Decloaking chamber

Formalin-fixed paraffin-embedded tumor sections (4 μm) were deparafinized and rehydrated in a series of decreasing concentrations of ethanol. Antigen retrieval was performed at 110°C for 10 minutes using 10 mM sodium citrate buffer pH 6.0 (Decloaking Chamber, Biocare Medical). The samples were then incubated in 3% H₂O₂ (10 minutes). Tissue sections were blocked using Background Snipper (Biocare Medical) and incubated with CD73 (1:200, Cell Signaling (D7F9A, #13160) overnight at 4°C. A universal Dako-Labeled Streptavidin-Biotin2 System, Horseradish Peroxidase (LSAB2) System (#K0675), and Dako DAB (#K0673) were used to develop the staining. Tumor images (9–36 per sample, 10X magnification) were captured using a BX41 Olympus microscope. CD73 staining was quantified using QuPath software (57 ). Classifiers identifying tumor and stromal cells were built by manual selection of training data, then analyzed. Thresholds were set at 0.15 and DAB OD mean measured.

Tumor tissue analogs grown for 10 days under hypoxia received 5 Gy irradiation and then were stained for vimentin on day 5 after treatment. The 4–5 μm TTA sections were placed on the positively charged plus slides (Fisher Scientific, Pittsburgh, PA) and probed with monoclonal mouse anti-vimentin V9 antibody using the automated IHC (Dako, Inc. FLEX System). Briefly, sections were deparaffinized in xylene and hydrated stepwise through ethanol to water. Antigen retrieval was performed using a high pH antigen retrieval buffer (Dako, Inc.) in a decloaking chamber (Biocare Medical, Concord, CA) according to the manufacturer's instructions. Slides were incubated in pre-diluted monoclonal mouse anti-vimentin antibody clone 9 (1:400) for 10 min at room temperature (no. IR630; Dako, Inc.) followed by cell labeling with the EnVision+ kit (Dako, Inc.) according to the manufacturer's instructions. Signal was visualized with 3,3′-diaminobenzidine (DAB) followed by a light hematoxylin counterstain. Immunoprobed sections were then coverslipped with Permount™ and air dried before images were captured with a digital camera (AxioCam, Carl Zeiss Microscopy GmbH, Gottingen, Germany) mounted on an Axioskop 2 Plus microscope (Carl Zeiss Microscopy) at 40× magnification.

Processing of xenograft specimens, including paraffin embedding, sectioning, and all staining, was performed by the Mutant Mouse Pathology Laboratory at the Center for Comparative Medicine at the University of California, Davis. Four micrometer thick paraffin sections were stained with Mayer's hemotoxylin and eosin or immunostained as described previously,[42 (link)] with some antibody-dependent and empirical based modifications; e.g., lot-based differences in antibody dilutions. Antigen retrieval was performed at 125°C in pH 6.0 citrate buffer, using a Decloaking Chamber (Biocare Medical, Concord, CA) pressurized to 15psi. The total incubation time was 45 min. Antibodies used for immunohistochemistry are provided in Supplementary Supplementary Table 1. Specimens were incubated with primary antibodies overnight at room temperature in a humidified chamber. Slides were scanned at 20× magnification using an Aperio^® XT slide scanner (Leica Biosystems), imported into the Aperio Spectrum database, and visualized with Aperio^® Imagescope software.