Extended Data Fig. 8: Pseudotime analysis of basal keratinocytes denotes distinctive early tumour state; (Corresponds to Fig. 3).
From: Precancerous niche remodelling dictates nascent tumour persistence
a, Left (inset), basal keratinocyte cell distribution in the UMAP space annotated by condition (right, UMAP consisting of all cell types). b, UMAP of basal keratinocyte cell distribution representing cells in each condition. c, UMAPs showing expression of representative basal keratinocytes genes (Col17a1, basal; Top2a, cycling basal; Krt4, committed). Colour bars of UMAPs indicate log-transformed normalised expression levels. d, UMAP showing cell clusters as defined by Seurat. e, Mapped RNA velocity of basal keratinocytes colour coded by condition. Black arrows are velocity vectors that show predicted cellular state change overtime. f, Basal keratinocyte two-dimensional pseudotime (PST) UMAP (top) and trajectories (black line) (bottom) inferred by Monocle 3. Legend shows the range of pseudotime values g, Heatmap (left) of the top 1500 genes identified as differentially expressed between cells in the tumour and basal trajectories. Log-transformed expression levels were scaled in a gene-wise fashion from −4 to 4 (scale) and are shown after hierarchical clustering along trajectories from committed to basal (blue to orange) and from committed to tumour (blue to magenta). The average expression patterns of the genes contained in each cluster are depicted on the right, with two modules (1 and 12) identified as tumour unique based on their preferential upregulation at the end of the tumour trajectory (Δ_tumour > 2, Δ_basal <1). (Blue, committed; Magenta, tumour; Orange, basal). Dashed vertical lines label the highest PST score of committed. h, Tumour cell populations representing Tumour 1 and Tumour 12 clustering in UMAP space. i, Violin plots highlighting Krt6a and Krt17 expression between Tumour 12 and Tumour 1 states. j, Volcano plot showing differential gene expression between Tumour 12 and Tumour 1 states. Non-significant genes (p value higher than p = 0.05), green. Significant genes with FC ≤ 1, red. Genes driving stress and extracellular matrix related processes are labelled. k, Gene Set Enrichment Analysis of Tumour 12 versus Tumour 1 differentially expressed genes. Terms ranked by FDR (false discovery rate); representative genes listed below the bar plot. GO:BP (Gene Ontology Biological Processes); KEGG (Kyoto Encyclopedia of Genes and Genomes). Cartoon (top right) representing Tumour 12 keratinocyte communicates with stroma. l, Violin plots highlighting differences between Tumour 12 and Tumour 1 states. m, Representative confocal images from 6 mice showing expression of tumour markers: SOX9 and EGR1 (cyan); AREG and RUNX1, (magenta); KRT6A, red; KRT17 (yellow). DEN images were acquired 10 d after DEN treatment in regions adjacent to tumours. DAPI (blue). Scale bars, 10 µm. n, Relative frequency (%) (top) of tumours expressing cells positive for SOX9 (Tumour 12 state marker) per 1,000 µm2, tumours 10 d post-DEN, n- number of tumours=119 from 3 biological replicates. o, Representative confocal image from 10 mice demonstrating SOX9+ (cyan) keratinocytes are situated in direct contact to stroma (PDGFRα, grey). KRT6A (red), DAPI (blue). Scale bar 50 µm. Days (d), months (m). Illustration in k was created in BioRender; Alcolea, M. https://BioRender.com/mhoqqbw (2026). Source data (n).
