Epithelial-mesenchymal transition (EMT) enables epithelial cancer cells to acquire mesenchymal-associated traits that can promote invasion and metastasis. Although distinct EMT-associated states have been linked to invasive and metastatic behavior, it remains unclear when these states arise during primary tumor progression, how they diversify, and whether metastatic competence is restricted to a particular EMT phenotype. Using single-cell RNA sequencing in a genetically engineered mouse model of triple-negative breast cancer (TNBC), together with functional studies of tumor organoids, we reconstructed the emergence of EMT-associated heterogeneity during tumor progression. We found that early malignant cells first lost mammary lineage identity, generating lineage-altered epithelial states with increased intrinsic plasticity. Rather than progressing through a single EMT program, these plastic states diversified through ERK1/2-low and ERK1/2-high EMT-associated programs. These programs generated distinct hybrid epithelial-mesenchymal states in early tumors and more uniform mesenchymal-like subpopulations at later stages, with canonical EMT features, diminished plasticity, and highly invasive behavior. Importantly, metastatic competence was not restricted to a single EMT-associated state: both heterogeneous hybrid cells and more uniform mesenchymal-like cells initiated metastases, with metastatic lesions retaining features of their initiating populations. Together, our results show that EMT-associated heterogeneity in TNBC emerges through early lineage-state disruption followed by parallel regulatory programs that generate distinct metastatic cell states rather than converging on a single highly metastatic phenotype.

Germline inactivating mutations of the SLC25A1 gene contribute to various human disorders, including Velocardiofacial (VCFS), DiGeorge (DGS) syndromes and combined D/L-2-hydroxyglutaric aciduria (D/L-2HGA), a severe systemic disease characterized by the accumulation of 2-hydroxyglutaric acid (2HG). The mechanisms by which SLC25A1 loss leads to these syndromes remain largely unclear. Here, we describe a mouse model of SLC25A1 deficiency that mimics human VCFS/DGS and D/L-2HGA. Surprisingly, inactivation of both Slc25a1 alleles results in alterations in the development of multiple organs, and in a severe proliferation defect by activating two senescence programs, oncogene-induced senescence (OIS) and mitochondrial dysfunction-induced senescence (MiDAS), which converge upon the induction of the p53 tumor suppressor. Mechanistically, cells and tissues with dysfunctional SLC25A1 protein undergo metabolic and transcriptional rewiring leading to the accumulation of 2HG via a non-canonical pathway and to the depletion of nicotinamide adenine dinucleotide, NAD+, which trigger senescence. Replenishing the pool of NAD+ or promoting the clearance of 2HG rescues the proliferation defect of cells with dysfunctional SLC25A1 in a cooperative fashion. Further, removal of p53 activity via RNA interference restores proliferation, indicating that p53 acts as a critical barrier to the expansion of cells lacking functional SLC25A1. These findings reveal unexpected pathogenic roles of senescence and of p53 in D/L-2HGA and identify potential therapeutic strategies to correct salient molecular alterations driving this disease.

MOV file - 1.5MB, Live-imaging of a MCFDCIS-mCherryCAAX spheroid and FITC-Collagen-1 when co-cultured with mammary fibroblasts.

A) FDR-corrected shared and unique ChIP peaks of AIB1 and AIB1Î"4 in MCF10A parental cell line (10A) versus AIB1Î"4-expressing isogenic line (10A-Î"4). Shared peaks have at least 1bp overlap. B) AIB1 ChIP peaks in MCF7 cells treated for 3 hours with estradiol (E2) or vehicle (V) overlapped with MCF10A and MCFDCIS (parental and AIB1Î"4 derivative lines). MCF7 data set obtained from Zwart et al(32). C) Motifs within 200 bp of the peak summit for significant ChIP-Seq peaks were analyzed for motif enrichment using HOMER. In red are the motifs unique to AIB1Î"4 in MCF10A cells. D) FDR-corrected histone modification peaks in DCIS and DCIS-Î"4 overlapping with enhancer sites from human mammary epithelial cells (hMEC enh.) (42). E) The distribution of AIB1, H3K27ac or H3K4me1 ChIP peaks 500 kb up- and downstream of gene transcription start site (TSS). Genomic distance was divided into 5kb bins.

A list of differentially expressed genes in tumors and cells.

<p>MOV file - 312K, Live-imaging of a HCC1806-H2B:GFP spheroid co-cultured with fibroblasts.</p>

PDF file - 2MB, Expression of Cdc42 in mammary fibroblasts transfected with siRNAs.

ABSTRACTBiallelic germline mutations in theSLC25A1gene lead to combined D/L-2-hydroxyglutaric aciduria (D/L-2HGA), a fatal systemic disease uniquely characterized by the accumulation of both enantiomers of 2-hydroxyglutaric acid (2HG). How SLC25A1 deficiency contributes to D/L-2HGA and the role played by 2HG is unclear and no therapy exists. Both enantiomers act as oncometabolites, but their activities in normal tissues remain understudied. Here we show that mice lacking bothSLC25A1alleles exhibit developmental abnormalities that mirror human D/L-2HGA. SLC25A1 deficient cells undergo premature senescence, suggesting that loss of proliferative capacity underlies the pathogenesis of D/L-2HGA. Remarkably, D- and L-2HG directly induce senescence and treatment of zebrafish embryos with the combination of D- and L-2HG phenocopies SLC25A1 loss, leading to developmental abnormalities in an additive fashion relative to either enantiomer alone. Metabolic analyses further demonstrate that cells with dysfunctional SLC25A1 undergo mitochondrial respiratory deficit and remodeling of the metabolism and we propose several strategies to correct these defects. These results reveal for the first time pathogenic and growth suppressive activities of 2HG in the context of SLC25A1 deficiency and suggest that targeting the 2HG pathway may be beneficial for the treatment of D/L-2HGA.

<p>MOV file - 314K, Live-imaging of a HCC1806-H2B:GFP spheroid.</p>

<p>MOV file - 73K, Live-imaging of a T47D-H2B:GFP spheroid co-cultured with fibroblasts.</p>

PDF file - 1.6MB, EGFR and ERK1/2 are necessary for HCC1806 cell morphogenic movement and fibroblast induced collective invasion.

<p>Supplementary tables related to reagents used.</p>

PDF file - 2MB, MDA-MB-231 breast cancer cells, but not MCFDCIS cells collectively invade in organotypic culture.

A) Quantification of the number of Zebrafish embryos with DCIS or DCIS-Î"4-hy cells that have extravasated out of the blood vessels and into the neighboring tissue. p=0.57. B) Quantification of the number of Zebrafish embryos with MCF10A, MCF10A-Î"4 or mixed cells that have extravasated out of the blood vessels and into the neighboring tissue. In the mixed cell population, only MCF10A parental line was fluorescently labeled and scored for extravasation. MCF10A and MCF10A-Î"4 are mixed at a 4:1 ratio. C) Invasion assay of an endothelial monolayer (HUVEC) using ECIS. MCFDCIS cells were treated with conditioned media (CM) from MCFDCIS-Î"4 cells for 4 hours before added to the endothelial monolayer, and vice versa. No cells added to the endothelial monolayer as a negative control. D) A schematic showing a three-chambered co-culture system. Top chamber has electrodes underneath a porous membrane to detect migrating cells. Middle chamber is only permeable to factors but not cells. Bottom chamber harbors cells that can crosstalk with migrating cells through secreted factors. E) Real-time migration rate of MCFDCIS and MCFDCIS-Î"4 in co-culture chambers. F) MCFDCIS and MCFDCIS-Î"4 cells were aggregated separately then embedded in 80% col I and 20% Matrigel together to monitor their crosstalk as separate spheres. Scale bar = 50μm.

MOV file - 1.1MB, Live-imaging of a MCFDCIS-H2B:mCherry spheroid co-cultured with mammary fibroblasts.

Natural killer (NK) cells play a critical role in physiologic and pathologic conditions such as pregnancy, infection, autoimmune disease and cancer. In cancer, numerous strategies have been designed to exploit the cytolytic properties of NK cells, with variable success. A major hurdle to NK-cell focused therapies is NK cell recruitment and infiltration into tumors. While the chemotaxis pathways regulating NK recruitment to different tissues are well delineated, the mechanisms human NK cells employ to physically migrate are ill-defined. We show for the first time that human NK cells express fibroblast activation protein (FAP), a cell surface protease previously thought to be primarily expressed by activated fibroblasts. FAP degrades the extracellular matrix to facilitate cell migration and tissue remodeling. We used novel in vivo zebrafish and in vitro 3D culture models to demonstrate that FAP knock out and pharmacologic inhibition restrict NK cell migration, extravasation, and invasion through tissue matrix. Notably, forced overexpression of FAP promotes NK cell invasion through matrix in both transwell and tumor spheroid assays, ultimately increasing tumor cell lysis. Additionally, FAP overexpression enhances NK cells invasion into a human tumor in immunodeficient mice. These findings demonstrate the necessity of FAP in NK cell migration and present a new approach to modulate NK cell trafficking and enhance cell-based therapy in solid tumors. Graphical Abstract

MOV file - 76K, Live-imaging of a T47D-H2B:GFP spheroid.

<p>MOV file - 125K, Live-imaging of a MCFDCIS-mCherryCAAX spheroid.</p>