Organoid cultures from normal and cancer-prone human breast tissues preserve complex epithelial lineages

Abstract

Recently, organoid technology has been used to generate a large repository of breast cancer organoids. Here we present an extensive evaluation of the ability of organoid culture technology to preserve complex stem/progenitor and differentiated cell types via long-term propagation of normal human mammary tissues. Basal/stem and luminal progenitor cells can differentiate in culture to generate mature basal and luminal cell types, including ER+ cells that have been challenging to maintain in culture. Cells associated with increased cancer risk can also be propagated. Single-cell analyses of matched organoid cultures and native tissues by mass cytometry for 38 markers provide a higher resolution representation of the multiple mammary epithelial cell types in the organoids, and demonstrate that protein expression patterns of the tissue of origin can be preserved in culture. These studies indicate that organoid cultures provide a valuable platform for studies of mammary differentiation, transformation, and breast cancer risk.

An Automated Organoid Platform with Inter-organoid Homogeneity and Inter-patient Heterogeneity

“…Manual manipulation of unpatterned cell-suspension volumes introduces significant batch-to-batch and organoid-to-organoid variability. 16 , 17 Though the exact causes are unclear, inconsistent cellular complexity among organoids and batch experiments is a contributing factor. Growing organoids from manually patterned cell-laden Matrigel volumes reduces the organoid-to-organoid variability, but it remains labor intensive and batch-to-batch variant.…”

A single cell atlas of the healthy breast tissues reveal clinically relevant clusters of breast epithelial cells

“…Since the number of clusters identified in this new clustering are different (23) from the first analysis (13), new clusters are named with prefix N (N0-N22). Consistent with our earlier report and a recent report on organoid-derived single cell data, there was inter-individual differences in proportion of cells in each cluster Rosenbluth et al, 2020). The UMAP cell embeddings and cell cluster information generated from Seurat analysis were imported into 10X genomics Loupe Browser.…”

“…There has been a limited number of studies that utilized scRNA-seq technology to subclassify breast epithelial cells. Two publications to our knowledge utilized tissues from reduction mammoplasty samples and cells were either purified by flow cytometry or grown under organoid cultures prior to single cell sequencing (Nguyen et al, 2018;Rosenbluth et al, 2020). Although our source of tissue and methodology differed significantly than these studies as we used breast tissues from healthy women and were able to prepare single cell cDNA within two hours of tissue collection, there were several overlapping observations.…”

“…Epithelial cluster specific gene signatures were then applied on TCGA and METABRIC datasets to determine the impact of putative "cell-of-origin" on breast cancer outcomes (Cancer Genome Atlas, 2012;Curtis et al, 2012). Since there is limited subclassification of estrogen receptor positive (ER+) breast cancers and it is exceeding difficult to characterize ER+ breast epithelial cells from the normal breasts to identify genes co-expressed with ER in normal and tumor cells (Rosenbluth et al, 2020), we performed additional studies on TBX3 and PDK4, two genes that are co-expressed at different levels in ER+ clusters of the normal breasts.…”

Optimal, Large-Scale Propagation of Mouse Mammary Tumor Organoids

“…A variety of growth factors and ligands can be used in this culture setting to promote growth, but a simple media composed of DMEM/F12, ITS-X, and 2.5 nM FGF is frequently used to maintain both normal mammary and tumor organoids [6,45,47,61]. However other supplements can be included to promote growth or alter the cellular composition of organoids [26], particularly when culturing human tumor organoids which often grow poorly ex vivo [50,51].…”