Imagescope analysis software

All IHC-stained slides were scanned at an apparent magnification of 20X [resolution of 0.494 μm/pixel (7,970,000 pix/in.)] using the Aperio ScanScope CS and XT systems (Aperio Technologies). The acquired digital images representing whole-tissue sections were viewed and analyzed using “ImageScope analysis software” (version 12; Aperio Technologies, Inc.). Tumor areas were blindly annotated with the assistance of a pathologist (JRJ), and staining was quantitated by applying the “Positive Pixel” algorithm package to IHC and histochemical staining. To allow comparisons between tumor samples of different size, the number of pixels with positive staining for DAB (CD20 or CD3) was expressed as relative to 100 pixels with positive staining for Hematoxylin (nuclei).

Slides were scanned at 20X magnification using a ScanScope XT slide scanner (Aperio Technologies, Vista, CA, USA). The Spectrum Analysis algorithm package, ImageScope analysis software and Color Deconvolution algorithm (version 9; Aperio Technologies.) were applied to quantify immunohistochemical (IHC) staining. These algorithms were used to calculate the average positive intensity (API), as well as the area of positive staining, and the percentage of weak (1+), medium (2+), and strong (3+) positive staining. The final API was subtracted from 255, as these intensity ranges on an 8-bit scale of 0 to 255 (black to white, respectively). The maximum value from both cores for each patient was used for statistical analysis.

IHC materials were first viewed at low power to judge overall quality and distribution of staining. Subsequently, staining frequency (total % stained cells) and staining intensity (intensity of stained cells; 0 = no staining, 1+ = weak staining, 2+ = moderate staining, 3+ = strong staining) were determined. Histoscores (H scores) were then calculated as follows: $H\mathrm{score}=1\times {\%}_{cells1+}+2\times {\%}_{cells2+}+3\times {\%}_{cells3+}$ The manual scoring was performed on images acquired with the Aperio ScanScope XT slide scanner (Aperio Technologies, Vista, CA) used at × 20 magnification with a maximum pixel resolution of 0.5 µm. ImageScope analysis software (Aperio Technologies, Vista, CA) was used for viewing and analysing digital images. Aperio Spectrum software was used to generate individual tissue spot images for automated analysis. The Colour Deconvolution algorithm (Aperio Technologies) was used to obtain quantitative values for average positive intensity (average intensity of pixels positively stained, graded from 0, 1, 2, 3) and total percent positive (percentage of positive stained area in relation to total area of the core). Histoscores were calculated as described above.

Immunohistochemical stains using the antibodies TOM20 (rabbit polyclonal, Santa Cruz Biotech., Dallas, TX; dilution 1:100), MTCO1 (mouse monoclonal, Abcam, Cambridge, MA; dilution 1:2000), and Anti-8 Hydroxyguanosine (mouse monoclonal, Abcam, Cambridge, MA; dilution 1:400) were performed on formalin-fixed, paraffin-embedded tissues using the manufactures’ protocols.
The stained sections were scanned using the Aperio ScanScope XT systems (Aperio Technologies, Vista, CA) at 20X magnification. Immunohistochemical expression was quantified in all digital slides with either nuclear (Anti-8 Hydroxyguanosine) or cytoplasmic (TOM20 and MTCO1) algorithms using ImageScope analysis software (version 12; Aperio Technologies, Inc) and an H–score was calculated (Luna, 1968 ).

Paraformaldehyde-fixed and paraffin-embedded tumor sections were stained using EnVision G2 doublestain system (Dako) according to the manufacturer's instructions. Antibodies used for immunohistochemistry were carbonic anhydrase IX (CA9; 1:800 dilution; product AB1001; BioScience), CD31 (1:50 dilution; product ab28364; Abcam), GLUT1 (1:200 dilution; product ab14683; Abcam), RAD51 (1:200 dilution; product GTX70230; GeneTex Inc), γH2AX (1:1000 dilution; product 05-636; Millipore), and cleaved caspase 3 (CC3; 1:600 dilution; product 9661; Cell Signaling Technology). Staining for CC3, γH2AX, RAD51 (3,3′-diaminobenzidine) and CA9 (Permanent Red) in whole-tumor sections (excluding necrotic areas) was analyzed using an Aperio CS scanner and ImageScope analysis software (Aperio Technologies). Three tumor samples were used per group. For γH2AX foci quantification, 200 nuclei were assessed from each tumor sample. EF5 binding was detected using the Cy3-conjugated antibody ELK3-51 (75 mM; University of Pennsylvania) as described previously (24) (link).