Fig. 2: Cracd KO increases SCLC tumor cell heterogeneity.

a–d Workflow for spatial transcriptomics. Spatial transcriptomic results are shown with transcripts per bin, using a bin size of 20 μm (b). Scale bars = 2 mm. Cells were segmented and clustered by transcriptomes. 33 clusters were identified in each sample (c), and tumor cell clusters of RPR2 and CRPR2 were highlighted (d). Representative Xenium spatial transcriptomic results are shown. The experiment was independently repeated twice on distinct tumor samples with similar results. e Pie charts displaying the cell cluster composition for each tumor cell clone in RPR2⁸⁰ and CRPR2 (bottom). f UMAP projection of scRNA-seq data from RPR2 and CRPR2 tumors. Cells are colored by unsupervised clusters; NE and non-NE populations are delineated by dashed lines. g Spatial localization of NE and non-NE clusters in representative RPR2 and CRPR2 tumors. Cluster identities were inferred via correlation-based mapping of Xenium transcriptomic profiles to matched scRNA-seq clusters. h Spatial mapping of cell cycle states projected onto the Xenium coordinate space. G1 (magenta), S (aqua), and G2/M (purple). i UMAPs of tumors within RPR2 (up) and CRPR2 (down). UMAP coordinates profiling and tumor number annotations (T1-8) were performed using Xenium Explorer. j IHC of RPR2 vs. CRPR for ASCL1. Scale bars: 50 μm. Representative images (n ≥ 3). k IF staining of RPR2 and CRPR2 cells with phalloidin. Scale bars: 20 μm. l IHC of RPR2 vs. CRPR2 for YAP1. Scale bars: 50 μm. Representative images (n ≥ 3). m Immunoblot⁵⁷ of RPR2 cells treated with vehicle or verteporfin (VP) 0.5 (+) or 1.0 ( + + ) μM. Representative immunoblot from three independent experiments. n IB of RPR2 vs. CRPR2 transduced with lentiviruses encoding N1ICD. Representative immunoblot from three independent experiments. o Working model: CRACD inactivation activates HIPPO signaling to phosphorylate and inhibit YAP1, leading to NOTCH1 downregulation and ASCL1-mediated NE lineage activation, thereby promoting NE cell plasticity and tumor heterogeneity. a, o were created with BioRender.com. Source data are provided as a Source Data file.