Fig. 4: Analysis of senescence and functional markers in pancreatic islets using multiplexed iCLAP-CyCIF.

a Using the iCLAP-CyCIF workflow to detect 12 markers in archival pancreatic tissue. The images show the detection of senescence markers (P16, P53, P21, 53BP1, Lamin B1, and HMGB1), as well as pancreatic cell markers (Insulin, Glucagon, Somatostatin 28, Lipase, Trypsin, and Carboxypeptidase A). b The stained islet cell markers (insulin and glucagon) were used to segment the islet region with image processing. c A heatmap illustrates the pairwise Pearson correlation coefficients rho (ρ) between senescence, pancreatic functional markers, and islet morphology at the islet level. Blue indicates positive correlations and red indicates negative correlations, as shown by the color scale (ranging from –1 to +1). Each variable is perfectly correlated with itself (r = 1) along the diagonal. d, e Scatter plots show islet-level correlations between P16 and insulin expression (in arbitrary units) (d) and between P16 expression (in arbitrary units) and islet area (e). Statistical analysis was performed using Pearson correlation (two-tailed). No significant correlation was observed between P16 and insulin expression (r = −0.17, P = 0.169), whereas P16 expression was significantly correlated with islet area (r = 0.41, P = 0.00057). N = 67 islets. f, g Violin plots show the occurrence of P16-positive and 53BP1+ cells across different pancreatic cell types (Insulin+, Glucagon+, and SST-28+) in individual islets. The white dot indicates the median, and the grey vertical line indicates the interquartile range (25th–75th percentile). Sample sizes are indicated in each panel. Of the 67 islets identified, only islets containing all three endocrine subtypes (insulin+, glucagon+, and SST-28+ cells) were included for analysis (n = 35). h, i Violin plots show the beta cell insulin levels (in arbitrary units) (h) and nuclear size distribution (i) across different P16 and 53BP1 expression groups. The white dot indicates the median, and the grey vertical line indicates the interquartile range (25th–75th percentile). Sample sizes are indicated in each panel. Summary statistics, including mean, median, standard deviation (SD), standard error of the mean (SEM), minimum and maximum values, and 5th–95th percentiles, are provided in the Source Data files.