Fig. 1: High plex imaging mass cytometry to characterize the immune landscape of metastatic melanoma.

a Illustration of the data acquisition workflow used for IMC: (1) tumor tissue derived from metastatic melanoma tissue was obtained prior to the start of anti-PD-1 treatment. (2) Three 500 × 500 μm regions of interest (ROI) were selected based on H&E-stained sections by a pathologist and used on sequential tissue sections for IMC stainings. (3) Tissue sections were stained with a myeloid or T-cell panel consisting of metal isotope-labeled antibodies. (4) ROI was ablated with a high-energy laser using IMC. (5) Each antibody resulted in a single image per sample with the metal isotope composition per pixel, together constructing a multi-channel image stack. b Multidimensional scaling (MDS) using the median intensity of each marker per image region as input variables. Each data point is an ROI, color-coded by tumor state. c The median intensity of each lineage marker was calculated per ROI. The violin plots show the median intensity of each lineage marker per patient (across ROIs) split for therapy response. Mann-Whitney U-test with Benjamini-Hochberg correction was used to calculate the statistical difference between non-responders and responders. Bars indicate the median with a 95% confidence interval. d Representative H&E and IMC images. The scale bar is 50 μm. e The median intensity of each immune checkpoint was calculated per ROI. The violin plots show the median intensity of each lineage marker per patient (across ROIs) split for therapy response. Patient ID is indicated in colors. Mann-Whitney U-test with Benjamini-Hochberg correction was used to calculate the statistical difference between non-responders and responders. Bars indicate the median with a 95% confidence interval.