Fig 2: Bulk RNA-seq of flow-sorted APC CD24+ progenitors and CD24-preadipocytes reveals transcriptomic signatures for adipogenic, mitochondrial, and energetics pathways by low n6/n3 FA ratios(A) Differential gene expression by n6/n3 exposure group within either progenitor or preadipocyte APC subtypes was analyzed using comparative analysis in Ingenuity Pathway Analysis. Significantly enriched canonical pathways in common between CD24+ progenitor and CD24-preadipocyte APCs were ordered by FDR p value (=0.01) and colored by the predicted activation Z score. Coloring indicates common pathways predicted to be “on” (yellow), “off” (navy), or “no prediction” (white) due to low n6/n3 exposure. Pathway activation Z score values are presented for either APC subtype, and values =2.0 or = -2.0 are considered significant.(B) Significantly different genes in either CD24+ progenitor or CD24-preadipocyte relative to the high n6/n3 FA ratio group colored by Log2 fold change and grouped into transcription factor genes (Regulatory) and signal transduction pathway genes (Ligands/Kinases).(C) Significantly different lipid metabolism, mitochondrial, and glycolytic genes indicating that the low n6/n3 FA ratio changes oxidative capacity potentially through aldehyde dehydrogenases (Aldh1a1 and Aldh1a3), uncoupling protein 2 (Ucp2), citrate synthase (CS), isocitrate dehydrogenase (Idh1 and 2), malic enzyme (Me1), malate dehydrogenase (Mdh1), phosphofructokinases (Pfkm, Pfkp, and Pfkfb1), and transketolase (Tk) (See also Figure S2).(D) Canonical Pathway enrichment within either CD24+ progenitor or CD24-preadipocyte APCs plotted by the percentage DE-Gs within a pathway (%) by the pathway enrichment significance (-log(p value)) and colored by the predicted pathway activation (Z score, >2.0 and < -2.0 was considered significant) (See also Data S3 for all pathways).(E) Immunoblots for NR2F2, UCP2, C/EBPa, and DNMT1 from all lineage negative, CD29+/CD34+, and Sca1+ APCs isolated by flow cytometry from pooled litters (n = 3 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter).(F) EVC005 murine APC cell line was treated with and without NR2F2 ligand 1-DSO for 48 h, which induced gene expression of Prdm16, P2rx5, Runx1, Ucp2, Dnmt1, Ppar?2, and C/ebpa.(G) TMRE live cell mitochondrial potential dye following 48 h of 1-DSO treatment in EVC005 cells, indicating an increased mitochondrial potential. Panels F and G are n = 3 wells per treatment group. Scale bar represents 50 µm. Data are represented as mean ± SEM.

Fig 3: SPRY2 knockdown in stromal fibroblasts promotes breast cancer stemness depending on glycolysis. A Sphere establishment in both primary and secondary passages compared with control CM and CM from shSpry2 stroma cells pretreated with 2-DG in sphere-formation assays in 4T1 cells. Graph showing sphere formation was increased in 4T1 cells treated with CM from shSpry2 stromal cells compared with that treated with control CM. 4T1 cells further formed bigger spheres in diameter under shSpry2 stromal cell CM treatment compared with controls. The enhanced sphere-forming abilities of 4T1 cells under treatment of shSpry2 stromal cell CM were inhibited when treated with CM from shSpry2 stroma cells pretreated with 2-DG. Scale bar = 100 µm. B Flow cytometry analysis showing the percentage in the CD44+ high; CD24- low cell subpopulation in 4T1 cells treated with CM from shSpry2 fibroblasts compared to the control and that pretreated with 2-DG. C Relative mRNA expression of MCT1 and MCT4 in fibroblasts and 4T1 cells treated with CM from fibroblasts. D Protein expression of MCT1 and MCT4 in fibroblasts and 4T1 tumor cells treated with CM from fibroblasts. E Immunofluorescence staining of CK18 (green), CD44 (red) and DAPI (blue) in the tumor of mice co-injected with shNC or shSpry2 fibroblasts with tumor cells. Scale bar = 50 µm. F Model depicting the decreased stromal SPRY2 expression enhance the stem cell ability of breast cancer cells via glycolysis and promotes breast cancer progression. P values based on unpaired Student’s t test: *P < 0.05, **P < 0.01, ***P < 0.001

Fig 4: Model of molecular and cellular diversity of APC subtypes regulated by low n6/n3 exposure during postnatal development(A) Integration of bulk- and scRNA-sequencing observations for adipogenesis-related genes that are up- (red) or down- (blue) regulated in vivo by low n6/n3 FA ratio exposure. This transcriptomic signature may establish a cellular metabolism with a greater fatty acid oxidation capacity prior to functional differentiation into a mature adipocyte.(B) Populations of APC subtypes, such as the balance among regulatory APCs and the number of mitochondrial high APCs, may influence the types of functional mature adipocytes that arise from the APCs. Additionally, the overall proportion of the committed CD24-preadipocyte population that expands from the CD24+ APCs provides a ready supply of preadipocytes that can functionally differentiate into smaller, more numerous mature adipocytes. Artwork created with BioRender.com.

Fig 5: Primary flow-sorted APCs programmed by low n6/n3 FA ratio exposure have reduced white adipose differentiation in vitro, greater FA oxidation, and increased mitochondrial potential(A) Subcutaneous WAT was enzymatically digested to produce a stromal vascular pellet and Fluorescence Assisted Cell Sorting (FACS) was used to isolate CD24+ progenitor from CD24-preadipocyte APCs. Deuterium incorporation into the DNA in vivo indicated the rate of APC proliferation is not different, whole SWAT tended to be lower (p = 0.07), and a large variance was observed in the whole blood DNA (n = 5-7 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter). APC population analysis indicated no overt difference in CD24+ progenitors and CD24-preadipocytes, and a trend toward the fraction of committed preadipocytes in the low n6/n3 PUFA group (n = 7-8 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter).(B) Sca-1+ APCs plated for live cell imaging using Incucyte grew more slowly and accumulated less neutral lipid when differentiated to white adipocytes (n = 3 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter), suggesting the potential for a soluble inhibitory factor that curtails white adipocyte differentiation under low n6/n3 in vivo programming.(C-F) Seahorse substrate oxidation assays for primary Sca1+ APCs isolated from low or high n6/n3 ratio exposure (n = 3 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter). Maximal and spare respiration rates from C) exogenous palmitate oxidation, D) glucose, and E) glutamine in low and high n6/n3 in vivo programmed primary APCs measured by Seahorse. F) Kinetic data of exogenous palmitate, glucose, and glutamine oxidation assays in C, D, and E.(G) TMRE live cell mitochondrial potential dye in primary APCs programmed in vivo by low and high n6/n3 FA ratio exposure at 24 h post plating (n = 3 litter dyads per n6/n3 FA ratio group; litters standardized to 6-8 pups/litter). Scale bar represents 75 µm. Data are represented as mean ± SEM.