Pregnancy Reduces Il33+ Hybrid Progenitor Accumulation in the Aged Mammary Gland

Abstract

Aging increases breast cancer risk while an early first pregnancy reduces a woman's life-long risk. Several studies have explored the effect of either aging or pregnancy on mammary epithelial cells (MECs), but the combined effect of both remains unclear. Here, we interrogate the functional and transcriptomic changes at single cell resolution in the mammary gland of aged nulliparous and parous mice to discover that pregnancy normalizes age-related imbalances in lineage composition, while also inducing a differentiated cell state. Importantly, we uncover a minority population of Il33-expressing hybrid MECs with high cellular potency that accumulate in aged nulliparous mice but is significantly reduced in aged parous mice. Functionally, IL33 treatment of basal, but not luminal, epithelial cells from young mice phenocopies aged nulliparous MECs and promotes formation of organoids with Trp53 knockdown. Collectively, our study demonstrates that pregnancy blocks the age-associated loss of lineage integrity in the basal layer through a decrease in Il33+ hybrid MECs, potentially contributing to pregnancy-induced breast cancer protection.

Figure 1.. Pregnancy induces persistent changes in cell fate decisions and reduces organoid formation.

(a) Schematic of flow cytometry analysis for 3-month nulliparous (3m NP), 18-month nulliparous (18m NP), and 18-month parous (18m P). Created with Biorender.com. (b) Representative flow cytometry plots of DAPI-/CD45-/CD31-/Ter119-single cells from mouse mammary tissues. Basal (EPCAM^med/lowCD49f^high) and luminal (EPCAM^highCD49f^med/low) cells are denoted by representative gates. (c) Quantification of flow cytometry data shown in (b) (n = 11 mice). (d) Representative flow cytometry plots of DAPI-/Lineage-/EPCAM^highCD49f^med/low single cells from mouse mammary tissues. (e) Quantification of flow cytometry data shown in (d) (n = 4 mice). (f) Representative images of primary organoids derived from 3m NP, 18m NP, and 18m P luminal cells. (g) Quantification of luminal organoid number. (h) Quantification of basal organoid number. (i) Representative images of primary organoids derived from 3m NP, 18m NP, and 18m P basal cells. n = 8 mice for experiments in f-i. Statistical significance was determined by performing ANOVA (d, e) or paired t tests (g, i). * p <0.05, ** p < 0.01, *** p < 0.001.

Figure 2.. Bulk RNA sequencing in aged nulliparous and parous mammary glands reveals long-lasting transcriptomic changes.

(a) Principal Component Analysis (PCA) of basal and luminal RNA-seq samples from 3m NP, 18m NP, and 18m P mice. (b) Bar plot depicting the expression (FPKM × 10³) of basal (Krt14, Krt15, Acta2) and luminal (Krt18, Krt8) marker genes across samples. (c) Heatmap of differentially expressed genes (DEGs) across 3m NP, 18m NP, and 18m P basal cells and (d) luminal cells. Relative expression reflects log2(FC+1) values. Significance was determined using an adj-p value of <= 0.1. (e, f) Dotplot of Gene Ontology (GO) results for 18m P basal (e) and luminal (f) cells, relative to 18m NP. GO results were selected from the top 15 hits with the lowest adj-p value denoted as q-value (< 0.03 for basal, < 0.08 for luminal), NES = Normalized Enrichment Score. Gene number refers to the number of genes from the input gene list that are present in the GO gene list.

Figure 3.. Single cell RNA sequencing identifies a hybrid population of MECs in aged nulliparous mice but is reduced in aged parous mice.

(a) UMAP plot of scRNA-seq data generated from the mammary glands of 18m nulliparous (NP, red) and parous (P, blue) mice (n = 3 mice) and integrated with Tabula Muris (n=10,001). UMAPs are colored by condition (left) and annotated cell type (right). (b) Bar plot showing the proportion of cell types by condition. (c) Dotplot of gene expression for selected marker genes of mammary cell lineages. (d) Basal, HR-low, and HR-high gene expression scores across epithelial clusters (black), including a minority population of hybrid MECs (pink). (e) Violin plot showing the distribution of inferred partition-based graph abstraction (PAGA) pseudotime scores across epithelial cell clusters. (f) Violin plot comparing predicted developmental potential (obtained through CytoTRACE 2) across epithelial cell clusters. (g) Representative immunofluorescence (IF) stains against KRT5 (basal cells, green), KRT6a (hybrid MECs, red), and KRT8 (luminal cells, magenta). Nuclei are visualized using DAPI (blue). Hybrid MECs localized to the basal layer are indicated by white arrows. (h) Quantification of all KRT6a+ hybrid MECs in the epithelium. (i) Quantification of the localization of KRT6a+ hybrid MECs (basal or luminal). Statistical significance was determined by performing one-way ANOVA with Tukey test (d) or unpaired t tests with Welch’s correction to account for unequal standard deviations (h). * p <0.05, ** p < 0.01, *** p < 0.001. Statistical tests for scRNA-sequencing are described in the methods section. n = 5 mice for IF experiments. Scale bar = 50 μm.

Figure 4.. IL33 treatment of young basal cells phenocopies aged nulliparous MECs.

(a) Representative images of primary basal organoids from 3m NP mice treated with no Il33 treatment (top left) and with IL33 treatment (bottom left). Quantification of the number of organoids formed with 0, 5, or 10ng/mL of IL33. (b) Representative flow cytometry plots of basal and luminal cells from IL33-treated organoids (left) and the quantified proportion of basal cells relative to the luminal population (right). (c) Representative IF stains (left) against KRT5 (basal cells), KRT6a (hybrid MECs), and KRT8 (luminal cells) on IL33-treated organoids. Nuclei are visualized using DAPI. Quantification of KRT6a+ area in basal organoids cultured with 0, 5, or 10ng/mL of IL33 (right). (d) Representative images of preneoplastic (shTp53) basal organoids treated with (top left, 10ng/mL) or without IL33 (bottom left). Quantification of the number of shTrp53- basal organoids formed with or without IL33 treatment after 12 days in culture (right). Statistical significance was determined by performing unpaired t tests (a-c) or 2-way ANOVA with Šídák’s multiple comparisons test. * p <0.05, ** p < 0.01, *** p < 0.001. n = 3 mice. Scale bar = 1000 μm (a, d) and 100 μm (c).