Fig. 4: RNA in situ hybridization validating intratumoral heterogeneity in high-risk neuroblastoma stage 4.

Overview of tile-scanned images (20x) high-risk neuroblastoma (K10, MYCN amplified) using RNAscope in situ hybridization. Scalebar of overview: 500 μm; zoom of boxed image: 10 μm. a Tumor labeled with RNAscope ISH for TH (green), MYCN (red), and ALK (white) mRNA, and counter stained with DAPI (blue). Dashed circles indicate cells with large nuclei in a region (box #1), that are TH and MYCN positive, but negative for ALK. Box #2 indicates part of the tumor with majority of cells double positive for MYCN and ALK but negative for TH. Box #4 indicates cells negative for all probes: TH, MYCN, and ALK. b Adjacent section from (a) labeled for TH (green), NTRK1 (red), and NTRK2 (white). Box #1 indicate cells with large nuclei in a region that are TH+ and NTRK1+, and negative for NTRK2. Surrounding cells with small nuclei are positive for NTRK2 only. Box #2,3 visualizes tumor region with majority of cells positive for NTRK2 that are negative for TH and NTRK1. c Adjacent section stained for PDGFRA (green), LGR5 (red), and NTRK2 (white) mRNA is highlighting cells double positive for LGR5 and NTRK2 (box #1) or double positive for PDGRFA, NTRK2 (box #2). Box #3 indicates a region of the tumor that is positive for NTRK2 only. d Adjacent section stained for mesenchymal markers PDGFRA (green), CLDN11 (red), and LGR5 (white). Similar to c: some tumor regions (box #1) highlight cells double positive for CLDN11 (red) and LGR5 (white) that are negative for PDGFRA (box #1), as were other regions show cells that are double positive for PDGFRA (green) and CLDN11 (red) (box #2, 3, and 4). For all RNAscope experiments, the signal distribution patterns and cell morphological features were shown by the different combination of probes and independently reproduced three times on different samples in (a) and (b), and independently reproduced four times on different samples in (c) and (d).