Professional Education

  • Bachelor of Science, Tulane University of Louisiana (2005)
  • Doctor of Philosophy, University of California Berkeley (2011)

Stanford Advisors

All Publications

  • Viscoplasticity Enables Mechanical Remodeling of Matrix by Cells BIOPHYSICAL JOURNAL Nam, S., Lee, J., Brownfield, D. G., Chaudhuri, O. 2016; 111 (10): 2296-2308


    Living tissues consist largely of cells and extracellular matrices (ECMs). The mechanical properties of ECM have been found to play a key role in regulating cell behaviors such as migration, proliferation, and differentiation. Although most studies to date have focused on elucidating the impact of matrix elasticity on cell behaviors, recent studies have revealed an impact of matrix viscoelasticity on cell behaviors and reported plastic remodeling of ECM by cells. In this study, we rigorously characterized the plasticity in materials commonly used for cell culture. This characterization of plasticity revealed time-dependent plasticity, or viscoplasticity, in collagen gels, reconstituted basement membrane matrix, agarose gels, alginate gels, and fibrin gels, but not in polyacrylamide gels. Viscoplasticity was associated with gels that contained weak bonds, and covalent cross-linking diminished viscoplasticity in collagen and alginate gels. Interestingly, the degree of plasticity was found to be nonlinear, or dependent on the magnitude of stress or strain, in collagen gels, but not in the other viscoplastic materials. Viscoplastic models were employed to describe plasticity in the viscoplastic materials. Relevance of matrix viscoplasticity to cell-matrix interactions was established through a quantitative assessment of plastic remodeling of collagen gels by cells. Plastic remodeling of collagen gels was found to be dependent on cellular force, mediated through integrin-based adhesions, and occurred even with inhibition of proteolytic degradation of the matrix. Together, these results reveal that matrix viscoplasticity facilitates plastic remodeling of matrix by cellular forces.

    View details for DOI 10.1016/j.bpj.2016.10.002

    View details for Web of Science ID 000388545600020

    View details for PubMedID 27851951

  • Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury. Nature Vaughan, A. E., Brumwell, A. N., Xi, Y., Gotts, J. E., Brownfield, D. G., Treutlein, B., Tan, K., Tan, V., Liu, F. C., Looney, M. R., Matthay, M. A., Rock, J. R., Chapman, H. A. 2015; 517 (7536): 621-625


    Broadly, tissue regeneration is achieved in two ways: by proliferation of common differentiated cells and/or by deployment of specialized stem/progenitor cells. Which of these pathways applies is both organ- and injury-specific. Current models in the lung posit that epithelial repair can be attributed to cells expressing mature lineage markers. By contrast, here we define the regenerative role of previously uncharacterized, rare lineage-negative epithelial stem/progenitor (LNEP) cells present within normal distal lung. Quiescent LNEPs activate a ΔNp63 (a p63 splice variant) and cytokeratin 5 remodelling program after influenza or bleomycin injury in mice. Activated cells proliferate and migrate widely to occupy heavily injured areas depleted of mature lineages, at which point they differentiate towards mature epithelium. Lineage tracing revealed scant contribution of pre-existing mature epithelial cells in such repair, whereas orthotopic transplantation of LNEPs, isolated by a definitive surface profile identified through single-cell sequencing, directly demonstrated the proliferative capacity and multipotency of this population. LNEPs require Notch signalling to activate the ΔNp63 and cytokeratin 5 program, and subsequent Notch blockade promotes an alveolar cell fate. Persistent Notch signalling after injury led to parenchymal 'micro-honeycombing' (alveolar cysts), indicative of failed regeneration. Lungs from patients with fibrosis show analogous honeycomb cysts with evidence of hyperactive Notch signalling. Our findings indicate that distinct stem/progenitor cell pools repopulate injured tissue depending on the extent of the injury, and the outcomes of regeneration or fibrosis may depend in part on the dynamics of LNEP Notch signalling.

    View details for DOI 10.1038/nature14112

    View details for PubMedID 25533958

  • Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq. Nature Treutlein, B., Brownfield, D. G., Wu, A. R., Neff, N. F., Mantalas, G. L., Espinoza, F. H., Desai, T. J., Krasnow, M. A., Quake, S. R. 2014; 509 (7500): 371-375


    The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

    View details for DOI 10.1038/nature13173

    View details for PubMedID 24739965

  • Alveolar progenitor and stem cells in lung development, renewal and cancer. Nature Desai, T. J., Brownfield, D. G., Krasnow, M. A. 2014; 507 (7491): 190-194


    Alveoli are gas-exchange sacs lined by squamous alveolar type (AT) 1 cells and cuboidal, surfactant-secreting AT2 cells. Classical studies suggested that AT1 arise from AT2 cells, but recent studies propose other sources. Here we use molecular markers, lineage tracing and clonal analysis to map alveolar progenitors throughout the mouse lifespan. We show that, during development, AT1 and AT2 cells arise directly from a bipotent progenitor, whereas after birth new AT1 cells derive from rare, self-renewing, long-lived, mature AT2 cells that produce slowly expanding clonal foci of alveolar renewal. This stem-cell function is broadly activated by AT1 injury, and AT2 self-renewal is selectively induced by EGFR (epidermal growth factor receptor) ligands in vitro and oncogenic Kras(G12D) in vivo, efficiently generating multifocal, clonal adenomas. Thus, there is a switch after birth, when AT2 cells function as stem cells that contribute to alveolar renewal, repair and cancer. We propose that local signals regulate AT2 stem-cell activity: a signal transduced by EGFR-KRAS controls self-renewal and is hijacked during oncogenesis, whereas another signal controls reprogramming to AT1 fate.

    View details for DOI 10.1038/nature12930

    View details for PubMedID 24499815

  • Patterned Collagen Fibers Orient Branching Mammary Epithelium through Distinct Signaling Modules. Current biology Brownfield, D. G., Venugopalan, G., Lo, A., Mori, H., Tanner, K., Fletcher, D. A., Bissell, M. J. 2013; 23 (8): 703-709


    For decades, the work of cell and developmental biologists has demonstrated the striking ability of the mesenchyme and the stroma to instruct epithelial form and function in the mammary gland [1-3], but the role of extracellular matrix (ECM) molecules in mammary pattern specification has not been elucidated. Here, we show that stromal collagen I (Col-I) fibers in the mammary fat pad are axially oriented prior to branching morphogenesis. Upon puberty, the branching epithelium orients along these fibers, thereby adopting a similar axial bias. To establish a causal relationship from Col-I fiber to epithelial orientation, we embedded mammary organoids within axially oriented Col-I fiber gels and observed dramatic epithelial co-orientation. Whereas a constitutively active form of Rac1, a molecule implicated in cell motility, prevented a directional epithelial response to Col-I fiber orientation, inhibition of the RhoA/Rho-associated kinase (ROCK) pathway did not. However, time-lapse studies revealed that, within randomly oriented Col-I matrices, the epithelium axially aligns fibers at branch sites via RhoA/ROCK-mediated contractions. Our data provide an explanation for how the stromal ECM encodes architectural cues for branch orientation as well as how the branching epithelium interprets and reinforces these cues through distinct signaling processes.

    View details for DOI 10.1016/j.cub.2013.03.032

    View details for PubMedID 23562267

  • Self-organization is a dynamic and lineage-intrinsic property of mammary epithelial cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chanson, L., Brownfield, D., Garbe, J. C., Kuhn, I., Stampfer, M. R., Bissell, M. J., LaBarge, M. A. 2011; 108 (8): 3264-3269


    Loss of organization is a principle feature of cancers; therefore it is important to understand how normal adult multilineage tissues, such as bilayered secretory epithelia, establish and maintain their architectures. The self-organization process that drives heterogeneous mixtures of cells to form organized tissues is well studied in embryology and with mammalian cell lines that were abnormal or engineered. Here we used a micropatterning approach that confined cells to a cylindrical geometry combined with an algorithm to quantify changes of cellular distribution over time to measure the ability of different cell types to self-organize relative to each other. Using normal human mammary epithelial cells enriched into pools of the two principal lineages, luminal and myoepithelial cells, we demonstrated that bilayered organization in mammary epithelium was driven mainly by lineage-specific differential E-cadherin expression, but that P-cadherin contributed specifically to organization of the myoepithelial layer. Disruption of the actomyosin network or of adherens junction proteins resulted in either prevention of bilayer formation or loss of preformed bilayers, consistent with continual sampling of the local microenvironment by cadherins. Together these data show that self-organization is an innate and reversible property of communities of normal adult human mammary epithelial cells.

    View details for DOI 10.1073/pnas.1019556108

    View details for Web of Science ID 000287580400038

    View details for PubMedID 21300877

  • Collective epithelial cell invasion overcomes mechanical barriers of collagenous extracellular matrix by a narrow tube-like geometry and MMP14-dependent local softening INTEGRATIVE BIOLOGY Alcaraz, J., Mori, H., Ghajar, C. M., Brownfield, D., Galgoczy, R., Bissell, M. J. 2011; 3 (12): 1153-1166


    Collective cell invasion (CCI) through interstitial collagenous extracellular matrix (ECM) is crucial to the initial stages of branching morphogenesis, and a hallmark of tissue repair and dissemination of certain tumors. The collagenous ECM acts as a mechanical barrier against CCI. However, the physical nature of this barrier and how it is overcome by cells remains incompletely understood. To address these questions, we performed theoretical and experimental analysis of mammary epithelial branching morphogenesis in 3D type I collagen (collagen-I) gels. We found that the mechanical resistance of collagen-I is largely due to its elastic rather than its viscous properties. We also identified two strategies utilized by mammary epithelial cells that can independently minimize ECM mechanical resistance during CCI. First, cells adopt a narrow tube-like geometry during invasion, which minimizes the elastic opposition from the ECM as revealed by theoretical modeling of the most frequent invasive shapes and sizes. Second, the stiffness of the collagenous ECM is reduced at invasive fronts due to its degradation by matrix metalloproteinases (MMPs), as indicated by direct measurements of collagen-I microelasticity by atomic force microscopy. Molecular techniques further specified that the membrane-bound MMP14 mediates degradation of collagen-I at invasive fronts. Thus, our findings reveal that MMP14 is necessary to efficiently reduce the physical restraints imposed by collagen-I during branching morphogenesis, and help our overall understanding of how forces are balanced between cells and their surrounding ECM to maintain collective geometry and mechanical stability during CCI.

    View details for DOI 10.1039/c1ib00073j

    View details for Web of Science ID 000297407400001

    View details for PubMedID 21993836

  • Laminin and biomimetic extracellular elasticity enhance functional differentiation in mammary epithelia EMBO JOURNAL Alcaraz, J., Xu, R., Mori, H., Nelson, C. M., Mroue, R., Spencer, V. A., Brownfield, D., Radisky, D. C., Bustamante, C., Bissell, M. J. 2008; 27 (21): 2829-2838


    In the mammary gland, epithelial cells are embedded in a 'soft' environment and become functionally differentiated in culture when exposed to a laminin-rich extracellular matrix gel. Here, we define the processes by which mammary epithelial cells integrate biochemical and mechanical extracellular cues to maintain their differentiated phenotype. We used single cells cultured on top of gels in conditions permissive for beta-casein expression using atomic force microscopy to measure the elasticity of the cells and their underlying substrata. We found that maintenance of beta-casein expression required both laminin signalling and a 'soft' extracellular matrix, as is the case in normal tissues in vivo, and biomimetic intracellular elasticity, as is the case in primary mammary epithelial organoids. Conversely, two hallmarks of breast cancer development, stiffening of the extracellular matrix and loss of laminin signalling, led to the loss of beta-casein expression and non-biomimetic intracellular elasticity. Our data indicate that tissue-specific gene expression is controlled by both the tissues' unique biochemical milieu and mechanical properties, processes involved in maintenance of tissue integrity and protection against tumorigenesis.

    View details for DOI 10.1038/emboj.2008.206

    View details for Web of Science ID 000260638300003

    View details for PubMedID 18843297