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Confinement induces an undifferentiated neuronal gene program. a, Schematic detailing the workflow of spatial transcriptomics and scRNA-seq experiments performed on zebrafish melanomas. b, Uniform manifold approximation and projection (UMAP) of human melanoma scRNA-seq dataset from Jerby-Arnon et al.7. Cluster annotations from the original paper are labelled. Tumour cell clusters are outlined. c, Gene module scoring for interface genes extracted from zebrafish spatial transcriptomics and scRNA-seq data, projected onto tumour cells outlined in b. The red arrow denotes the subpopulation with the highest expression of interface genes. d, Cell state classification for melanoma differentiation states identified by Tsoi et al.2. Cells were classified on the basis of the highest expression of the gene modules indicated. e, Module scores for melanoma cell state genes from Tsoi et al.2 in interface cells. f, Normalized expression per cell in UMAP space for the indicated genes. The red arrow indicates the interface cluster identified in b. g, Top 20 most highly upregulated genes in the human interface cluster. Neuronal genes are labelled in purple. h,i, GSEA barcode plot for the Gene Ontology (GO) pathways ‘cell fate specification’ (h) and ‘regulation of neuron differentiation’ (i). Normalized enrichment score (NES) and false discovery rate (FDR) are labelled. j,k, Immunofluorescence of adult zebrafish tissue sections highlighting the centre of the tumour (j) and tumour–TME interface (k). Individual nuclei are pseudocoloured and displayed without image overlay at right. l, Schematic of in vitro confinement workflow using a polydimethylsiloxane (PDMS) piston. m, Principal component analysis plot for each RNA sequencing (RNA-seq) replicate. Percentage variance for each principal component (PC) is labelled. n = 3 biological replicates for each condition. n, Top 10 most highly upregulated pathways from GSEA of confined cells relative to unconfined cells. NES and FDR are indicated. Scale bars, 100 μm (a), 10 µm (j,k).
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