Mucinous breast cancer organoids: an in vitro research model

Abstract

Background: Pure mucinous breast cancer is an uncommon form of cancer characterized by a low metastatic rate and a generally favorable prognosis. However, some patients may experience lymph node metastasis, leading to a worse prognosis. Currently, there is no reliable in vitro model available to effectively address the heterogeneity of pure mucinous breast cancer.

Methods: We obtained surgical tumor samples from a 64-year-old Chinese female patient diagnosed with pure mucinous breast cancer to establish patient-derived organoids. Using these organoids, we performed histological staining, drug testing and single-cell RNA-Seq analysis.

Results: We accomplished the establishment of a patient-derived mucinous breast cancer organoid model from a Chinese female. Hematoxylin and eosin staining, along with immunohistochemistry, revealed histology and protein expression (ER, PR, HER2 and Ki-67) at early passages similar to the original breast cancer tissue. Single-cell RNA sequencing at passage 7 identified 17 cell clusters, which were assigned to three cell types based on marker genes. This showed that most ER-positive luminal cells had been replaced by ER-negative basal-like cells at passage 7. We tested drug sensitivity to five antitumor drugs at passage 5. The organoids showed the highest sensitivity to Epirubicin and the lowest sensitivity to Carboplatin.

Conclusions: This is the first reported case of a mucinous breast cancer organoid. Our experimental results indicate that this model exhibits similar characteristics to the original tissue at early passages. Organoids at early passages could be a promising tool for clinical drug screening and further scientific research.

Keywords: Heterogeneity; Mucinous breast cancer; Organoid; Single-cell RNA-Seq analysis.

Fig. 1

Establishing patient-derived PMBC organoids. a The patient’s CT scan revealed an irregular shadow in the left breast, confirmed as cancerous tissue following modified radical mastectomy. b Representative images of the third, sixth, ninth and twelfth generations on the tenth day of incubation. c Representative images of H&E and IHC of third-generation organoids. ER estrogen receptor, PR progesterone receptor, HER2 human epidermal growth factor receptor-2, Ki-67 Antigen Ki67

Fig. 2

The PMBC organoids exhibit stability. Representative images of H&E and IHC for fifth-generation organoids before and after cryopreservation

Fig. 3

Drug sensitivity of the PMBC organoids. The x-axis represents drug concentration, the y-axis represents cell viability, and different colors represent different drugs. The organoids of fifth passage were treated, the duration of drug exposure was 5 days (n = 3 wells per condition, n = 3 separate experiments). Error bars represent standard deviation. The IC50 is the half-maximal inhibitory concentration for different drugs

Fig. 4

Single-cell RNA sequencing analysis of the PMBC organoids. Single-cell RNA sequencing was performed on the 7th generation organoids. a The t-SNE visualization of all cells in PMBC organoid sample, with clusters color-coded. The number in brackets is the cell number of the cluster. b Cells are annotated into three main types, with different colors representing different cell types. The bar chart shows the proportion of each cell type in the total cell count. Basal, Basal cells; Lumina epi, Luminal epithelial cells; Pro basal, Proliferating basal cells. c Dot plot of marker genes display. The FindAllMarkers module of Seurat software was used to identify marker genes for all clusters. The dot plot is horizontally represented by cluster ID and vertically by gene ID. Larger circles indicate a higher proportion of gene expression in the cluster, and redder colors indicate higher average expression levels of the gene. d The expression values of ESR1, PGR, and ERBB2 genes are mapped onto the t-SNE plot to show their expression levels in each cell. Darker red indicates higher expression