Fig. 5

Spatial analysis of multiple markers provides insight into gonadotroph intratumoural heterogeneity and unravels spatial associations. A Representative images of scans (upper panel), enlarged views of IHC staining (middle panel), and resulting segmentations (lower panel), performed for S100B, FSH, LH, Ki67, SSTR2, and ERα, on serial tumour-sections. Scale bar = 50 µm. B For each of the five tumours (Gon#3, #5, #11, #12 and #18), we annotated the same tumour areas as previously done for the S100B marker (four areas per tumour). The graphs show the distribution of the S100B + , FSH + , LH + , Ki67 + SSTR2 + , and ERα + cells, in the resulting 20 independent tumour areas, revealing that these markers were either homogeneously, or heterogeneously distributed within each tumour. Each plotted symbol represents a tumour area. C Percentage of FSH + cells in S100B^High (median percentage of FSH + cells = 76.32%) versus S100B^Low areas (median percentage of FSH + cells = 18.93%), p = 0.0001 (***). D Percentage of ERα + cells in S100B^High (median percentage of ERα + cells = 19.78%) versus S100B^Low areas (median percentage of ERα + cells = 1.47%), p = 0.008 (**). E Percentage of Ki67 + cells in S100B^High (mean percentage of Ki67 + cells = 0.22%) versus S100B^Low areas (mean percentage of Ki67 + cells = 0.67%), p = 0.02 (*). S100B^High and S100B^Low expressing areas were separated by the median percentage of S100B + cells for the 20 areas, n = 10 for each group. Graphs are as follows: median with range (C and D), mean with range (E). Statistical tests: Mann–Whitney (C and D), Student’s t-test with Welch’s correction (E). Abbreviations: immunohistochemistry (IHC), follicle-stimulating hormone (FSH), luteinizing hormone (LH), somatostatin receptor type 2 (SSTR2), oestrogen receptor alpha (ERα)