Most human breast cancers have diversified genomically and biologically by the time they become clinically evident. Early events involved in their genesis and the cellular context in which these events occur have thus been difficult to characterize. Here we present the first formal evidence of the shared and independent ability of basal cells and luminal progenitors, isolated from normal human mammary tissue and transduced with a single oncogene (KRAS(G12D)), to produce serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. DNA barcoding of the initial cells revealed a dramatic change in the numbers and sizes of clones generated from them within 2 weeks, and the first appearance of many 'new' clones in tumours passaged into secondary recipients. Both primary and secondary tumours were phenotypically heterogeneous and primary tumours were categorized transcriptionally as 'normal-like'. This system challenges previous concepts that carcinogenesis in normal human epithelia is necessarily a slow process requiring the acquisition of multiple driver mutations. It also presents the first description of initial events that accompany the genesis and evolution of malignant human mammary cell populations, thereby contributing new understanding of the rapidity with which heterogeneity in their properties can develop.

Cellular barcoding offers a powerful approach to characterize the growth and differentiation activity of large numbers of cotransplanted stem cells. Here, we describe a lentiviral genomic-barcoding and analysis strategy and its use to compare the clonal outputs of transplants of purified mouse and human basal mammary epithelial cells. We found that both sources of transplanted cells produced many bilineage mammary epithelial clones in primary recipients, although primary clones containing only one detectable mammary lineage were also common. Interestingly, regardless of the species of origin, many clones evident in secondary recipients were not detected in the primary hosts, and others that were changed from appearing luminal-restricted to appearing bilineage. This barcoding methodology has thus revealed conservation between mice and humans of a previously unknown diversity in the growth and differentiation activities of their basal mammary epithelial cells stimulated to grow in transplanted hosts.

Mechanisms that control the levels and activities of reactive oxygen species (ROS) in normal human mammary cells are poorly understood. We show that purified normal human basal mammary epithelial cells maintain low levels of ROS primarily by a glutathione-dependent but inefficient antioxidant mechanism that uses mitochondrial glutathione peroxidase 2. In contrast, the matching purified luminal progenitor cells contain higher levels of ROS, multiple glutathione-independent antioxidants and oxidative nucleotide damage-controlling proteins and consume O 2 at a higher rate. The luminal progenitor cells are more resistant to glutathione depletion than the basal cells, including those with in vivo and in vitro proliferation and differentiation activity. The luminal progenitors also are more resistant to H 2 O 2 or ionizing radiation. Importantly, even freshly isolated "steady-state" normal luminal progenitors show elevated levels of unrepaired oxidative DNA damage. Distinct ROS control mechanisms operating in different subsets of normal human mammary cells could have differentiation state-specific functions and long-term consequences.human epithelial stem and progenitor cells | mammary differentiation | 3D clonogenic assay | superoxide dismutase | peroxiredoxin C ellular synthesis of different reactive oxygen species (ROS) results primarily from the incomplete reduction of molecular oxygen in mitochondria to generate free radical superoxide anions. ROS also are produced when cells are exposed to different environmental sources of oxidative stressors, including ionizing radiation. Together, these regulate many normal cellular processes and also contribute to DNA damage, tumorigenesis, and cell death (1-3).In mice, the inner layer of "luminal" epithelial cells of the normal adult mammary gland have been found to contain higher levels of ROS than the outer "basal" layer of epithelial cells (4). The basis for these differences in ROS levels in the two cell types has been attributed to differences in their content of mitochondria; however, their stress response mechanisms have not been defined. Even less is known about ROS levels and their control in the normal adult human mammary gland, which consists of a similar continuous bilayered epithelial network of ducts and terminal alveolae. MCF10A cells are an immortal but nontumorigenic human mammary cell line that, when grown in 3D Matrigel cultures, generates multilayered spheres in which a lumen forms owing to the acquisition of lethal levels of ROS by the inner cells (5). These studies have suggested that ROS regulation plays an important role in the structural morphogenesis and homeostasis of normal adult human mammary tissue.Here we report the results of experiments designed to investigate the levels of ROS and their control in highly purified populations of viable luminal progenitors (LPs) and basal cells (BCs) isolated from normal human breast tissue (6-8). Our results confirm the different levels of ROS identified in the corresponding subsets of mouse mammary cells an...

Mouse fetal mammary cells display greater regenerative activity than do adult mammary cells when stimulated to proliferate in a new system that supports the production of transplantable mammary stem cells ex vivo.

In the current era of precision medicine, the identification of genomic alterations has revolutionised the management of patients with solid tumours. Recent advances in the detection and characterisation of circulating tumour DNA (ctDNA) have enabled the integration of liquid biopsy into clinical practice for molecular profiling. ctDNA has also emerged as a promising biomarker for prognostication, monitoring disease response, detection of minimal residual disease and early diagnosis. In this Review, we discuss current and future clinical applications of ctDNA primarily in non-small cell lung cancer in addition to other solid tumours.