2 | Nature | www.nature.com Article 1. Patient inclusion ≤24 mo RFS >60 mo RFS Stage III n = 5–6 ≤48 mo RFS >96 mo RFS Stage II n = 5 2. ROI selection Peritumoural Cortex Paracortex Cortex Paracortex 3. Acquisition Laser Mass spec a Control n = 2 4. Image stacks ×40 7. Cell composition Percentage Sample 8. Spatial analysis 5. Segmentation 6. Annotation Intratumoural B cells Naive CD4 + T Non-naive CD4 + T Naive CD8 + T Non-naive CD8 + T BECs LECs CD163 + mac CD169 + mac Melanophages Neutrophils Fibroblasts Collagen Tumour HLA-DR hi DCs HLA-DR lo DCs Non-naive T reg Naive T reg b d R DF Marker expression Cell annotation NB Tumour Macrophages CD8 + non-naive T CD8 + naive T T reg DCs Other 1 2 3 4 5 6 7 8 9 10 CD8a CD11c CD68 FOXP3 DNA Dyad Dyad-adjecent macrophage <15–30 μ m DC CD8 Mac i g z -score –4 0 4 CSK-III SOX10 CD169 CD163 CD11c CD68 CD14 TCF1 CD4 CD3 TOX CD45 CTLA4 PD-1 HLA-DR FOXP3 CD8a Ki-67 CD45RA CD20 D2-40 CD15 Granzyme B CD31 Col-I 15.0 0 B cells BECs Naive CD4 + T Non-naive CD4 + T Non-naive CD8 + T Naive CD8 + T Fibroblasts HLA-DR hi DCs HLA-DR lo DCs LECs CD163 + mac CD169 + mac Melanophages Neutrophils Collagen Naive T reg Non-naive T reg Tumour Cells (×1,000) c Avoid Interact Macrophages R CD8 + T cells DF DCs Proportion of ROIs f Cell annotation: Tumour Stroma CD8 + T CD4 + T T reg Fibroblasts Endothelial B cells Neutrophils DCs Macrophages Undefined Distance to CD8–DC dyad: >30 μ m 15–30 μ m 0 μ m <15 μ m CD8a CD11c CD68 h j e Min Max Cells (%) B cells BECs Naive CD4 + T Non-naive CD4 + T Naive CD8 + T Non-naive CD8 + T Fibroblasts HLA-DR hi DCs HLA-DR lo DCs LECs CD163 + mac CD169 + mac Melanophages Neutrophils Stroma Naive T reg Non-naive T reg R DF R DF R DF B cells CD4 + T cells CD8 + T cells DCs Endothelial Fibroblasts Macrophages Neutrophils Stroma T reg cells 0 2 4 –4 –2 0 2 −log 10 ( P ) Cortex –4 –2 0 2 Intratumoural –5.0 –2.5 0 2.5 5.0 Paracortex Tumour Naive T reg CD169 + mac Naive CD4 + T HLA-DR hi DCs HLA-DR lo DCs log 2 -transformed fold change CD169 + mac Non-naive T reg B cells Non-naive CD4 + T Naive T reg BECs –3 –2 –1 0 1 2 3 Peritumoural CD169 + mac Stroma Fibroblasts R DF R DF R DF R DF Area of NB2 cells ( μ m 2 per mm 2 ) **** 0 1 × 10 5 2 × 10 5 3 × 10 5 4 × 10 5 R DF DF R Macrophages near dyad per ROI (%) <15 μ m <30 μ m Distance from macrophage to dyad Cells in dyad with DC (%) Non-naive *** 0 25 50 75 100 Naive CD8 + T cells 0 25 50 75 100 **** **** **** Fig. 1 | See next page for caption.