Basal cell of origin resolves neuroendocrine-tuft lineage plasticity in cancer [Single cell RNA-seq]

Neuroendocrine and tuft cells are rare, chemosensory epithelial lineages defined by expression of ASCL1 and POU2F3 transcription factors, respectively. Neuroendocrine cancers, including small cell lung cancer (SCLC), frequently display tuft-like subsets, a feature linked to poor patient outcomes. The mechanisms driving neuroendocrine–tuft tumour heterogeneity, and the origins of tuft-like cancers are unknown. Using multiple genetically-engineered animal models of SCLC, we demonstrate that a basal cell of origin (but not the accepted neuroendocrine origin) generates neuroendocrine–tuft-like tumours that highly recapitulate human SCLC. Single-cell clonal analyses of basal-derived SCLC further uncovers unexpected transcriptional states and lineage trajectories underlying neuroendocrine–tuft plasticity. Uniquely in basal cells, introduction of genetic alterations enriched in human tuft-like SCLC, including high MYC, PTEN loss, and ASCL1 suppression, cooperate to promote tuft-like tumours. Transcriptomics of 944 human SCLCs reveal a basal-like subset and a tuft-ionocyte-like state that altogether demonstrate remarkable conservation between cancer states and normal basal cell injury response mechanisms. Together, these data suggest that the basal cell is a plausible origin for SCLC and other neuroendocrine-tuft cancers that can explain neuroendocrine–tuft heterogeneity—offering new insights for targeting lineage plasticity. Overall design: For basal organoids, mouse tracheas from Rb1 fl/fl; Trp53 fl/fl; Rbl1/p130 fl/fl (RPR2), Rb1 fl/fl; Trp53 fl/fl;H11b-LSL-MycT58A-Ires-Luc (RPM), and Rb1 fl/fl; Trp53 fl/fl;H11b-LSL-MycT58A-Ires-Luc; Ascl1 fl/fl (RPMA) mouse models (not exposed to Cre recombinase) were isolated and ITGA6+ basal cells were live-sorted and propogated as organoids. Organoids from each genotype were lineage tagged with the CellTag V1 lentiviral library (Kong et al., Nature Protocols, 2020) pre-transformation ("CellTagged Pre-Cre"; to trace lineage from a single normal basal cell of origin) and one set of RPM organoids were rather "CellTagged Post-Cre" (to trace lineage from a single transformed basal cell of origin). Wildtype organoids (no CellTag information used) were subject to 10X single cell RNA-sequencing (scRNA-seq) and then recombined via Adenoviral-CMV-Cre. "Celltagged Pre-Cre" recombined organoids were subject to scRNA-seq and were submitted as one sample, but multiplexed using 10X Genomics CellPlex Kit (CellPlex oligos included as metadata information). RPM "CellTagged Post-Cre" recombined organoids were also subject to 10X scRNA-seq. On the same day of scRNA-seq sample preparation of transformed organoids, RPM ("CellTagged Pre-Cre" and "CellTagged Post-Cre"), RPR2, and RPMA organoids were allografted into flanks of scid/beige mouse hosts. Resulting allograft tumors (n=2 RPM "CellTagged Post-Cre", n=1 RPM "CellTagged Pre-Cre", n=1 RPMA, and n=1 RPR2) were subject to mechanical and enzymatic digestion into a single-cell suspension, then underwent 10X scRNA-seq library prep. For primary tumors, Rb1 fl/fl; Trp53 fl/fl;H11b-LSL-MycT58A-Ires-Luc (RPM) mice were treated with naphthalene to stimulate basal cell proliferation, then 72 hours later received KRT5-Cre to initiate tumors from basal cells in the mouse lung. Upon tumor formation, n=2 tumors from n=2 K5-Cre initiated mice were isolated, digested, pooled (one pool multiplexed), then subject to scRNA-seq. In addition, n=1 primary RPM tumor and n=1 primary RPM-Cas9-GFP tumor, initiated by CGRP-Cre were isolated (one tumor per animal), digested, pooled and multiplexed using 10X CellPlex oligos, and subject to scRNA-seq. See associated GEO metadata and manuscript methods for additional detail. Source code and additional metadata used to reproduce these analyses are also publicly available on Github (https://github.com/TGOliver-lab/Ireland_Basal_SCLC_2025). CellTag Experiment "A"= RPM matching organoids and resulting allograft, CellTagged pre-Cre; CellTag Experiment "B"= RPM matching organoids and resulting allografts (n=2), CellTagged post-Cre; CellTag Experiment "C"= RPMA matching organoids and resulting allograft, CellTagged pre-Cre.