Fig 2: Aberrant Pfkfb3 expression drives emergence from metastatic dormancy and increased BCSC frequency. a D2.OR and D2.A1 cells were incubated in the absence or presence of the Pfkfb3 inhibitor, 3PO (0–500 µM), and differences in cell viability were quantified by CellTiter-Glo Assay. Data are the mean (±SD; n = 3). **P = 0.01, ***P = 0.001, and ****P = 0.0001. This experiment was independently repeated for a total of three experiments, all with similar results. b Pfkfb3 overexpression drives D2.OR organoid growth in 3D cultures, while Pfkfb3-deficiency suppresses the growth of D2.A1 organoids in 3D culture, c, d Parental and Pfkfb3-deficient D2.A1 cells (1 × 106 cells/injection) were inoculated into the lateral tail veins of BALB/c mice (n = 5). Pulmonary tumor growth was monitored by BLI and normalized to Day 0 readings. IHC images (×20 magnification) show loss of Pfkfb3 knockdown in tumors excised on Day 72. e Parental and Pfkfb3-overexpressing D2.OR cells or f parental and Pfkfb3-deficient D2.A1 cells were monitored for mammosphere forming ability by ELDA (n = 8). Stem cell frequency was evaluated using the ELDA: Extreme Limiting Dilution Analysis (http://bioinf.wehi.edu.au/software/elda/). These experiments were independently repeated for a total of three experiments, all with similar results. g Evaluating CD49f/CD24 expression levels by flow cytometry in parental and Pfkfb3-overexpressing D2.OR cells and in parental and Pfkfb3-deficient D2.A1 cells

Fig 3: Reprogramming of PB 49f Cells(A) Diagrams illustrating the sorting strategy to isolate PB 49f cells, CMPs, and GMPs. Mononuclear cells were stained with a cocktail of lineage antibodies, CD34, CD38, CD123, CD45RA, Thy1, and CD49f, and with Rhodamine 123 and sorted as single cells in round-bottom 96-well plates. Dead cells were gated out using DAPI staining.(B) Individually sorted cells were transduced with Sendai virus on day 0, incubated with cytokines until day 3, without cytokines from day 3 to day 7, and switched to iPSC growing condition. OKSM, Oct4, KLF4, Sox2, Myc. MPP, multipotent progenitors. CLP, common lymphoid progenitor.(C) Bar graph illustrating the efficiency of reprogramming of PB 49f cells, CMPs, and GMPs transduced as described in (B). The results of experiments in which 20-40 individual 49f cells, CMPs, or GMPs from seven different individuals were transduced are summarized. 49f cells can be reprogrammed more efficiently than CMPs and GMPs. The error bars represent the SEM. * t test, p < 0.05; **t test, p < 0.01. Efficiency of reprogramming was assessed 3 weeks after transduction using alkaline phosphatase staining and confirmed by multiple assays (see text).(D) Individual PB 49f cells were sorted in individual wells and allowed to divide three to five times before being transduced as above. In these conditions nearly 100% of the wells yielded multiple colonies of iPSCs demonstrating that all the 49f cells that survive the sorting are competent to be reprogrammed. Two experiments in which either 10 or 20 49f cells were sorted are summarized.

Fig 4: Inactivation of autophagy induces BCSC expression of Pfkfb3 and emergence from metastatic dormancy. Inactivation of autophagy induces Pfkfb3 expression in D2.OR cells. Autophagy was inhibited by administration of chloroquine (CQ; 25 µM; a), and by shRNA-mediated depletion of either Atg3 (b), Atg7 (c), or p62 (d). Inactivation of either Atg3 (e, ***P = 0.001), Atg7 (f, **P = 0.01), or p62 (f, **P = 0.01; ****P = 0.0001) promotes D2.OR organoid outgrowth in 3D cultures. This experiment was independently repeated for a total of 3 experiments, all with similar results. g Dual inhibition of autophagy through Atg3 knockdown and Pfkfb3 by PFK-158 treatment inhibits D2.OR organoid outgrowth in 3D cultures. Data represent mean (±SD) of biological replicates performed in triplicate (ns P > 0.05, **P = 0.01, and ****P = 0.0001). h CD49f/CD24 flow cytometry evaluation in parental and Atg3-deficient D2.OR cells. i BCSC frequency as determined by an ELDA mammosphere assay. j Quantification of total cell numbers isolated following completion of the primary mammosphere assays depicted in i. Data demonstrate significantly larger spheres associated with Atg3-deficient D2.OR cells as compared with parental D2.OR cells. k Parental (i.e., NT) and Atg3-deficient D2.OR cells were inoculated into the lateral tail veins of BALB/c mice (2 × 106 cells/mouse) and pulmonary tumor development was monitored by BLI. Photomicrograph shows macroscopic tumors (black arrows) in 2/5 mice inoculated with Atg3-deficient D2.OR cells. l Microscopic nodule count from lung tumors in k. m Bioluminescence imaging (BLI) of Day 70 D2.OR and D2.OR shAtg3-derived tumors. Red arrows indicate the presence of tumor. Yellow circles indicate BLI signal that does not derive from lung tumors, but instead from nonspecific signals present in tail tumors. A scale bar is included where red represents the maximum of the figure (1000) while violet represents the minimum (50). n Representative IHC images show elevated Pfkfb3 expression in tumor nodules produced by Atg3-deficient D2.OR cells. Scale bar (×0.75 magnification) = 2.5 mm. Scale bar (×10 magnification) = 250 µM

Fig 5: Identification of three major epithelial cell types and their markers using scRNAseq. a Overview of scRNAseq approach using primary human breast tissue samples that were processed into single cell suspension, followed by FACS isolation of basal (CD49f-hi, EPCAM+) and luminal (CD49f+, EPCAM-hi), and scRNAseq analysis using the microfluidics-enabled scRNAseq. b Combined tSNE projection of cells from all three microfluidics-enabled scRNAseq datasets. The major basal cluster is highlighted in red; Luminal1 (L1) in green; Luminal2 (L2) in blue. c Heatmap displaying the scaled expression patterns of top marker genes within each cell type with selected marker genes highlighted; yellow indicating high expression of a particular gene, and purple indicating low expression. d Feature plots showing the scaled expression of TCF4 and ZEB1 marking a subpopulation of basal cells and gene plot showing co-expression of TCF4 and ZEB1 in the same cells. See Supplementary Fig. 1 capture site imaging, gene detection, individual principal component analysis, tSNE plot colored by individual-derived cells and feature plots of cell type-specific markers