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Breast tumors are inherently heterogeneous, but the evolving cellular organization through neoplastic progression is poorly understood. Here we report a rapid, large-scale single-cell resolution 3D imaging protocol based on a one-step clearing agent that allows visualization of normal tissue architecture and entire tumors at cellular resolution. Imaging of multicolor lineage-tracing models of breast cancer targeted to either basal or luminal progenitor cells revealed profound clonal restriction during progression. Expression profiling of clones arising in Pten/Trp53-deficient tumors identified distinct molecular signatures. Strikingly, most clones harbored cells that had undergone an epithelial-to-mesenchymal transition, indicating widespread, inherent plasticity. Hence, an integrative pipeline that combines lineage tracing, 3D imaging, and clonal RNA sequencing technologies offers a comprehensive path for studying mechanisms underlying heterogeneity in whole tumors. • A single-step, non-toxic clearing agent for 3D imaging of whole organs and tumors • Derivation of an integrative platform to interrogate intratumoral heterogeneity • Profound clonal restriction occurs during neoplastic progression • The epithelial-mesenchymal transition occurs clonally as a frequent event Rios et al. develop a rapid, large-scale single-cell resolution 3D imaging protocol and use the protocol together with RNA sequencing to explore the cellular dynamics of mammary tumorigenesis and show that a molecular epithelial-to-mesenchymal transition is a prominent feature of tumor clones. [ABSTRACT FROM AUTHOR]

Summary Long-lived quiescent mammary stem cells (MaSCs) are presumed to coordinate the dramatic expansion of ductal epithelium that occurs through the different phases of postnatal development, but little is known about the molecular regulators that underpin their activation. We show that ablation of the transcription factor Foxp1 in the mammary gland profoundly impairs ductal morphogenesis, resulting in a rudimentary tree throughout life. Foxp1 -deficient glands were highly enriched for quiescent Tspan8hi MaSCs, which failed to become activated even in competitive transplantation assays, thus highlighting a cell-intrinsic defect. Foxp1 deletion also resulted in aberrant expression of basal genes in luminal cells, inferring a role in cell-fate decisions. Notably, Foxp1 was uncovered as a direct repressor of Tspan8 in basal cells, and deletion of Tspan8 rescued the defects in ductal morphogenesis elicited by Foxp1 loss. Thus, a single transcriptional regulator Foxp1 can control the exit of MaSCs from dormancy to orchestrate differentiation and development. Graphical Abstract Highlights • Foxp1 is an essential transcription factor for mammary gland development • Foxp1 controls the activation of quiescent MaSCs marked by Tspan8 • Foxp1 directly represses the transcription of Tspan8 in basal, but not luminal, cells • Tspan8 deletion rescues the Foxp1 -deficient mammary phenotype Fu et al. show that the transcriptional repressor Foxp1 is crucial for governing the exit of mammary stem cells from quiescence. Moreover, Tspan8 was identified as a direct Foxp1 target that plays a functional role in regulating the stem cell state, and its deletion reversed the effects of Foxp1 loss. [ABSTRACT FROM AUTHOR]