Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.

MicroRNAs (miRNAs) are potent post‐transcriptional regulators of protein coding genes. Patterns of misexpression of miRNAs in cancer suggest key functions of miRNAs in tumorigenesis. However, current bioinformatics tools do not entirely support the identification and characterization of the mode of action of such miRNAs. Here, we used a novel functional genetic approach and identified miR‐221 and miR‐222 (miR‐221&222) as potent regulators of p27Kip1, a cell cycle inhibitor and tumor suppressor. Using miRNA inhibitors, we demonstrate that certain cancer cell lines require high activity of miR‐221&222 to maintain low p27Kip1 levels and continuous proliferation. Interestingly, high levels of miR‐221&222 appear in glioblastomas and correlate with low levels of p27Kip1 protein. Thus, deregulated expression of miR‐221&222 promotes cancerous growth by inhibiting the expression of p27Kip1.

Every cancer originates from a single cell. During expansion of the neoplastic cell population, individual cells acquire genetic and phenotypic differences from each other. Here, to investigate the nature and extent of intra-tumour diversification, we characterized organoids derived from multiple single cells from three colorectal cancers as well as from adjacent normal intestinal crypts. Colorectal cancer cells showed extensive mutational diversification and carried several times more somatic mutations than normal colorectal cells. Most mutations were acquired during the final dominant clonal expansion of the cancer and resulted from mutational processes that are absent from normal colorectal cells. Intra-tumour diversification of DNA methylation and transcriptome states also occurred; these alterations were cell-autonomous, stable, and followed the phylogenetic tree of each cancer. There were marked differences in responses to anticancer drugs between even closely related cells of the same tumour. The results indicate that colorectal cancer cells experience substantial increases in somatic mutation rate compared to normal colorectal cells, and that genetic diversification of each cancer is accompanied by pervasive, stable and inherited differences in the biological states of individual cancer cells.