Highlights d PDAC regional heterogeneity stems from sub-tumor microenvironments (subTMEs) d SubTMEs exhibit distinct immune phenotypes and CAF differentiation states d SubTMEs execute distinct tumor-promoting and chemoprotective functions d Intratumoral subTME co-occurrence links stromal heterogeneity to patient outcome

Cancer metabolism adapts the metabolic network of its tissue-of-origin. However, breast cancer is not a disease of a singular origin. Multiple epithelial populations serve as the culprit cell-of-origin for specific breast cancer subtypes, yet knowledge surrounding the metabolic network of normal mammary epithelial cells is limited. Here, we show that mammary populations have cell type-specific metabolic programs. Primary human breast cell proteomes of basal, luminal progenitor, and mature luminal populations revealed their unique enrichment of metabolic proteins. Luminal progenitors had higher abundance of electron transport chain subunits and capacity for oxidative phosphorylation, whereas basal cells were more glycolytic. Targeting oxidative phosphorylation and glycolysis with inhibitors exposed distinct metabolic vulnerabilities of the mammary lineages.Computational analysis indicated that breast cancer subtypes retain metabolic features of their putative cell-of-origin. Lineage-restricted metabolic identities of normal mammary cells partly explain breast cancer metabolic heterogeneity and rationalize targeting subtype-specific metabolic vulnerabilities to advance breast cancer therapy..

1Cancer metabolism adapts the metabolic network of its tissue-of-origin. However, breast 2 cancer is not a disease of a singular origin. Multiple epithelial populations serve as the 3 culprit cell-of-origin for specific breast cancer subtypes, yet knowledge surrounding the 4 metabolic network of normal mammary epithelial cells is limited. Here, we show that 5 mammary populations have cell type-specific metabolic programs. Primary human breast 6 cell proteomes of basal, luminal progenitor, and mature luminal populations revealed their 7 unique enrichment of metabolic proteins. Luminal progenitors had higher abundance of 8 electron transport chain subunits and capacity for oxidative phosphorylation, whereas 9 basal cells were more glycolytic. Targeting oxidative phosphorylation and glycolysis with 10 inhibitors exposed distinct metabolic vulnerabilities of the mammary lineages. 11 Computational analysis indicated that breast cancer subtypes retain metabolic features 12 of their putative cell-of-origin. Lineage-restricted metabolic identities of normal mammary 13 cells partly explain breast cancer metabolic heterogeneity and rationalize targeting 14 subtype-specific metabolic vulnerabilities to advance breast cancer therapy. 15 16 17 18 RESULTS Abundance 3 To discover protein distinctions of primary human MEC populations we generated their 4 global proteomes. We performed mass spectrometry-based shotgun proteomics on 5 equivalent numbers of FACS-purified basal (CD45 -CD31 -CD49f hi EpCAM lo-med ; color-6 coded as red in all figures), luminal progenitor (CD45 -CD31 -CD49f lo EpCAM med ; light 7 blue), and mature luminal (CD45 -CD31 -CD49f hi EpCAM lo ; dark blue) cells from 10 normal 8 human breast samples obtained from reduction mammoplasties (Figure 1B, S1A). Our 9 patient cohort represented diverse physiologies, covering a wide age range (28-67 years 10 old) and sex hormone status (3 luteal, 3 follicular, 4 post-menopausal). We detected 6034 11 unique proteins ( Figure 1B). Expression of known markers for each mammary cell type 12 was accurately captured by our proteomics data (Figure S1B); higher abundance of 13 Vimentin and ITGA6 (Integrin α6, CD49f) was seen in basal cells, higher KIT and 14 ALDH1A3 levels in luminal progenitors, and higher GATA3, FOXA1 and KRT8/18 15 (Cytokeratin 8/18) in the mature luminal. Principal component analysis highlighted the 16 distinct proteomes of mammary cells; the dominant clustering feature was mammary cell 17 type with a minor segregation of post-menopausal samples within each cluster (Figure 181C). Out of the 6034 proteins, 5881 were detected in all three cell types ( Figure S1C).