Extended Data Fig. 1. Workflow of the analysis of 7-plex Immunofluorescence images.

Representative pictures of analysis workflow in HALO® quantitative pathology software (Indica Labs) of selected patients (n = 2). a, H&E staining of whole-tumor tissue and corresponding multispectral images (MSI, 1872x 1404) fused in HALO, 500 µm bar, 1x magnification. 3–6 equally sized areas, consisting of 50% tumor stroma and 50% tumor islands in case of available tumor islands, were selected from a representative patient tumor tissue sample (n = 1). b, Overview of the automated image analysis: Multispectral imaging, tissue segmentation in tumor and tumor stroma, cell phenotyping, infiltration analysis (tumor margin was set as the interface (red line) and an inside/outside range of 50 µm was split into 10 bands) and density analysis, 100 µm bar, 5$\times$ magnification from a representative patient tumor tissue sample (n = 1). c, Immune cell subsets are presented in merged and single stainings (PanCK / rose; CD4 / green; Foxp3 / magenta; CD8 / red; CD20 / blue; CD68 / yellow), 100 µm bar, 5$\times$ magnification. Analysis markup classified with the nuclei segmentation classifier Nuclei Seg (Plugin) - FL v1.0.0) and analyzed (Indica Labs – HighPlex FL v4.1.3 algorithm) from a representative patient tumor tissue sample (n = 1). The analysis was repeated once to ensure reproducibility.