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High YAP activity is associated with poor prognosis human breast cancers, but its role during the initial stage of mammary cell transformation is unknown. To address this question, we designed experiments that exploit the ability of KRASG12D-transduced subsets of freshly isolated normal human mammary cells to form invasive tumors rapidly and efficiently when transplanted into immunodeficient mice. Initial examination of the newly developing tumors thus generated revealed a consistent marked loss of nuclear YAP, independent of the initial primary human mammary cell type transduced. Conversely, co-transduction of the same subsets of primary human mammary cells with KRASG12D plus the constitutively active YAPS127A prevented tumor formation. These findings contrast with the enhanced display of transformed properties obtained when the immortalized, but non-tumorigenic MCF10A cells are transduced just with YAPS127A. In addition, we show that YAPS127A-transduction of the human MDA-MB-231 breast cancer cell line (that carry a similar KRAS mutation) enhances their metastatic activity in vivo. We also discover that the KRASG12D-induced early loss of YAP in primary human mammary cells is associated with their induced secretion of amphiregulin. Collectively, these findings suggest that YAP can differentially affect the acquisition of malignant properties by human mammary cells at different stages of their transformation.

Assessment of autophagy activity has historically been limited to investigations of fixed tissue or bulk cell populations. To address questions of heterogeneity and relate measurements to functional properties of viable cells isolated from primary tissue, we created a lentiviral (RFP-GFP-MAP1LC3B) vector that allows the autophagosome and autolysosome content of transduced cells to be monitored at the single-cell level. Use of this strategy to analyze purified subsets of normal human mammary cells showed that both the luminal progenitor-containing (LP) subset and the basal cells (BCs) display highly variable but overall similar autophagic flux activity despite differences suggested by measurements of the proteins responsible (i.e., LC3B, ATG7 and BECLIN1) in bulk lysates. Autophagosome content was also highly variable in the clonogenic cells within both the LPs and BCs, but the proliferative response of the BCs was more sensitive to autophagy inhibition. In addition, use of this vector showed cells with the lowest autophagosome content elicited the fastest tumor growth in 2 different models of human mammary tumorigenesis. These results illustrate the utility of this vector to define differences in the autophagy properties of individual cells in primary tissue and couple these with their responses to proliferative and oncogenic stimuli.

Modelling the genesis of human cancers is at a scientific turning point. Starting from primary sources of normal human cells, it is now possible to reproducibly generate several types of malignant cell populations. Powerful methods for clonally tracking and manipulating their appearance and progression in serially transplanted immunodeficient mice are also in place. These developments circumvent historic drawbacks inherent in analyses of cancers produced in model organisms, established human malignant cell lines, or highly heterogeneous patient samples. In this review, we survey the advantages, contributions and limitations of current de novo human tumorigenesis strategies and note several exciting prospects on the horizon., A better understanding of the earliest stages of human cancer formation can enable future improvements in early detection, diagnosis and treatment. In this review, the authors summarize the methods enabling de novo tumorigenesis protocols to be applied to human cells and the insights derived from them to date, as well as the exciting and relevant technical developments anticipated to extend even further the utility of these strategies.

Genomic and phenotypic analyses indicate extensive intra- as well as intertumoral heterogeneity in primary human malignant cell populations despite their clonal origin. Cellular DNA barcoding offers a powerful and unbiased alternative to track the number and size of multiple subclones within a single human tumour xenograft and their response to continued in vivo passaging. Using this approach we find clone-initiating cell frequencies that vary from ~1/10 to ~1/10,000 cells transplanted for two human breast cancer cell lines and breast cancer xenografts derived from three different patients. For the cell lines, these frequencies are negatively affected in transplants of more than 20,000 cells. Serial transplants reveal five clonal growth patterns (unchanging, expanding, diminishing, fluctuating or of delayed onset), whose predominance is highly variable both between and within original samples. This study thus demonstrates the high growth potential and diverse growth properties of xenografted human breast cancer cells., Cancer cells within the same tumour are heterogeneous in their tumorigenic potential, differentiation status and sensitivity to treatments. Here Nguyen et al. use a sensitive DNA barcoding method to characterize the diversity of clonal growth behaviour within human breast tumours.

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.

Most human breast cancers have diversified genomically and biologically by the time they become clinically evident and little is known about their origin from normal human mammary cells, or the cellular and molecular mechanisms that lead to their genesis and evolution. We have developed methods to quantify, purify and characterize different subsets of normal human mammary cells and have used these to identify properties that may influence their propensity for transformation. We have also developed methods for inducing the rapid transformation in vivo of these purified subsets following their transplantation into immunodeficient mice. The results demonstrate the ability of a single oncogene (KRASG12D) to induce the formation of serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. Both primary and secondary tumors are phenotypically heterogeneous and transcriptome analyses of primary tumors assign them to a “normal-like” category. DNA barcoding of the cells at the time of their initial transduction with KRASG12D has revealed a dramatic change in the numbers and sizes of clones they generate after 2 weeks in vivo. DNA barcoding also showed the unexpected appearance of many “new” clones in tumors generated upon passage into secondary recipients, thus recapitulating some features of in vivo passaged human breast cancer cell lines and patients’ tumor xenografts. This system challenges previous concepts about the process of human mammary oncogenesis and provides a new system for analyzing factors that can influence its speed, efficiency and heterogeneity of outcomes. Citation Format: Connie J. Eaves, Long Nguyen, Davide Pellacani, Nagarajan Kannan, Sylvan Lefort, Sneha Balani, Claire Cox, Tomo Osako, Samuel Aparicio, Martin Hirst. Clonal dynamics of normal and malignant human mammary cell growth in xenografts. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr IA13.

Knowledge gap: Fatal breast cancers are characterized by biological, genomic and extensive treatment heterogeneity. Although many breast cancers can now be cured by established therapies, treatment failure remains a major problem and is difficult to predict. In the current era of “personalized medicine”, a possible solution is to develop a large-scale system for quantifying responses to candidate treatments of individual malignant human mammary cells with in vivo clonogenic activity. Such cells can be detected by their ability to produce uniquely barcoded clones of progeny in xenografted immunodeficient mice and the clones obtained can be assessed for their size and number using next generation sequencing of tumor extracts. However, to pursue this approach it is first critical to establish how the clone content of a tumor may vary according to the number or type of competent tumorigenic and/or other cells that are present in the inoculum used to initiate tumor formation, and hence whether and how these parameters may influence assessment of the treatment responsiveness of these cells. Approach/methods: Here we describe the development and initial testing of a method to measure the treatment responsiveness of large numbers of tumorigenic cells using radiation as a prototypic treatment. Treatment sensitivity of in vitro colony-forming cells (CFCs) will then be compared with future measurements of in vivo clone-initiating tumorigenic cells obtained by sequencing the progeny of DNA-barcoded input cells. Results: In an initial series of experiments we showed that normal human luminal progenitor (LP) CFCs are ~1.5-fold more radioresistant than basal cell (BC) CFCs, and both are more sensitive than either type of mouse mammary CFCs. In vitro CFC assays of 2 human breast cancer cell lines (MDA MB231 and SUM149, with in vitro CFC frequencies of 70% and 40%, respectively) showed these to be 1.2- and 1.5-fold more radioresistant than normal LPs. Limiting dilution analysis showed the corresponding frequency of in vivo tumor-initiating cells in these 2 cell lines to be 1/6 and 1/47. Assessment of their response to radiation is complicated by the finding that the barcoded clone content of tumors initiated with >20,000 of these cells (untreated) is inversely related to the number injected and, at these input cell doses, very heterogeneous clone dynamics are also seen in successive passages. However, evidence of a positive linear cell dose-clone yield relationship is seen at input transplants of Conclusion: These results highlight the complex clonal dynamics already operative in the growth of tumorigenic cells present in relatively homogeneous established human mammary cell lines and set the stage for future measurements of clone yields from irradiated cells derived from mammary tumors of different origins. Citation Format: Sneha Balani, Nagarajan Kannan, Long V. Nguyen, Sylvain Lefort, Davide Pellacani, Connie J. Eaves. Clonal analysis of normal and malignant human mammary epithelial cell responsiveness to radiation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A63.