Long V. Nguyen

Analysis of the clonal growth and differentiation dynamics of primitive barcoded human cord blood cells in NSG mice

Human cord blood (CB) offers an attractive source of cells for clinical transplants because of its rich content of cells with sustained repopulating ability in spite of an apparent deficiency of cells with rapid reconstituting ability. Nevertheless, the clonal dynamics of nonlimiting CB transplants remain poorly understood. To begin to address this question, we exposed CD34+ CB cells to a library of barcoded lentiviruses and used massively parallel sequencing to quantify the clonal distributions of lymphoid and myeloid cells subsequently detected in sequential marrow aspirates obtained from 2 primary NOD/SCID-IL2Rγ(-/-) mice, each transplanted with ∼10(5) of these cells, and for another 6 months in 2 secondary recipients. Of the 196 clones identified, 68 were detected at 4 weeks posttransplant and were often lympho-myeloid. The rest were detected later, after variable periods up to 13 months posttransplant, but with generally increasing stability throughout time, and they included clones in which different lineages were detected. However, definitive evidence of individual cells capable of generating T-, B-, and myeloid cells, for over a year, and self-renewal of this potential was also obtained. These findings highlight the caveats and utility of this model to analyze human hematopoietic stem cell control in vivo.

Clonal Analysis via Barcoding Reveals Diverse Growth and Differentiation of Transplanted Mouse and Human Mammary Stem Cells

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.

Developmental changes in the in vitro activated regenerative activity of primitive mammary epithelial cells.

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.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare ‘outlier’ clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

DNA barcoding reveals diverse growth kinetics of human breast tumour subclones in serially passaged xenografts

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.

Glutathione-dependent and -independent oxidative stress-control mechanisms distinguish normal human mammary epithelial cell subsets

Significance Our study reveals lineage-specific mechanisms of ROS control and associated sensitivity to oxidative DNA damage in the basal and luminal progenitor-enriched subsets of normal human mammary cells. We show that the primitive luminal cells contain more mitochondria, show greater uptake of O2, sustain and withstand higher levels of ROS, and have mechanisms that allow them to accrue mutagenic levels of oxidative DNA damage. These findings support a growing body of data suggesting the involvement of primitive luminal cells in the generation of human breast cancers. 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 O2 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 H2O2 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.

Integrin β3 links therapy resistance and cancer stem cell properties.

Heterogeneity in tumour cell properties underlies many treatment failures. Understanding the sources of such heterogeneity has proved to be challenging, but remains critical to improving patient outcomes. Integrin αvβ3 expression in multiple types of solid tumour stem cells is now shown to control a pro-survival pathway that contributes to therapy resistance.

Modeling the process of human tumorigenesis

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.

Abstract A63: Clonal analysis of normal and malignant human mammary epithelial cell responsiveness to radiation

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.