Generation of benign mammary organoids in vitro
We generated an in vitro 3D organoid system using human benign breast tissues from patients undergoing reduction mammoplasty (RM) and prophylactic mastectomy from women with germline BRCA1 mutations (Fig. 1a). Detailed patient information is provided in Additional file 8: Table S1. Organoids derived from breast tissues from RM which are BRCA1 wildtype (WT) will be referred to as “non-carrier” and organoids derived from BRCA1-mutated tissues will be referred to as “BRCA1mut”. Individual patient-derived organoids are referred to as PT#. To mimic the human menstrual cycle, breast organoids were treated with a 28-day stepwise menstrual cycle hormone regimen replacing the medium every 2–3 days (Fig. 1a, bottom). During the last 14 days (luteal phase), when adding P4, telapristone acetate (TPA) was also added to identify PR target genes (Fig. 1a, bottom).
Immunofluorescent staining for ER and PR showed a focal subset of cells expressing ER or PR in both non-carrier and BRCA1mut organoids (Fig. 1b). Co-staining with the luminal epithelial cell marker, CK18, showed co-localized with ER or PR (red arrows) but not with basal/myoepithelial cells (α smooth muscle actin, aSMA; Additional file 1: Figure S1). Negative controls for immunofluorescent staining are shown in Additional file 2: Figure S2.
As shown in Fig. 1c, all the patient-derived organoids expressed ER and PR mRNA at varying levels after the 28-day hormone regimen. These results demonstrate that mammary organoids grown in vitro in a scaffold-free 3D system for 28 days retain the expression of HRs which are expressed in a subset of luminal epithelial cells.
Characterization of mammary organoids
Immunofluorescent staining was done to visualize the architecture and composition of the breast organoids using specific cellular markers including pan cytokeratin (panCK; epithelial cell), CK18 (luminal epithelial cell), vimentin (Vim; fibroblast cell), and αSMA (basal/myoepithelial cell). As a comparative control, native breast tissues from non-carrier or BRCA1mut patients were immunostained for the same markers. No distinct differences in cellular composition in the BRCA1mut and non-carrier benign breast tissues were observed using these markers (Fig. 2a). Non-carrier and BRCA1mut organoids both preserved extensive intercellular contacts and contained multiple cell types, shown by hematoxylin and eosin (H&E) staining (Fig. 2b). Trichrome staining which stains the collagen blue showed the presence of collagen within the BRCA1mut and non-carrier organoids (Fig. 2b) suggesting that the fibroblasts are actively producing and depositing collagen to maintain the organoid structure. Proliferation of the organoids was assessed with Ki67 staining, and it was observed that at the end of the 28-day hormone treatment, some cells were proliferating (Additional file 3: Figure S3). In addition, immunofluorescent staining of E-cadherin, an epithelial cell-cell adhesion molecule, and Integrin (ITGB4), a protein that facilitates cell-ECM adhesion (Fig. 2c), were expressed in the BRCA1mut and non-carrier organoids after the 28-day hormone cycle demonstrating intercellular contacts and communication. Furthermore, immunofluorescent staining for luminal, basal, and fibroblast markers (Fig. 2c, d) showed that the organoids contained multiple cell types. Together, these data show that BRCA1mut and non-carrier organoids, derived from benign breast tissues preserve intercellular contacts, maintain an organoid structure and contain multiple cell types.
Hormone response in non-carrier and BRCA1mut organoids
It is unclear how PR functions in the background of BRCA1 mutations in non-cancerous benign mammary cells before breast cancer develops. In order to assess the hormonal response in the non-carriers and BRCA1mut organoids at the transcriptomic level, RNA-seq was performed. Breast organoids from BRCA1mut (N = 6) and non-carrier (N = 6) patients were treated with the 28-day stepwise menstrual cycle hormones, with or without TPA (Fig. 1a). RNA-seq data was analyzed using Artificial Intelligene (AI), an online integrated analysis system tool. Principal component analysis (PCA) plot showed BRCAmut and non-carrier organoids clustering separately with expected heterogeneity among patient samples (Fig. 3a).
Four differential gene expression comparisons were done to assess the role of E2 and P4 in the non-carrier vs BRCA1mut, as well as the role of PR, specifically with the TPA for non-carrier and BRCA1mut (non-carrier vs BRCA1mut, non-carrier vs non-carrier+TPA, BRCA1mut vs BRCA1mut+TPA, and non-carrier+TPA vs BRCA1mut+TPA). Correlation of top 3% of most variant genes between the various treatment groups is shown in Fig. 3b and Additional file 4: Figure S4. The PR-regulated genes in the non-carrier+TPA versus BRCA1mut+TPA showed that 393 genes were equally expressed in both sample sets (Fig. 3b center, gray dots), 115 genes were highly expressed in both non-carrier TPA and BRCA1mut TPA groups (top right, green), 9 genes had high expression in BRCA1mut+TPA group only (top left, red), 44 genes had low expression in both groups (bottom left, green), and 30 genes had low expression in BRCA1mut+TPA only (bottom right, red) (Fig. 3b). This data demonstrates that while some PR-responsive genes are similar between the two groups, there are genes that are differentially regulated in BRCA1mut compare to non-carrier organoids.
When these genes were subjected to gene ontology analysis, distinct pathways were enriched between the non-carrier and BRCA1mut groups (Fig. 3c and Additional file 10: Table S3). Genes involved in extracellular matrix (ECM) organization were enriched in response to menstrual cycle hormones (E2+P4) in BRCA1mut compared to non-carrier organoids. In addition, these ECM-specific genes were differentially regulated by TPA in the BRCA1mut group but not in the non-carrier group further suggesting different PR activity in BRCA1mut organoids compared to non-carrier. Interestingly, known PR target genes that regulate the cell cycle were responsive to TPA in the non-carrier group only (Fig. 3c).
Gene set enrichment analysis (GSEA) showed that BRCA1mut organoids treated with E2+P4+TPA exhibited a significant downregulation of ECM organization genes compared to the BRCA1mut organoids treated with E2+P4 only (Fig. 3d). Shown in Fig. 3e, are the expression of specific ECM genes in E2+P4-treated BRCA1mut organoids in response to TPA. Taken together, these data demonstrate that BRCA1 mutation influences hormone response and, in particular, ECM organization genes.
Confirmation and validation of the effect on ECM genes
To confirm the RNA-seq data, quantitative real-time PCR was done for ECM genes in each patient-derived organoids from the RNA-seq samples referred to as PT#. The E2+P4+TPA data are represented as fold changes of E2+P4 treatment (Fig. 4a, dotted red line is at 1). As shown in Fig. 4a left, the ECM genes MMP1, MMP10, COL1A1, COL3A1, A2M, and FN1 were significantly downregulated in individual BRCA1mut organoids in response to TPA treatment. These genes were not downregulated with TPA in the non-carrier organoids with an exception of MMP1, COL1A1, and FN1 genes in organoids P278 and P282 (Fig. 4a, right).
To validate our RNA-seq data, additional patient-derived organoids from BRCA1mut and non-carriers were exposed to the same hormonal treatments (E2+P4) in the absence or presence of TPA. Similar to the RNA-seq confirmation data in Fig. 4a, ECM genes, MMP1, MMP10, COL1A1, COL3A1, and DCN from the new BRCA1mut organoids (N = 3), were significantly downregulated by TPA, but not in the non-carrier organoids (N = 3) (Fig. 4b).
IHC staining of ECM proteins MMP10, COL1A1, COL3A1, and FN1 were performed in BRCA1mut and non-carrier organoids treated with hormones (E2+P4) (Fig. 4c). Higher levels of the ECM proteins were observed in the hormone-treated BRCA1mut compared to the non-carriers.
Taken together, these data validate our RNA-seq data and demonstrate that in the background of BRCA1 mutation PR regulates ECM gene and protein expression.
Identification of cell type marker genes in mammary organoids
RNA-seq data also demonstrated a differential regulation of the aldehyde dehydrogenase-1 (ALDH1) gene which has been shown to be a breast stem cell marker [25, 26]. Using immunofluorescent staining, we observed that ALDH1 protein levels were increased in the BRCA1mut organoids treated with E2+P4 (Fig. 5a). Furthermore, the ALDH1 mRNA expression was increased in BRCA1mut organoids compared to the non-carrier organoids (Fig. 5b). Interestingly, ALDH1 was present in the stroma of the organoids, which is consistent with a study showing the presence of ALDH1 in intralobular stroma and could be involved in breast stem cell renewal and differentiation [27].
The RNA-seq data was further mined to assess the expression of specific cell type markers in the BRCA1mut and non-carrier mammary organoids. In a recent bioRxiv study, Murrow et al. performed single-cell (sc)-RNA sequencing in human breast tissues that were in the luteal and follicular phases of the menstrual cycle and identified seven different cell clusters [28]. The three major epithelial groups were hormone responsive luminal-hormone receptor positive (HR+), hormone-insensitive luminal-hormone receptor negative (HR−), and vascular accessory cells [28]. Each group had specific and distinct transcriptional profiles and differed depending on the menstrual cycle phase. Therefore, we looked at the expression of various genes specific to these cell types that were shown to be upregulated in the luteal phase. First, the expression of the luminal HR+ marker genes in hormone-treated non-carrier organoids were increased compared to hormone-treated BRCA1mut organoids with TFF1 and KRT8 reaching statistical significance (Fig. 5c, left). When these marker genes were compared in BRCA1mut and non-carrier organoids that were treated with TPA, the luminal HR+ marker genes were not significantly downregulated by TPA (Fig. 5c, right) which indicates that these genes are not regulated by PR. Second, the luminal HR-marker genes in hormone-treated BRCA1mut and non-carrier organoids were analyzed (Fig. 5d, left). Luminal HR− marker genes were decreased in non-carrier hormone-treated organoids and MMP3 was significantly downregulated compared to hormone-treated BRCA1mut organoids. When we compared the luminal HR− marker genes in the TPA-treated BRCA1mut and non-carrier groups, no significant downregulation by TPA was observed (Fig. 5d, right), again, suggesting that these genes are not PR target genes. Third, the myoepithelial (basal) marker genes in hormone-treated BRCA1mut and non-carrier organoids were analyzed (Fig. 5e, left). Most of these marker genes were downregulated in hormone-treated non-carrier, and ACTA2 was significantly downregulated compared to hormone-treated BRCA1mut organoids. Interestingly, when we compared the myoepithelial marker genes in the TPA-treated BRCA1mut and non-carrier groups, IGFBP3 and VEGFA were significantly downregulated by TPA in BRCA1mut organoids but not in the non-carrier organoids (Fig. 5e, right). This data suggests that IGFBP3 and VEGFA are regulated by PR in the BRCA1mut organoids through paracrine action but not in the non-carrier organoids. Finally, the fibroblast marker genes, mostly ECM genes in hormone-treated BRCA1mut and non-carrier organoids were evaluated (Fig. 5f, left). As expected, most of the fibroblast marker genes were significantly increased in the hormone-treated BRCA1mut organoids compared to the hormone-treated non-carrier organoids. Similarly, most of the fibroblast marker genes were significantly downregulated in response to TPA in hormone-treated BRCA1mut organoids but not in the hormone-treated non-carrier organoids (Fig. 5f, right). Collectively, these data support that hormone responses and PR-regulated genes for certain breast cell type-specific genes are altered in BRCA1mut organoids.