Extended Data Fig. 2: Basal cells give rise to SCLC with expansive subtype heterogeneity. Related to Fig. 1 and Supplementary Tables 1 and 2.

a, Survival of RPM mice infected with indicated Ad-Cre viruses. Mouse numbers in figure. Mantel-Cox log-rank test vs. K5-Cre + naphthalene (purple); exact p-values in figure. b, IHC images for indicated proteins from in situ (3–5 wks post-infection) or invasive (> 6 weeks post-infection) RPM K5-Cre tumours. Invasive tumours are most often negative for basal markers (middle row), but rare tumours have sporadic basal marker expression (bottom row). Representative of tumours from n = 10 mice. c, POU2F3 IHC H-scores by airway location and Ad-Cre virus. Each dot = 1 tumour. Kruskal-Wallis with post-hoc Dunn’s test (p-values in figure). N = 11 Cgrp, 43 Cmv, 101 K5-Cre tumours quantified (as in Fig. 1e), but split by airway location. Error represents mean ± SD. d, YAP1 IHC in RPM tumours by indicated Ad-Cre viruses (left) and H-score quantification (right). Median (red bar) and quartiles (dotted liness) indicated. KWwith post-hoc Dunn’s multiple comparisons (exact p-values in figure). Number of tumours quantified indicated on figure. e, Co-immunofluorescent (co-IF) staining for DAPI (nuclei, blue), ASCL1 (green), NEUROD1 (purple), or POU2F3 (red) in RPM K5-Cre tumours, representative of n = 5 mice. Tumour regions outlined with dashed white line. Yellow arrows indicate co-expressing cells. Scale bars, 75 μm. f, Quantification of co-expression by immunofluorescence staining for SCLC subtype markers ASCL1 (A), NEUROD1 (N), or POU2F3 (P) from n = 10 K5-Cre-initiated RPM tumours from n = 5 mice, where tumours have 1 or >1 subtype-defining transcription factor(s) (TF) detected (left). Student’s unpaired two-tailed t-test. Box and whiskers represent min, median, and max values. Percent of RPM tumour cells co-expressing the indicated subtype markers (right). One-way ANOVA with post-hoc Tukey’s. Exact p-values in figure. Each dot represents one field of a tumour. Error, mean with SEM. g, UMAP of scRNA-seq from RPM tumours: NE-derived (Cgrp-Cre, purple, n = 6 mice, 7 tumours) vs basal-derived (K5-Cre, orange, n = 2 mice, 4 tumours). Cells coloured by sample on right with number of cells analyzed indicated. h, UMAP (top) and violin plots (bottom) of selected gene expression in Cgrp- vs K5-Cre–derived tumour cells. Each dot is one cell. Two-sided Wilcoxon rank sum tests. Exact p-values indicated in figure. i, UMAP in (g) annotated by Leiden cluster (left) (Supplementary Table 1). Proportion of cells from Cgrp- vs K5-Cre tumours per Leiden cluster, as % of all cells per sample (right). j,m, Signature scores in tumour cells from (g): j, ChIP-seq targets, k, NE score, l, SCLC archetypes, or m, human SCLC subtype-specific signatures. Yellow/red diamond = mean. Two-sided Wilcoxon rank sum tests. Exact p-values indicated in figure. See Supplementary Table 2 for gene signatures. n, Stacked bar graph depicting percent of Leiden clusters as in (i) dominated by Cgrp- (purple) vs. K5-Cre (orange) initiated RPM tumour cells (left). Dot plot of top 10 differentially-expressed marker genes per Leiden cluster (Supplementary Table 1) (right). o, Violin plots of SCLC-A2 archetype or basal- or luminal-hillock signatures by Leiden cluster as in (i). Red diamond =mean. KW tests (p-value indicated on figure) and post-hoc Dunn’s with Bonferroni correction; ****p < 2e-16, other exact p-values in figure. Scale bars, 50 μm unless otherwise noted.

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