E1l3n

Slides (3 slides per sample) from formalin-fixed paraffin-embedded lung adenocarcinoma tissues were used for the histologic evaluation of PKM2 and PD-L1 expression by IHC. Briefly, 4-μm tissue sections were deparaffinized and subjected to heat-induced antigen retrieval. The slides were incubated overnight at 4 °C with a rabbit anti-human PKM2 mAb (1:100; D78A4, Cell Signaling Technology, Danvers, MA, USA) or rabbit anti-human PD-L1 mAb (1:100; E1L3N, Cell Signaling Technology). After incubation with horseradish peroxidase-conjugated secondary antibody (Envision™ Detection Kit, GK500705, Genetech), diaminobenzidine was used to visualize the staining.

All tumor samples from the MIND cohort were analyzed by next-generation sequencing (NGS) before ICI treatment [23 (link)]. The test method was performed on the U.S. Food and Drug Administration-licensed Memorial Sloan Kettering Cancer Center’s Integrated Mutation Profiling of Actionable Cancer Targets platform, which includes somatic mutations, copy number alterations, and fusions of 341–468 genes most commonly associated with cancer. Based on NGS profiling in patients from the MIND cohort, we defined a TMB of ≥ 10 mutations (muts)/Mb as “high TMB” and TMB < 10 muts/Mb as “low TMB.” PD-L1 immunohistochemistry was performed on formalin-fixed and paraffin-embedded tumor tissue sections using standard PD-L1 antibody (E1L3N; Cell Signaling Technology, Danvers, Massachusetts, USA). The tumor cells were considered to have a high PD-L1 expression level when ≥ 50% of the cells stained positively.

WES and targeted NGS of tumor and blood samples were performed before ICB. DNA and circulating tumor DNA (ctDNA) were separately extracted from formalin-fixed paraffin- embedded (FFPE) tumor masses and peripheral blood samples from the patients. The different sequencing assays, including WES and targeted NGS (MSK-IMPACT and OncoPlus), were performed as described in Supplementary Methods.
Based on the results of WES and targeted NGS profiling, we defined a high TMB as ≥20/Mb or total somatic nonsynonymous as ≥ 200, and low TMB as<20/Mb or total nonsynonymous mutations as<200. The E1L3N (Cell Signaling, Danvers, MA, USA), 22C3 (DAKO), and 28-8 (DAKO) were used to determine the PD-L1 expression of tumor cells. Positive PD-L1 expression was defined as > 1% staining.

Recently, TILs, PD-1, and PD-L1 were investigated in this STS cohort [9 (link)]. We used those results to explore the relationship between VISTA and the PD-1/PD-L1 pathway in STS. TILs between tumour cells were counted per high-power field (HPF) (400× magnification, field of view 0.237 mm²) in H&E-stained TMA slides, as routinely carried out by the pathologist.
As described previously [9 (link)], slides were pre-treated with heat and Target Retrieval solution (S1699, Agilent, Santa Clara, CA, USA) before incubation with the monoclonal primary anti-PD-1 mouse antibody (315M; 1:80; Cell Marque, Rocklin, CA, USA) for 60 min at room temperature. The Vectastain Elite ABC HRP Kit (Vector Laboratories, Burlingame, CA, USA) and the chromogen DAB+ (Agilent) were used for detection and Hematoxylin (Vector Laboratories) for counterstaining.
For PD-L1 staining, slides were pre-treated with heat and the Epitope Retrieval Solution pH8 Novocastra (Leica Biosystems, Wetzlar, Germany) before incubation with the monoclonal primary anti-PD-L1 rabbit antibody (E1L3N; 1:50; Cell Signalling Technology) for 60 min at room temperature.
For CD3 staining, a monoclonal antibody raised in rabbit (SP7; 1:150; Zytomed, Berlin, Germany) was employed according to standard procedures. We used the SignalStain Boost IHC Detection Reagent (Cell Signalling Technology) and the chromogen DAB+ (Agilent) for detection.