Visiomorph software

Paraformaldehyde fixed paraffin embedded sections were sectioned at 5 μm (rotary microtome) onto superfrost slides (VWR International, United States). Slides were deparaffinized, re-hydrated and then stained using picrosirius red solution and differentiated using acid water. Slides were then dehydrated using ethanol and xylene, mounted using cytoseal resin and scanned at 40× magnification using a NanoZoomer-XR (Hamamatsu, Japan) to produce whole-slide images, which were then analyzed using the Visiomorph software (Visiopharm, Denmark) in the VIS program suite. Total area of Picrosirius Red staining was quantified as a percentage of total tissue area using custom thresholds at 20× magnification. Correct quantification of the staining was confirmed by visual examination by a trained individual who was blinded to the treatment groups.

All 89 slides were scanned on a Hamamatsu NanoZoomer 2.0–HT whole slide scanner (Hamamatsu Photonics K.K., Japan) at 20x magnification. Digital image analysis and quantitation were performed using Visiomorph software (Visiopharm, Denmark). Regions of interests containing tumor tissue were manually outlined followed by a systematic uniform random meander sampling using a sampling fraction of 10% and a 20x objective. The optimal sampling fraction was determined in a pilot study on 10 GBM specimens as previously described [30 (link), 31 ]. Sample images were reviewed and excluded according to the following criteria: viable tumor tissue < 50%, staining artefacts > 50%, necrotic foci > 50%, and normal brain tissue > 50%.
MMP-2 was quantified using a trained pixel classifier based on Bayesian classification. The mean MMP-2 intensity was measured in a three μm perimeter around the detected nuclei covering the cytoplasm/membrane or parts of the cytoplasm/membrane (Fig 1). The output data were given in RGB color model values ranging between 0–255 with low values equaling darker staining and high values equaling weaker staining. The average MMP-2 expression was calculated for all 89 tumors. Next, data was normalized for each patient by subtracting the measured RGB value from 255. The normalized values were then used in subsequent analyses. Patient data are available in S1 File.

The volume of the lesion and the volume of astrogliosis were determined from the area of every tenth LFB- or GFAP/Nissl/DAPI-stained section sampled by systematic uniform random sampling. The area of the lesion site was estimated in LFB-stained sections as previously described [13 (link)] using the VisioMorph software (Visiopharm, Hørsholm, Denmark) and the Cavalieri principle for volume estimation. For estimation of the lesion area in GFAP/Nissl/DAPI-stained sections, photomicrographs were acquired using an Olympus BX51 microscope with an Olympus DP73 camera connected to a PC setup with the Olympus cellSens Imaging software (Ballerup, Denmark). Lesion size was then estimated using Image J analysis software (NIH) as per directions of the Image J developers (http://rsb.info.nih.gov/ij) [13 (link)]. Analysis performed on digital images was carried out on un-manipulated pictures. On the presented pictures the contrast and curves have been adjusted to allow readers to appreciate the details on small-scale figures.