Fig. 6: Immune tumor microenvironment analysis by spatial proteomics.

Immunostaining performed on bladder cancer tissue microarrays from 184 patients. All protein measurements were performed once for each distinct sample. a Staining results shown for multiplex immunofluorescence (mIF) panel 1 and PD-1 with corresponding image analysis application (APP) of four tumors representing each immune subtype and high PD-1 expression, respectively. Red dashed lines divide tissue into intratumoral and peritumoral regions of interest. Scale bar: 20 µm. b Spatial organisation of immune cells and immune evasion mechanisms stratified by immune subtypes. Heatmap shows z-scores and the barplot the mean + SD immune scores (IS), cell percentages, or H-score [1 × (% cells low intensity) + 2 × (% cells moderate intensity) + 3 × (% cells high intensity)] for MHC-expression on carcinoma cells. Asterisks denote the barplot representing the H-score. Points represent the corresponding data points. c Immune subtypes compared to chemotherapy response. d Kaplan–Meier survival curves showing overall survival (OS) stratified by immune subtypes. e–g Intratumoral and peritumoral fractions of immune evasion mechanisms (PD-1, PD-L1, MHC) compared to chemotherapy response. h Intratumoral combined PD-1/PD-L1 expression stratified by chemotherapy response. PD-1 high/PD-L1 low was compared to PD-1 high/PD-L1 high, PD-1 low/ PD-L1 low and PD-1 low/PD-L1 high combined. i–k Relationship between immune evasion mechanisms and immune subtypes. Statistical significance was assessed using a chi-square test for categorical variables, a Wilcoxon rank-sum test for continuous variables and a log-rank test for comparing survival curves. For all boxplots, the center line represents the median, box hinges represent first and third quartiles, whiskers represent ±1.5 × interquartile range (IQR) and points represent outliers. Source data are provided as a Source data file.