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. 2023 Apr 25;14:2364. doi: 10.1038/s41467-023-37905-y

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Composite spectral mixing images from multiplex immunofluorescence (mIF; 20× magnification, scale bars represent 50 µm on each image) is shown for (A) panel 1: cytokeratin (CK), CD3, CD8, PD-1, PD-L1, and CD68; (B) panel 2: CK, CD3, CD8, CD45RO, granzyme B (GZB), and FOXP3; (C) panel 3: CK, CD3, PD-L1, B7-H3, B7-H4, IDO-1; and VISTA; (D) panel 4: CK, CD3, ICOS, LAG3, OX40, TIM3, and CD20; (E) panel 5: CK, Arg-1, CD11b, CD14, CD33, CD66b, and CD68. F Chord diagram visualization showing the diversity of inter-relationships between markers’ co-expression, including all the markers in the five mIF panels. Data from 225 samples was used. Experiments and quantifications related to the presented results were conducted once. The images were generated using Vectra-Polaris 1.0.13 scanner system and InForm 2.4.8 image analysis software (Akoya Biosciences). The chord diagram was generated using all tumor cores from all mIF panels by R studio software version 3.6.1. (Source data is provided as a source data file).