Fig 2: Characterisation of the RMS immune microenvironment.a UMAP projection of myeloid cells, coloured by cluster assignment. b Dot plot depicting the average expression of selected cell type-specific genes (Mq = differentiated macrophages, M0 = undifferentiated macrophages, cDC = conventional dendritic cells and pDC = plasmacytoid dendritic cells). Dot size corresponds to the percentage of cells expressing each marker. Colour bar on the x-axis indicates for which cluster each gene is specific. c Combined Violin/Box and UMAP plots showing the distribution of M1 (left panel) and M2 (right panel) signature scores in undifferentiated (M0, coloured yellow in the violin/box plots) and differentiated (Mq, coloured blue in the violin/box plots) macrophages (n = 637 biologically independent cells). ns = not significant (p > 0.05, Student’s T test), **** indicates p < 2.2e-16 (two-sided student’s T test). Mean is used as the centre measurement for each box, which encloses the range between the first and third quartiles. Whiskers extend to the largest (or smallest) values no further than 1.5× the IQR from the box hinges. Non-macrophage cells are coloured grey in UMAP plots. d Representative immunofluorescence (IF) microscopy images depicting the expression of CD206 (green) and DAPI counterstaining (blue), in RMS tissue sections from FN and FP tumours. Scale bars equivalent to 50 µm. e UMAP projection of T and NK cells, coloured by cluster assignment. f Dot plot depicting the average expression of selected cell type-specific genes (Naïve T = Naïve T cells, GD T = Gamma delta T cells, CD8 + T = Cytotoxic T cells, ILR7 + CD4 + T = IL7R + T helper cells, ISG + CD4 + T = Interferon stimulated T helper cells, Treg = T regulatory cells, NK = Natural Killer cells). Dot size corresponds to the percentage of cells expressing each marker. Colour bar on the x-axis indicates the cluster specificity for each gene. g Normalised enrichment scores (NES) of selected gene sets, as determined by gene set enrichment analysis (GSEA) comparing CD8 + T cells between RMS subtypes. Codes in parenthesis indicate the database from which the gene set derives (H, C2 and C7 correspond to MSigDB collections). Colour corresponds to a positive (pink) or negative (blue) NES. h Representative IF microscopy images depicting the expression of TIGIT (red) and NECTIN3 (green), along with DAPI counterstaining (blue), in RMS tissue sections from FN and FP tumours. White arrows highlight TIGIT+ cells. Scale bars equivalent to 50 µm. Source data are provided as a Source Data file.