Mantra quantitative pathology workstation

TMA blocks were cut into 5-micron slices and placed onto charged slides for processing. Slides were baked in a hybridization chamber for 1 hour at 60°C. Once baked, TMA slides were subjected to deparaffinization and rehydration, then fixed with formalin. Following our established protocol (35 (link)), multiplex staining was conducted on the slides through six rounds of staining. The slides were prepared for each round of staining using either an antigen retrieval buffer with pH 9 or pH 6 (AR9 and AR6 Akoya Biosciences) preceded by a primary antibody. The following primary antibodies were used—CD3, CD8, CD163, PD-L1, pancytokeratin, and FoxP3 (Supplementary Table 1) followed by secondary antibody application (Opal Polymer, Akoya Biosciences) and fluorescent tyramide signal amplification (TSA, Akoya Biosciences). Slides were counter stained using 4’,6-diamidino-2-phenylindole (DAPI), mounted, cover-slipped and left to dry overnight. Using the Mantra Quantitative Pathology WorkStation (Akoya Biosciences), cores were imaged at 20x magnification in all channels: DAPI, FITC, CY3, CY5, CY7, Texas Red, and Qdot, with an exposure of 250 ms. Composite images were created by automatically merging images from each channel, then taken for further analysis. More detailed descriptions of staining and imaging methods can be found in prior publications (35 (link), 41 (link)).

Extracted tumors were fixed with 10% formalin, embedded in paraffin and sliced in 4 μm thickness. The multiplex IHC was performed with an Opal 7-Color Automation IHC Kit (Akoya Biosciences, Hopkinton, MA, USA) and BOND RXm automated stainer (Leica Biosystems, Wetzlar, Germany) using the following antibodies: anti-pan-cytokeratin (CK) (rabbit poly; Bioss, Woburn, MA, USA), anti-CD8α (EPR20305; Abcam, Cambridge, United Kingdom), anti-CD44 (IM7; Bio X Cell, Lebanon, NH, USA), anti-F4/80 (D2S9R; Cell Signaling Technology, Danvers, MA, USA), anti-Ly6G (1A8; Bio X Cell, Lebanon, NH, USA), anti-CD11b (EPR1344; Abcam, Cambridge, United Kingdom) and anti-Gzmb (rabbit polyclonal; Abcam, Cambridge, United Kingdom). The slides were imaged in the Mantra Quantitative Pathology Workstation (Akoya Biosciences, Menlo Park, CA, USA). The multiplex IHC images were analyzed using inForm Tissue Finder software (Akoya Biosciences, Menlo Park, CA, USA). Cell phenotype was defined based on the antigen expressions as the following: CD8+ = CD8+ T cell, F4/80+ = macrophage, Ly6G+CD11b+ = neutrophil, Gzmb+CD3− = NK cell. Tissue phenotype was determined as the following: pan-cytokeratin positive = tumor. CD8+ T cells in the tumor area were counted as tumor-infiltrating CD8+ T cells.