Figure 3

Cellular heterogeneity in OXPHOS protein abundance in prostate cancer tissue. (A) A region of interest was selected from an overview pan-cytokeratin map of a prostate cancer tissue section (patient P13) labelled using the final optimised multispectral staining protocol. (B) Composite image generated from multispectral imaging and spectral deconvolution noted spatial variations in raw NDUFB8 and MTCO1 abundance. Single cell level OD-NDUFB8 and OD-MTCO1 were obtained following automated tissue and cell segmentation. Scale bar represents 100 μm. (C) Immunofluorescence and pseudo-colour Z-score images of each mitochondrial marker demonstrates spatial heterogeneity in protein abundance. (D) Cropped regions of interest from panel (B) represent three cell phenotypes: normal mitochondrial protein abundance (ROI1), isolated NDUFB8 defects (ROI2), and combined NDUFB8 and MTCO1 defects (ROI3). (E) Mitochondrial respiratory chain (MRC) graph. Single cell level OD-NDUFB8 and OD-MTCO1 data were log-transformed and normalised for variable mitochondrial mass using OD-TOMM20. Z-scores were calculated for each of the mitochondrial markers, using data from benign prostate tissue from patients aged ≤ 45 years as controls. Each spot represents an individual epithelial cell (n = 1320 cells) from the region of interest in panel (B). The size of each spot correlates with cell area (in pixels). MRC graphs stratified by individual patients are presented in Supplementary Fig. 5.