Highlights d Generate combined scATAC-seq and scRNA-seq libraries of adult mouse MECs d Uncover luminal cell heterogeneity and molecular underpinning driving cell type/state d Identify the pre-committed lactation progenitor within the luminal progenitor compartment d Regulatory the grammar of MEC types and putative enhancer identification
Women with germline mutations in BRCA1 (BRCA1+/mut) have increased risk for developing hereditary breast cancer. Cancer initiation in BRCA1+/mut is associated with pre-malignant changes in the breast epithelium including altered differentiation, proliferative stress and genomic instability. However, the role of the epitheliumassociated stromal niche during BRCA1-driven tumor initiation remains unclear. Here, we show that the pre-malignant stromal niche promotes epithelial proliferation and BRCA1-driven cancer initiation in trans. Using single-cell RNAseq (scRNAseq) analysis of human pre-neoplastic BRCA1+/mut and control breast tissues, we show that stromal cells provide numerous pro-proliferative paracrine signals inducing epithelial proliferation. We identify a subpopulation of pre-cancer associated fibroblasts (pre-CAFs) that produces copious amounts of pro-tumorigenic factors including matrix metalloproteinase 3 (MMP3), and promotes BRCA1-driven tumorigenesis in vivo. Our gene-signature analysis and mathematical modeling of epithelial differentiation reveals that stromal-induced proliferation leads to the accumulation of luminal progenitor cells with altered differentiation, and thus contributes to increased breast cancer risk in BRCA1+/mut. Our results demonstrate how alterations in cell-cell communication can induce imbalances in epithelial homeostasis ultimately leading to cancer initiation. We anticipate our results to form the foundation for novel disease monitoring and therapeutic strategies to improve patient management in hereditary breast cancer. For example, pre-CAF specific proteins may serve as biomarkers for pre-cancerous disease initiation to inform whether radical bilateral mastectomy is needed. In addition, MMP inhibitors could be re-indicated for primary cancer prevention treatment in women with high-risk BRCA1 mutations.
Metastasis is a fatal disease where research progress has been hindered by a lack of authentic experimental models. Here, we develop a 3D tumor sphere culture-transplant system that facilitates the growth and engineering of patient-derived xenograft (PDX) tumor cells for functional metastasis assays in vivo. Orthotopic transplantation and RNA sequencing (RNA-seq) analyses show that PDX tumor spheres maintain tumorigenic potential, and the molecular marker and global transcriptome signatures of native tumor cells. Tumor spheres display robust capacity for lentiviral engineering and dissemination in spontaneous and experimental metastasis assays in vivo. Inhibition of pathways previously reported to attenuate metastasis also inhibit metastasis after sphere culture, validating our approach for authentic investigations of metastasis. Finally, we demonstrate a new role for the metabolic enzyme NME1 in promoting breast cancer metastasis, providing proof-of-principle that our culture-transplant system can be used for authentic propagation and engineering of patient tumor cells for functional studies of metastasis.
Metastatic disease carries a poor prognosis and its study is limited by the availability of authentic experimental models. Here, we present a 3D tumor sphere culture-transplant system that facilitates expansion and modulation of patient-derived xenograft (PDX) cells for use in functional experimental metastasis assays. RNA sequencing shows PDX tumor spheres maintain fundamental properties of native tumor cells. In vivo transplantation studies demonstrate cultured cells yield robust spontaneous and experimental metastasis following orthotopic and intravenous or intracardiac delivery, respectively. Pharmacologic inhibition of previously reported metastasis-promoting pathways also inhibits metastasis of PDX cells after culture, validating our approach. Finally, we used our culture-transplant system to demonstrate a new role of the metabolic enzyme NME1 in promoting breast cancer metastasis. Thus, we present a novel method for authentic propagation, engineering and transplantation of PDX models for functional studies of metastasis using bona fide patient tumor cells.
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