Figure S1: Tissue detection in StrataQuest software via creation of a digital mask; Figure S2: StrataQuest workflow for detection of SOX2+ nuclei and thresholding.

<p><strong>Figure S1: Tissue detection in StrataQuest software via creation of a digital mask</strong><strong>.</strong> A digital &lsquo;mask&rsquo; is created to measure the area of tissue for quantification of cell densities. The tissue mask is automatically generated by StrataQuest software via conversion of the scanned image from RGB to grayscale and then application of an intensity threshold.&nbsp; Manual adjustments are made to the tissue mask to remove necrotic areas and/or staining artefacts. Only nuclei present within the tissue mask are quantified. Representative tumour cores with low (A) and high (B) SOX2 densities, respectively, with corresponding overlaid tissue masks are shown (C-D; purple colour).&nbsp; Tissue mask generation is based on haematoxylin staining so is not affected by the level of DAB staining. Scale bars 200&micro;m.</p> <p><strong>Figure S2: StrataQuest workflow for detection of SOX2+ nuclei and thresholding. </strong>Inbuilt colour deconvolution algorithms within StrataQuest software separate SOX2 DAB (brown) staining from haematoxylin (blue) staining to produce grayscale images for each channel. Nuclear segmentation was then performed on the resulting grayscale DAB image to detect brown-stained nuclei.&nbsp; This is possible as SOX2 expression is localised to the nucleus. Segmented nuclear masks overlaid onto the DAB (brown) channel (A) and the original colour image (B) allow visualisation of the segmentation algorithm. The software then calculates parameters such as nuclear size, haematoxylin intensity and DAB intensity for each nuclear mask, which are reported on a scattergram, with each dot representing a single nuclear mask. A scattergram displaying DAB intensity vs haematoxylin intensity is used to threshold and accurately detect SOX2+ stained nuclei (C). Gated nuclei from the scattergram (C) can be visualised on an image of segmented nuclear masks overlaid onto the RGB image (D). Red nuclei represent SOX2+ cells (red gate on C) and green nuclei are SOX2- cells (green gate on C). Scale bars 50&mu;m.</p>