intraductal-injection-for-localized-drug-delivery-to-mouse-mammary

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

intraductal-delivery-to-the-rabbit-mammary-gland

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Harvard University

The University of Texas at Austin

A novel trap for dynamic protein interactions in cytokine signaling.

Directed cell migration is critical for tissue morphogenesis and wound healing, but the mechanism of directional control is poorly understood. Here we show that the direction in which cells extend their leading edge can be controlled by constraining cell shape using micrometer-sized extracellular matrix (ECM) islands. When cultured on square ECM islands in the presence of motility factors, cells preferentially extended lamellipodia, filopodia, and microspikes from their corners. Square cells reoriented their stress fibers and focal adhesions so that tractional forces were concentrated in these corner regions. When cell tension was dissipated, lamellipodia extension ceased. Mechanical interactions between cells and ECM that modulate cytoskeletal tension may therefore play a key role in the control of directional cell motility.

Directional control of lamellipodia extension by constraining cell shape and orienting cell tractional forces.

Interactions between mRNA and the cytoskeleton are critical for the localization of a number of transcripts in eukaryotic somatic cells. To characterize additional transcripts that may be subject to this form of regulation, we developed a two-step approach that utilizes biochemical fractionation of cells to isolate transcripts from different subcellular compartments followed by microarray analysis to examine and compare these subpopulations of transcripts in a massively-parallel manner.

Identification of a distinct class of cytoskeleton-associated mRNAs using microarray technology.

Mammalian cells redirect their movement in response to changes in the physical properties of their extracellular matrix (ECM) adhesive scaffolds, including changes in available substrate area, shape, or flexibility. Yet, little is known about the cell's ability to discriminate between different types of spatial signals. Here we utilize a soft-lithography-based, microcontact printing technology in combination with automated computerized image analysis to explore the relationship between ECM geometry and directional motility. When fibroblast cells were cultured on fibronectin-coated adhesive islands with the same area (900 micrometers2) but different geometric forms (square, triangle, pentagon, hexagon, trapezoid, various parallelograms) and aspect ratios, cells preferentially extended new lamellipodia from their corners. In addition, by imposing these simple geometric constraints through ECM, cells were directed to deposit new fibronectin fibrils in these same corner regions. These data indicate that mammalian cells can sense edges within ECM patterns that exhibit a wide range of angularity and that they use these spatial cues to guide where they will deposit ECM and extend new motile processes during the process of directional migration.

Geometric determinants of directional cell motility revealed using microcontact printing.

Structural features found in biomolecular networks that are absent in random networks produced by simple algorithms can provide insight into the function and evolution of cell regulatory networks. Here we analyze "betweenness" of network nodes, a graph theoretical centrality measure, in the yeast protein interaction network. Proteins that have high betweenness, but low connectivity (degree), were found to be abundant in the yeast proteome. This finding is not explained by algorithms proposed to explain the scale-free property of protein interaction networks, where low-connectivity proteins also have low betweenness. These data suggest the existence of some modular organization of the network, and that the high-betweenness, low-connectivity proteins may act as important links between these modules. We found that proteins with high betweenness are more likely to be essential and that evolutionary age of proteins is positively correlated with betweenness. By comparing different models of genome evolution that generate scale-free networks, we show that rewiring of interactions via mutation is an important factor in the production of such proteins. The evolutionary and functional significance of these observations are discussed.

High-betweenness proteins in the yeast protein interaction network.

Clonal populations of mammalian cells are inherently heterogeneous. They contain cells that display non-genetic variability resulting from gene expression noise and the fact that gene networks have multiple stable states. These stable, heritable variants within one cell type can exhibit different levels of responsiveness to environmental conditions. Hence, they could in principle serve as a temporary substrate for natural selection in the absence of mutations. We suggest that such ubiquitous but non-genetic variability can contribute to the somatic evolution of cancer cells, hence accelerating tumour progression independently of genetic mutations.

Non-genetic heterogeneity--a mutation-independent driving force for the somatic evolution of tumours.

C&eacute;lulas pluripotentes inducidas mediante reprogramaci&oacute;n celular ahora est&aacute;n encontrando m&uacute;ltiples aplicaciones en la tuber&iacute;a de desarrollo de investigaci&oacute;n y drogas farmac&eacute;utica. En las etapas precl&iacute;nicas, sirven como sistemas modelo para la investigaci&oacute;n b&aacute;sica sobre enfermedades espec&iacute;ficas y luego como claves herramientas experimentales para probar y desarrollar terapias. Aqu&iacute; examinamos el estado actual de la tecnolog&iacute;a de reprogramaci&oacute;n celular, con especial &eacute;nfasis en los enfoques que recapitular previamente insuperables enfermedades humanas in vitro. Discutimos los desaf&iacute;os t&eacute;cnicos y operativos que deben abordarse como c&eacute;lulas reprogramadas incorporarse a la rutinario descubrimiento de investigaci&oacute;n y drogas farmac&eacute;utico.

Reprogramaci&oacute;n Celular: una nueva frontera de la tecnolog&iacute;a en la investigaci&oacute;n farmac&eacute;utica.

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Cellule staminali pluripotenti indotte tramite riprogrammazione cellulare ora stanno trovando applicazioni multiple nella pipeline di sviluppo ricerca e droga farmaceutica. Nelle fasi precliniche, essi servono come modelli di sistemi per la ricerca di base su malattie specifiche e poi come strumenti chiave sperimentali per il testing e lo sviluppo di terapie. Qui esaminiamo lo stato attuale della tecnologia di riprogrammazione cellulare, con una particolare enfasi sugli approcci che riassumere in precedenza intrattabile malattie umane in vitro. Discutiamo le sfide tecniche ed operative che devono essere affrontate come cellule riprogrammate diventano incorporate routine farmaceutica ricerca e droga scoperta.

Riprogrammazione cellulare: una nuova frontiera della tecnologia nella ricerca farmaceutica.

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Induced pluripotent stem cells via cellular reprogramming are now finding multiple applications in the pharmaceutical research and drug development pipeline. In the pre-clinical stages, they serve as model systems for basic research on specific diseases and then as key experimental tools for testing and developing therapeutics. Here we examine the current state of cellular reprogramming technology, with a special emphasis on approaches that recapitulate previously intractable human diseases in vitro. We discuss the technical and operational challenges that must be tackled as reprogrammed cells become incorporated into routine pharmaceutical research and drug discovery.

Cellular reprogramming: a new technology frontier in pharmaceutical research.

Cellules souches pluripotentes induites par la reprogrammation cellulaire sont maintenant de trouver des applications multiples dans le pipeline de d&eacute;veloppement pharmaceutique en recherche et de la drogue. Dans les stades pr&eacute;cliniques, ils servent comme syst&egrave;mes mod&egrave;les pour la recherche fondamentale sur les maladies sp&eacute;cifiques, puis comme principaux outils exp&eacute;rimentaux pour les essais et le d&eacute;veloppement de th&eacute;rapies. Nous examinons ici l&#39;&eacute;tat actuel de la technologie de reprogrammation cellulaire, avec un accent particulier sur les approches qui r&eacute;capitulent auparavant insolubles maladies humaines in vitro. Nous discutons les d&eacute;fis techniques et op&eacute;rationnelles qui doivent &ecirc;tre abord&eacute;s comme cellules reprogramm&eacute;s soit int&eacute;gr&eacute;s &agrave; la routine pharmaceutique recherche et d&eacute;couverte de m&eacute;dicaments.

Reprogrammation cellulaire : une nouvelle fronti&egrave;re de la technologie dans la recherche pharmaceutique.

Induzierten pluripotenten Stammzellen &uuml;ber zellul&auml;re Neuprogrammierung sind nun mehrere Anwendungen in der pharmazeutischen Forschung und Drogen-Entwicklungs-Pipeline finden. In der pr&auml;klinischen Phase dienen sie als Modellsysteme f&uuml;r die Grundlagenforschung auf bestimmte Krankheiten und dann als experimentelle Schl&uuml;sselinstrumente f&uuml;r Tests und Entwicklung von Therapeutika. Hier untersuchen wir den aktuellen Zustand der Neuprogrammierung Mobilfunktechnologie, mit besonderem Schwerpunkt auf Ans&auml;tze, die zuvor unheilbare menschliche Krankheiten, die in-vitro zu rekapitulieren. Wir besprechen die technischen und betrieblichen Herausforderungen, die angegangen werden m&uuml;ssen, wie routinem&auml;&szlig;ige pharmazeutische Forschung und Droge-Entdeckung umprogrammiert Zellen einverleibt werden.

Zellul&auml;re Neuprogrammierung: eine neue Technologie-Grenze in der pharmazeutischen Forschung.

The development of drug resistance, the prime cause of failure in cancer therapy, is commonly explained by the selection of resistant mutant cancer cells. However, dynamic non-genetic heterogeneity of clonal cell populations continuously produces metastable phenotypic variants (persisters), some of which represent stem-like states that confer resistance. Even without genetic mutations, Darwinian selection can expand these resistant variants, which would explain the invariably rapid emergence of stem-like resistant cells. Here, by using quantitative measurements and modelling, we show that appearance of multidrug resistance in HL60 leukemic cells following treatment with vincristine is not explained by Darwinian selection but by Lamarckian induction. Single-cell longitudinal monitoring confirms the induction of multidrug resistance in individual cells. Associated transcriptome changes indicate a lasting stress response consistent with a drug-induced switch between high-dimensional cancer attractors. Resistance induction correlates with Wnt pathway upregulation and is suppressed by Î²-catenin knockdown, revealing a new opportunity for early therapeutic intervention against the development of drug resistance.

Non-Darwinian dynamics in therapy-induced cancer drug resistance.

With advances in screening, the incidence of detection of premalignant breast lesions has increased in recent decades; however, treatment options remain limited to surveillance or surgical removal by lumpectomy or mastectomy. We hypothesized that disease progression could be blocked by RNA interference (RNAi) therapy and set out to develop a targeted therapeutic delivery strategy. Using computational gene network modeling, we identified HoxA1 as a putative driver of early mammary cancer progression in transgenic C3(1)-SV40TAg mice. Silencing this gene in cultured mouse or human mammary tumor spheroids resulted in increased acinar lumen formation, reduced tumor cell proliferation, and restoration of normal epithelial polarization. When the HoxA1 gene was silenced in vivo via intraductal delivery of nanoparticle-formulated small interfering RNA (siRNA) through the nipple of transgenic mice with early-stage disease, mammary epithelial cell proliferation rates were suppressed, loss of estrogen and progesterone receptor expression was prevented, and tumor incidence was reduced by 75%. This approach that leverages new advances in systems biology and nanotechnology offers a novel noninvasive strategy to block breast cancer progression through targeted silencing of critical genes directly within the mammary epithelium.

Silencing HoxA1 by intraductal injection of siRNA lipidoid nanoparticles prevents mammary tumor progression in mice.

Nanoparticle-based therapeutics are poised to become a leading delivery strategy for cancer treatment because they potentially offer higher selectivity, reduced toxicity, longer clearance times, and increased efficacy compared to conventional systemic therapeutic approaches. This article reviews existing nanoparticle technologies and methods that are used to target drugs to treat cancer by altering signal transduction or modulating the tumor microenvironment. We also consider the implications of recent advances in the nanotherapeutics field for the future of cancer therapy.

Nanoparticle targeting of anti-cancer drugs that alter intracellular signaling or influence the tumor microenvironment.

Differentiation therapies that induce malignant cells to stop growing and revert to normal tissue-specific differentiated cell types are successful in the treatment of a few specific haematological tumours. However, this approach has not been widely applied to solid tumours because their developmental origins are less well understood. Recent advances suggest that understanding tumour cell plasticity and how intrinsic factors (such as genetic noise and microenvironmental signals, including physical cues from the extracellular matrix) govern cell state switches will help in the development of clinically relevant differentiation therapies for solid cancers.

Amy Brock

Wyss Institute for Biologically Inspired Engineering

Harvard University

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Intraductal Delivery to the Rabbit Mammary Gland

Amy Brock

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Wyss Institute for Biologically Inspired Engineering

Harvard University

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Intraductal Delivery to the Rabbit Mammary Gland

Wyss Institute for Biologically Inspired Engineering