Data represented as mean ± SD.
Fig 4: DAPK1-dependent proteomics signature. (A) GO-term network of all 70 proteins annotated with a GO-term subset. The node color, ranging from blue to red, represents the log2(FC) value of the respective protein, whereas the thickness of the node border represents the p-value. The node for DAPK1 (yellow) was added manually to the analysis. (B) GO-term IDs and definitions of the selected GO-term subset including the number of annotated proteins. (C) Schematic representation of the role of uPAR in the plasminogen-signaling cascade resulting in ECM degradation (Figure adapted from Brungs et al., 2017 [34], CC-BY for unrestricted use). (D) Validation of uPAR up-regulation upon DAPK1 loss in HCT116 WT cells using an ELISA assay (Mann-Whitney test: * p < 0.05). Data represented as mean ± SEM.
Supplier Page from Abcam for Human uPAR ELISA Kit