Human Milk Protein Production in Xenografts of Genetically Engineered Bovine Mammary Epithelial Stem Cells

BACKGROUND: In the bovine species milk production is well known to correlate with mammary tissue mass. However, most advances in optimizing milk production relied on improvements of breeding and husbandry practices. A better understanding of the cells that generate bovine mammary tissue could facilitate important advances in milk production and have global economic impact. With this possibility in mind, we show that a mammary stem cell population can be functionally identified and isolated from the bovine mammary gland. We also demonstrate that this stem cell population may be a promising target for manipulating the composition of cow's milk using gene transfer. METHODS AND FINDINGS: We show that the in vitro colony-forming cell assay for detecting normal primitive bipotent and lineage-restricted human mammary clonogenic progenitors are applicable to bovine mammary cells. Similarly, the ability of normal human mammary stem cells to regenerate functional bilayered structures in collagen gels placed under the kidney capsule of immunodeficient mice is shared by a subset of bovine mammary cells that lack aldehyde dehydrogenase activity. We also find that this activity is a distinguishing feature of luminal-restricted bovine progenitors. The regenerated structures recapitulate the organization of bovine mammary tissue, and milk could be readily detected in these structures when they were assessed by immunohistochemical analysis. Transplantation of the bovine cells transduced with a lentivirus encoding human β-CASEIN led to expression of the transgene and secretion of the product by their progeny regenerated in vivo. CONCLUSIONS: These findings point to a common developmental hierarchy shared by human and bovine mammary glands, providing strong evidence of common mechanisms regulating the maintenance and differentiation of mammary stem cells from both species. These results highlight the potential of novel engineering and transplant strategies for a variety of commercial applications including the production of modified milk components for human consumption

A Biobank of Breast Cancer Explants with Preserved Intra-tumor Heterogeneity to Screen Anticancer Compounds.

The inter- and intra-tumor heterogeneity of breast cancer needs to be adequately captured in pre-clinical models. We have created a large collection of breast cancer patient-derived tumor xenografts (PDTXs), in which the morphological and molecular characteristics of the originating tumor are preserved through passaging in the mouse. An integrated platform combining in vivo maintenance of these PDTXs along with short-term cultures of PDTX-derived tumor cells (PDTCs) was optimized. Remarkably, the intra-tumor genomic clonal architecture present in the originating breast cancers was mostly preserved upon serial passaging in xenografts and in short-term cultured PDTCs. We assessed drug responses in PDTCs on a high-throughput platform and validated several ex vivo responses in vivo. The biobank represents a powerful resource for pre-clinical breast cancer pharmacogenomic studies (http://caldaslab.cruk.cam.ac.uk/bcape), including identification of biomarkers of response or resistance.This research was supported with funding from Cancer Research UK and from the European Union to the EUROCAN Network of Excellence (FP7; grant numnumber 260791). M.C. has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sk1odowska-Curie grant agreement no. 660060 and was supported by the Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. R.N.B. is supported by the Wellcome Trust PhD Programme in Mathematical Genomics and Medicine. S-J.S. is supported by the Wellcome Trust PhD Programme for Clinicians in Cambridge. A.Bruna, O.M.R., E.M., V.S., and C.C. are members of the EurOPDX Consortium. Weare very grateful for the generosity of all the patients that donated samples for implantation. We are also deeply indebted to all the staff (surgeons, pathologists, oncologists, theatre staff, and other ancillary personnel) at the Cambridge Breast Unit, Cambridge University Hospital NHS Foundation Trust, for facilitating the timely collection of samples. We thank the Cancer Research UK Cambridge Institute Genomics, Bioinformatics, Histopathology, Flow Cytometry, Biological Resource, and Bio-repository Core Facilities for support during the execution of this project.This is the final version of the article. It first appeared from Elsevier at http://dx.doi.org/10.1016/j.cell.2016.08.041

Targeting breast cancer stem cells

The cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self‐renewing CSC population that is also capable of differentiating into non‐self‐renewing cell populations that constitute the bulk of the tumor. Although, the CSC hypothesis does not directly address the cell of origin of cancer, it is postulated that tissue‐resident stem or progenitor cells are the most common targets of transformation. Clinically, CSCs are predicted to mediate tumor recurrence after chemo‐ and radiation‐therapy due to the relative inability of these modalities to effectively target CSCs. If this is the case, then CSC must be efficiently targeted to achieve a true cure. Similarities between normal and malignant stem cells, at the levels of cell‐surface proteins, molecular pathways, cell cycle quiescence, and microRNA signaling present challenges in developing CSC‐specific therapeutics. Approaches to targeting CSCs include the development of agents targeting known stem cell regulatory pathways as well as unbiased high‐throughput siRNA or small molecule screening. Based on studies of pathways present in normal stem cells, recent work has identified potential “Achilles heals” of CSC, whereas unbiased screening provides opportunities to identify new pathways utilized by CSC as well as develop potential therapeutic agents. Here, we review both approaches and their potential to effectively target breast CSC.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135704/1/mol2201045404.pd

Patient-derived xenograft (PDX) models in basic and translational breast cancer research

Patient-derived xenograft (PDX) models of a growing spectrum of cancers are rapidly supplanting long-established traditional cell lines as preferred models for conducting basic and translational preclinical research. In breast cancer, to complement the now curated collection of approximately 45 long-established human breast cancer cell lines, a newly formed consortium of academic laboratories, currently from Europe, Australia, and North America, herein summarizes data on over 500 stably transplantable PDX models representing all three clinical subtypes of breast cancer (ER+, HER2+, and "Triple-negative" (TNBC)). Many of these models are well-characterized with respect to genomic, transcriptomic, and proteomic features, metastatic behavior, and treatment response to a variety of standard-of-care and experimental therapeutics. These stably transplantable PDX lines are generally available for dissemination to laboratories conducting translational research, and contact information for each collection is provided. This review summarizes current experiences related to PDX generation across participating groups, efforts to develop data standards for annotation and dissemination of patient clinical information that does not compromise patient privacy, efforts to develop complementary data standards for annotation of PDX characteristics and biology, and progress toward "credentialing" of PDX models as surrogates to represent individual patients for use in preclinical and co-clinical translational research. In addition, this review highlights important unresolved questions, as well as current limitations, that have hampered more efficient generation of PDX lines and more rapid adoption of PDX use in translational breast cancer research

Detection and characterization of human mammary stem cells

The mammary gland of adult female mice contains undifferentiated epithelial stem cells with in vivo regenerative and self-renewal properties. A biologically similar population likely exists in the human breast but a specific and quantitative methodology to identify and characterize these cells has been lacking. In this study I show that human mammary structures are reproducibly generated when dissociated suspensions of primary human mammary cells are propagated in collagen gels that have been implanted under the renal capsule of highly immunodeficient, hormone-treated mice. These structures contain differentiated cells of both mammary lineages in a spatial organization similar to normal mammary tissue, and display functional maturation into milk-producing glands when the hosts become pregnant. In vitro assays of single cell suspensions prepared from these regenerated glands revealed the consistent presence of mammary progenitor cells able to form adherent bi-lineage as well as pure luminal and myoepithelial colonies. In addition, when these cells are suspended in new gels and transplanted into secondary immunodeficient mice, similar progenitor-containing structures are demonstrable, indicative of a regenerative process that recreates the normal developmental hierarchy. This daughter progenitor production endpoint allows the frequency of these self-renewing human mammary stem cells to be derived from limiting dilution transplant assays as 1 per 10³–10⁴ cells in normal adult human reduction mammoplasty samples, and their phenotype to be established as CD49f⁺EpCAM⁻⁄low CD31⁻CD45⁻. I have also developed methodologies to isolate fractions of cells from mammoplasty tissue that are enriched in cells in different phases of the cell cycle (G0/G1/S/G2/M). Application of functional assays to these fractions indicates that a proportion of stem and progenitor cells in normal adult breast tissue exhibit phenotypes that are associated with actively proliferating cells. These studies support a model of mammary cell production that includes a significant rate of normal turnover of primitive cells, and sets the stage for further work to identify the factors and molecular interactions that regulate this process.Medicine, Faculty ofMedical Genetics, Department ofGraduat

Human <i>β-CASEIN</i> expression in regenerated bovine alveoli.

<p>Representative photomicrographs showing in bovine lactating mammary tissue the presence of milk proteins in the lumen of an alveolus (a) and the absence of crossreactivity of the antibody used to detect human <i>β-CASEIN</i> (b). In (c) the expression and localization in the lumen of recombinant human <i>β-CASEIN</i> is evident in outgrowths formed by cells transduced with the human β-cas-pWPI lentiviral vector. Scale bar is 25 µm.</p

FACS profiles of bovine mammary cells stained for ALDH activity.

<p>Cells were stained with ALDEFLUOR with (a) or without (b) diethylamino-benzaldehyde (DEAB), an ALDH inhibitor. Panel c shows the proportional distribution of total clonogenic luminal and myoepithelial progenitors between the Aldehyde Dehydrogenase (ALDH)^high and ALDH^low fractions (% of all progenitors of a given type in the fraction shown ± SEM, n = 3).</p