Fig. 4.

Post mortem tumor cryosection from prostate cancer (PC-3) xenografts. a Overview mosaic image of a whole PC-3 tumor tissue section co-stained with the peptide FnBPA5 Alexa-488 (red), together with polyclonal antibodies against Fn (green) and endothelial cell marker CD-31 (PECAM-1) (blue). Scale bar, 1 mm b Zoomed-in high-resolution z-projection images of different indicated regions showing superpositions of the stains FnBPA5 (red), fibronectin (green), and CD-31 (blue), as well as the mature collagen fibers in the same regions as visualized by SHG (cyan). Analysis of neighboring pixels (indicated by the small box showing the 5 × 5 pixel matrix surrounding each analyzed pixel) between antibody-stained Fn, FnBPA5 peptide and collagen bundles (SHG) showed a clear spatial proximity of FnBPA5 with Fn (99.9% of all FnBPA5 pixels have at least one neighboring Fn pixel). A total of 87% of pixels from mature collagen bundles were found to be in close proximity (within the 5 × 5 matrix of neighboring pixels) of FnBPA5-positive pixels (19 images analyzed). Scale bar, 20 μm. c Representative z-projection images of PC-3 tumor tissue sections stained for α-SMA and FnBPA5 and merged with nuclei stainings using DAPI. Analysis of neighboring pixels (indicated by the small box showing the 5 × 5 pixel matrix surrounding each analyzed pixel) reveals that 94% of the α-SMA-positive pixels are in close proximity (within the 5 × 5 matrix of neighboring pixels) to FnBPA5-positive regions (60 images analyzed). Lower thresholds were set based on individual channels to exclude unspecific pixels from the analysis, and a 5 × 5 matrix was used for the spatial proximity analysis of each pixel in 60 images, respectively, with each of the analyzed pixels being in the center (Methods section for more details). Scale bar, 20 μm