Fig 2: Stratification of samples reveals prostate tissue changes associated with tumor and advanced disease(A) UMAP of 23,200 cells (400 per patient) colored by expression of indicated marker.(B) Representative tissue sample stained for CD3 from a tissue microarray generated from prostate samples from the same cohort analyzed by mass cytometry.(C) Densities of T cells as determined by CD3 staining (p = 0.042).(D) Representative tissue sample stained for CD15 from a tissue microarray generated from prostate samples from the same cohort analyzed by mass cytometry.(E) Densities of granulocytes as determined by CD15 staining (p = 0.058). Scale bar, 50 µm.(F) Proportion of T cell metaclusters in ABPT and tumor samples across patients with paired samples (p = 0.066, 0.169, 0.023, 0.002, and 0.332 for TC01–05, respectively). N = 17.(G) Summary table of clusters that were significant when comparing ABPT and tumor samples (N = 17 for both groups). Metaclusters enriched in ABPT are colored in blue, while those enriched in tumor samples are colored in red. Comparison between intermediate- and high-grade patient samples (for combined tumor/ABPT; intermediate N = 46 and high grade N = 12). Metaclusters enriched in patients with high-grade disease are colored in dark red. Only significant relationships are colored, and remaining comparisons are shown in gray.(H) Proportion of macrophage metaclusters in ABPT and tumor samples across patients with paired samples (p = 0.095, 0.169, 0.515, 0.0004, and 0.014 for MA01–05, respectively). N = 17. In panels (F) and (H), dots are colored by disease severity (intermediate versus high grade). In all boxplots, boxes illustrate the IQR (25th to 75th percentile), the median is shown as the middle band, and the whiskers extend to 1.5 times the IQR from the top (or bottom) of the box to the furthest datum within that distance. Statistical testing between dependent paired tumor and ABPT samples was done using a Wilcoxon signed rank paired-sample statistical tests (two-sided). Independent intermediate- and high-grade samples were tested using a two-sided Wilcoxon rank-sum test. Number of patients in each group is indicated by N.

Fig 3: Analysis of correlation between size and infiltration. (A) The cross-sectional area (CSA) of the metastases was measured using H&E or peroxidase staining for melanoma-specific markers as here exemplarily shown (measured area surrounded in black). CSA varied from 0.04 mm2 up to 436 mm2 with a median size of 68.5 mm2. (B) CSA was correlated to the amount of neutrophil (left: N0-N3) and NET infiltration (right: NET0-NET3). For both, the median CSA was strongly distinct between medium and high infiltrated groups compared to not-infiltrated metastases. N1-infiltrated metastases were significantly smaller than N2 (p = 0.0487) and N3 (p = 0.0034)-infiltrated metastases, but no difference was seen between NET1 and NET2 or NET3-infiltrated metastases. (C) However, comparing CSA between metastases with CD15-positive neutrophils that contained NETs and those that did not, no association with size could be identified. (D) Left: Example of a massively neutrophil (N3) and NET (NET3)-infiltrated liver metastasis. Right: Lung metastasis with neutrophils (N3) but without NETs. Scale bars = 100 µm. Scale bar for (A) = 1 mm. * p < 0.05, ** p < 0.002, *** p < 0.001, **** p < 0.0001, ns = not significant. Error bars indicate mean ± SD.

Fig 4: Immunofluorescence co-staining of neutrophils by CD15 (green) and NETs by citrullinated histone 3 (H3Cit, red). (A) Neutrophils (yellow arrows) and NETs (white arrows) inside a melanoma metastasis. Isotype controls were used to distinguish specific from background staining. (B) The neutrophil staining was further cross-validated using different neutrophil markers, myeloperoxidase (MPO), neutrophil elastase (NE) and CD15 in an immunoperoxidase staining to confirm the staining pattern especially in necrotic areas (black and white arrows) independent of autofluorescence. Scale bars = 100 µm (A) and 500 µm (B).

Fig 5: Infiltration is associated with necrosis. Necrosis was evaluated in corresponding H&E or IHC sections of the metastases and categorized as present or absent. (A) Left: Of the necrotic metastases (n = 36), 83.3% (n = 30) were infiltrated with neutrophils, whereas the proportion of neutrophil infiltration in non-necrotic metastasis (n = 45) was 22.2% (n = 10) (Fisher’s exact test, p < 0.0001). Right: A similar pattern could be seen for NETs. A total of 66.6% (n = 24) of necrotic metastases and only one non-necrotic (2.2%) metastasis contained NETs (Fisher’s exact test, p < 0.0001). Within the infiltrated metastases, the maximal neutrophil and NET density was predominantly found in necrotic areas. (B) Example of a necrotic cutaneous metastasis. Left: H&E overview. Box indicates magnified area shown in the other images presented in (B). Middle: H&E of necrotic areas (marked). Right: Corresponding image of an IF staining for DAPI (blue), CD15 (green) and H3Cit (red) shows neutrophils and NETs in the necrotic areas. Bottom: Single channels of the same image. (C) Analysis of CSAs of infiltrated and non-infiltrated metastases in necrotic and non-necrotic subgroups. CD15 neutrophil-positive metastases were significantly larger than neutrophil negative metastases in both necrotic and non-necrotic subgroups. Interestingly, for NETs, no significant difference regarding size occurred. (D) shows a necrotic region in a liver metastasis containing neutrophils with intact cell morphology (white arrows) and scattered CD15 signal (green), showing neutrophil debris after NETosis (yellow arrows) that could only be found in larger scales within necrotic areas. (E) shows a vessel near to a liver metastasis with accumulating neutrophils that form NETs (yellow arrows). V = vessel lumina. Scale bars: (B) overview: 1 mm; close-up: 500 µm. * p < 0.05, **** p < 0.0001, ns = not significant. The error bars in (A) indicate the 95% confidence interval. (C) presents the data with mean ± SD.