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. 2025 Jan 16;6(1):51–66. doi: 10.1038/s43018-024-00879-x

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© The Author(s) 2025

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, 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 licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

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Fig. 2 — a, Representative images of a baseline sample with merged seven-plex immunofluorescence staining (PanCK (rose), CD4 (green), CD8 (red), Foxp3 (magenta), CD20 (blue) and CD68 (yellow) markers were targeted in addition to nuclear counterstaining with 4,6-diamidino-2-phenylindole (DAPI)) image (left) and tissue segmentation in tumor, tumor stroma and epidermis are displayed (right). Scale bar 100 µm. b, The immune cell infiltrate of patients with treatment-naive BCCs (n = 16) is presented separately in tumor and tumor stroma by box plots (√cells per mm²) (CD4⁺, P = 0.0041; CD4⁺Foxp3⁺, P = 0.0290; CD8⁺, P = 0.0092; CD68⁺, P = 0.0042; CD20⁺, P = 0.0004) (two-sided Wilcoxon signed-rank test, statistical significance determined with P < 0.05). Data are presented as mean ± s.e.m. Each box extends from the 25th (Q1, lower bound of box) to the 75th (Q3, upper bound of box) percentile, the horizontal line in the center of the box represents the median value (Q2), the lower bound of lower whiskers mark the 5th (min) and the upper bound of whiskers the 95th (max) percentiles, dots represent individual values. c, Distribution of immune cells (relative values, %) on an individual patient level pre- and post-treatment, in accordance with the surgical outcome (with or without (w/o) skin flap/graft) and pathological response (pCR and non-pCR) are displayed in bar plots. Immune cells (%) pre- versus post-treatment are displayed with bar plots (two-sided Wilcoxon signed-rank test, statistical significance determined with P < 0.05). d, Representative post-treatment histopathological picture (seven-plex immunofluorescence panel), 500 µm and 100 µm bar, from one patient (n = 1). Tumor cell density is presented in box plots pre- and post-treatment from patients with a non-pCR (n = 10). Data are presented as mean ± s.e.m. (two-sided Wilcoxon signed-rank test, statistical significance determined with P < 0.05). Each box extends from the 25th (Q1, lower bound of box) to the 75th (Q3, upper bound of box) percentile, the horizontal line in the center of the box represents the median value (Q2), the lower bound of lower whiskers mark the 5th (min) and the upper bound of whiskers the 95th (max) percentiles; dots represent individual values.

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