Breast cancer evolves from normal epithelium through the accumulation of mutations that result in tumours with complex genomic and biological heterogeneity. Deciphering the critical early events in tumorigenesis that results in this complex biology is essential for understanding the key driver mutations in different breast cancer subtypes. Recently, human breast organoid technology has been developed to enable genetic modification, long-term expansion and transplantation of breast cancer cells. Here we test the feasibility of generating breast cancer organoids from normal human breast epithelium through transduction with three key tumour suppressor genes to evaluate their contribution to breast cancer tumorigenesis.
We generated breast organoids from single sorted epithelial cells of normal human reduction mammoplasty samples based on CD49f and EpCAM expression. Confocal 3D imaging revealed that organoids consist of E-cadherin luminal cells with an outer layer of Keratin 5-positive basal cells. Using CRISPR-Cas9-mediated gene editing, we deleted TP53, PTEN and RB1 in breast organoids and investigated their in vitro growth properties. As expected, mutated organoids demonstrated increased proliferation rates and gained long-term culturing capacity compared to wild-type controls. To evaluate the influence of deleting different gene combinations on tumour development, engineered breast tumour organoids were transplanted into the mammary fat pad of immunodeficient NOD-SCID-IL2Rg–/– mice. Notably, transplanted organoids from TP53-PTEN-RB1- but not TP53-PTEN-knockout cells gave rise to tumours that could be serially transplanted. Next generation sequencing analysis indicated that clonal heterogeneity was maintained in organoids in vitro and in tumours propagated in vivo. Based on immunohistochemical staining, tumours (which were ER+PR+HER2–) exhibited features consistent with the luminal subtype of breast cancer. Drug therapy to assess response to endocrine agents is currently underway. Our study highlights the potential for generating of human breast cancer organoids from normal epithelium using CRISPR-Cas9 gene editing that recapitulates the luminal subtype of breast cancer and a system that can be applied to understand critical events in early breast cancer tumorigenesis.