Dendritic cells (DCs) are important regulators of the mammalian immune system and motility is critical to their proper function. Technologies such as cancer immunotherapy critically depend on DC migration. DCs possess multiple chemokine receptors and crawl in response to chemokine gradients, which direct DC positioning throughout the immune system. Ultimately, DCs must integrate multiple signals in order to move in a single direction. The goal of this project is to use a novel biointerfacial tool, micropost array detectors (mPADs), coupled with microfluidic gradient chambers, to apply a time- invariant chemokine gradient to cells, and measure the traction forces exerted by DCs during migration. Our recently published work shows that mPAD arrays are sufficiently sensitive to measure the low traction stresses (0.5 nN per filopod and 20 nN per cell) of migrating DCs. We now use these arrays to understand the components within cells that give rise to directed cell motion and to understand how DCs integrate chemokine signals and convert them to traction stresses and directional motion. The specific aims of the proposal are: 1) to use novel micropost force detector to measure DC motility in well- defined gradients of single chemokines on multiple adhesive ligands; 2) to measure the effects of regulatory proteins HS1 and WASp on DC migration in single chemokine gradients; and 3) to measure the forces of DC migration during turning when the gradient rapidly changes direction. In all aims, post arrays will be calibrated to ensure force maps are independent of post architecture, and we will correlate the direction of motion to the spatio-temporal map of forces that DCs exert. Furthermore, by varying the length of posts, we will determine the relationship between substrate elasticity and directional motion. This project is aided by a wealth of molecular and cellular tools including knock out mice in which chemokine receptors, actin regulatory proteins such as WASp, HS1 and myosin II, and various molecular knockdowns and pharmacological agents. The methods we establish here will yield a comprehensive picture of the forces exerted during DC motility, and the methods established here will have a significant impact on the elucidation of the mechanisms of motility of other fast moving amoeboid cells of the immune system that generate low forces, including T-lymphocytes.

The goal of this project is to develop novel tools to understand and quantify the behavior of dendritic cells. Dendritic cells are key cellular regulators of the immune response, and they coordinate the activity of many other immune cells. Also, they are the key cell that is activated in cancer immunotherapy, and motility is critical to their ability to effectively stimulate the immune system. In this proposal, we use novel devices to measure the forces that dendritic cells exert while crawling, and study the chemical and physical cues that drive their motility and thus affect their ability to move throughout the body. Fundamental insight into dendritic cell motility will allow the development of methods to manipulate dendritic cell motility and the design of devices to coordinate the interaction of dendritic cells with other immune cells. Just In Time Report IRB Confirmation: Human Subjects Assurance Number: Human Subjects Education: IACUC Confirmation: Page 1 Report submitted on : 08/06/2013 03:19 PM No IRB Certification was required No Human Subjects Assurance was required No Human Subjects Education was provided 1R01GM104287-01A1 Using micropost arrays to measure traction forces during dendritic cell motility Daniel A. Hammer (PD), Christopher S. Chen (co-I), Janis K. Burkhardt (co-I) Specific Aims (revised) Dendritic cells (DCs) are important controllers of the immune response. DC trafficking throughout the body and infiltration of immune cell organs and other tissues is central to the coordination both native and adaptive immunity, and for technologies such as cancer immune therapy. The elucidation of the fundamental mechanisms dendritic cells employ for directional migration and homing would help develop strategies for manipulating immune responses both in vivo and in vitro. We propose to use a novel biointerfacial tool - a microstamped micropost array force detector embedded within a microfluidic gradient generator - to elucidate key biochemical and mechanical controllers of dendritic cell motility in gradients of chemokine. This device is sufficiently sensitive to measure the weak forces exerted by dendritic cells during motility, allows us to measure sub nanoNewton forces at the level of a single filopodium and determine the precise spatio-temporal distribution of traction stresses during directional cell motility. We will systematically characterize and calibrate our device using an established, positive control (mature murine DCs crawling in a gradient of the chemokine CCL19), and use the device to relate the differential chemokine occupancy to the spatiotemporal dynamics of force generation for multiple adhesion molecules. Using our fully calibrated device, we will use selected knock-out mice to make molecular modifications in DCs and measure the resulting effects on the mechanics of cell motility, including the role of cytoskeletal regulators WASp, HS1, and the force generator myosin II in generating traction forces during chemotaxis. To understand the regulation of forces during turning, we will switch the spatial direction of the gradient rapidly and measure the changes in force during turning. The specific aims of this proposed work are: Aim 1. Using a novel micropost force detector to measure DC motility in well-defined chemokine gradients. DCs will be induced to crawl on fibronectin or ICAM-1-coated elastomeric posts of polydimethyoxysilane (PDMS) under the imposition of a gradient of a single chemokine, and the forces of DC motility will be measured. We will extend this work by measuring force profiles during directional DC motility as a function of post spacing, diameter and elasticity, and measure DC motility in response to chemokines CCL19, CCL21 and CxCL12 and multiple adhesion ligands. Under different magnitude chemokine gradients, the relationship between the force dynamics and cell motion will be determined. Aim 2. Measure the effects of individual actin regulatory proteins on DC migration in chemokine gradients. DC migration depends on actin-driven formation of filopodia at the leading edge, integrin- mediated adhesion, and actomyosin contraction. In DCs, adhesion is mediated in part by podosomes, dynamic contacts containing integrins, F-actin, actin binding proteins and signaling molecules. In this Aim, we will use knock-out mice and siRNA knock-down strategies to dissect the contributions of individual cytoskeletal regulators on DC motion and force generation, focusing on the role of WASp and HS1, which are known to be involved in protrusion and podosomes. In addition, we will image the actin cytoskeleton during protrusion using LifeAct labeled DCs. In this aim, mPAD methodologies established in aim 1 will be used throughout. Aim 3. Measure the forces of DC migration during DC turning. We will use mPADs measure forces exerted during DC migration when there are temporal changes in gradients of chemokines. We will measure the turning dynamics and changes in force generation measured when a single gradient is temporally reversed. Knock-downs and mutants from aim 2 will be employed to understand the intracellular regulators of DC turning. Just In Time Report IRB Confirmation: Human Subjects Assurance Number: Human Subjects Education: IACUC Confirmation: Page 1 Report submitted on : 04/09/2013 04:55 PM No IRB Certification was required No Human Subjects Assurance was required No Human Subjects Education was provided 03/26/2013 For New and Renewal Applications (PHS 398) - DO NOT SUBMIT UNLESS REQUESTED For Non-competing Progress Reports (PHS 2590) - Submit only Active Support for Key Personnel PHS 398/2590 OTHER SUPPORT HAMMER, DANIEL A. ACTIVE PO1 HL-18208 (R.E. Waugh, PD; Hammer PI of Project 1) 9/1/10-6/30/15 2.4 Calendar months NIH/HLBI $ 139,782 (Hammer Current Direct Cost) Title of Program Project: "Vascular Relations of Blood Cells and Proteins" Title of Hammer Grant: "Mechano-dynamics of the Transition to Firm Adhesion and Motility in Neutrophils" Specific aims of Hammer project are to 1) simulate the progressive activation of neutrophils under flow; 2) to perform measurements of neutrophil and HL-60 cell adhesion adhesion under flow; and 3) to measure the mechano-dynamics of neutrophil crawling using traction microscopy. Overlap: There is no overlap with the currently proposed project. DMR11-20901 (A.Yodh, PD; Hammer, Investigator) 09/15/11 - 08/31/17 0.5 calendar months National Science Foundation $ 55,000 (Hammer Current Direct Cost) Materials Science Center & Research Center The aim of the grant is to foster developments in materials science by through interdisciplinary science. The specific aim for Hammer is to make and characterize complex soft materials made of vesicles, colloidal particles, and polymersomes. Overlap: There is no overlap with the currently proposed project DE-SC0007063 (D. Hammer, PI; D.Lee, Collaborator) 7/01/11 - 6/30/14 0.5 Calendar months U.S. Department of Energy $117,728 (Hammer Current Direct Cost) Designing Smart, Responsive Communicating Microcapsules from Polymersomes The goal of this work is to design vesicles that can emit nanoparticles and make autonomous communication systems in response to light. Overlap: There is no overlap with the currently proposed project R01-AI082292 (D. Hammer, PI, Gary T Koretzky, Co-PI) 11/01/12 - 10/31/17 1.8 Calendar months NIH $132,521 (Hammer Current Direct Cost) Integrated Multi-scale Adhesive Dynamics Modeling of T-lymphocyte Homing The goal of this proposal is to develop a computational framework for modeling the interplay between adhesion and lymphocyte signal transduction to gain a better understanding of the factors that control lymphocyte homing and hence the proper response of the immune system. Overlap: There is no overlap with the currently proposed project PENDING DMR-1309556 (D. Hammer, PI) 05/01/13 - 06/30/15 0.5 Calendar months National Science Foundation $96,190 (Hammer Current Direct Cost) Responsive Vesicles from Recombinant Oleosin Objective of this proposal is to use molecular biology to engineer vesicles made from a recombinant protein, oleosin. Overlap: There is no overlap with the currently proposed project R01-GM104287 (Daniel A. Hammer, PI) 09/1/12 - 08/31/17 2.4 Calendar months NIH $138,328 (Hammer Current Direct Cost) Using micropost arrays to measure traction forces during dendritic cell motility The goal of this project is to develop novel tools to understand and quantify the behavior of dendritic cells. Dendritic cells are key cellular regulators of the immune response, and they coordinate the activity of many other immune cells. Overlap: This is the project under consideration. CMMI-1334443 (M. Lazzara, PI) 09/01/13 - 08/31/16 0.2 Calendar months National Science Foundation $30,623 (Hammer Current Direct Cost) Regulation of Receptor Trafficking and Signaling by Cell - Matrix Interactions Objective of this proposal is to study the effects of cell matrix materials properties on the endocytosis of cells and their effects on cell adhesion and signal transduction. Overlap: There is no overlap with the currently proposed project OTHER SUPPORT Chen, Christopher S. ACTIVE 1-R01-GM074048 Chen (PI) 05/01/06 - 04/30/14 2.0 Calendar NIGMS $210,000 (Current Annual Direct Cost) Mesenchymal Stem Cells and the Microenvironment The long term objective of this research is to characterize the cues within the local surrounding microenvironment that drive the lineage specification and differentiation of human mesenchymal stem cells (MSCs), and the molecular pathways involved. Overlap: There is no overlap with the currently proposed project. N/A Chen (co-I) 4/1/07 - 5/31/13 0.2 Calendar NIH EB001046 via Rutgers U. (Kohn PI) $83,643 (Current Annual Direct Cost) RESBIO - Integrated Technologies for Polymeric Biomaterials The major goal of this project is to develop integrated tools and technologies that advance the discovery of polymeric biomaterials for regenerative medicine, the delivery of biological agents, and the next generation of medical implants. Overlap: There is no overlap with the currently proposed project. 2-R01-EB000262 Chen (PI) 07/16/10 - 06/30/14 2.0 Calendar NIBIB $250,000 (Current Annual Direct Cost) Local regulation of Angiogenesis by Microenvironment These studies will define the mechanisms by which local structural and mechanical properties within ECM modulate endothelial cell function and capillary morphogenesis, and establish new strategies to promote angiogenesis in native ischemic tissues as well as in ex-vivo engineered tissues. Overlap: There is no overlap with the currently proposed project. 1-R01-EB008396 Chen & Bhatia (PI) 6/15/09 - 3/31/13 1.8 Calendar NIBIB No Cost Extension (Chen NCE Direct Cost: $37,351) Engineering multicellular tissue structure, function, and vascularization The goal of this project is to develop technologies for spatially organizing multiple cell types in a 3D scaffold in order to enhance the engineering of 3D vascularized liver tissue. The use of the liver as a model system focuses on the crosstalk between endothelium compartments and parenchymal (hepatocyte compartments) rather than the interaction between hepatocytes themselves or between endothelial cells by themselves (homotypic cell-cell interactions) or the impact of matrix cues on compartmentalized (zonal) hepatocyte or vascular functions. Overlap: There is no overlap with the currently proposed project. This project is under a no cost extension and will end before the proposal under evaluation is funded. 1R01 HL73305 Chen (PI) 7/5/07 - 6/30/13 2.5 Calendar National Heart Lung Blood Institute. No Cost Extension (Chen NCE Direct Cost: $44,026) Cadherins, Integrins, and Mechanochemical Signaling This project focuses on the mechanisms by which cadherins, integrins and the cytoskeleton cooperate to regulate proliferation in endothelial and smooth muscle cells in the context of atherosclerotic vessels. This work does not examine microvasculature, but rather, the primary effects on large artery wall remodeling in the face of hypertensive or inflammatory settings. Overlap: There is no overlap with the currently proposed project. This project is under a no cost extension and will end before the proposal under evaluation is funded. 1-R01-HL090747 Chen (co-I) 9/1/08 - 6/30/13 1.5 Calendar NIH R01 via Johns Hopkins University (Reich PI) No Cost Extension (Chen NCE Direct Cost: $22,491) Subcellular Response to Local Forces This project will apply recent advances in magnetic micro- and nanotechnology to study the response of vascular cells to force and mechanical stress. Overlap: There is no overlap with the currently proposed project. This project is under a no cost extension and will end before the proposal under evaluation is funded. SR00001766 Morrisey (UPenn PI) 08/01/11 - 07/31/13 0.6 Calendar University of Maryland - sub to NIH U01 FINAL PERIOD - Ends 7/31/13 $136,425 NHLBI Progenitor Cell Biology Consortium - Anciliary/Collaborative Project In this project, we will develop approaches to 1) obtain the correct type of cardiomyocyte, and 2) assess and enhance their electrical and mechanical maturation. Overlap: There is no overlap with the currently proposed project. This project is under a no cost extension and will end shortly after the proposal under evaluation is funded. 1-UH2-EB017103-01 Chen (Penn PI) 09/01/12 - 08/31/14 1.0 Calendar NIH via Columbia University $84,750 (Current Annual Direct Cost) Integrated Heart-Liver-Vascular Systems for Drug Testing in Human Health and Disease This project investigates an integrated multiorgan-on-chip system for drug screening. Overlap: There is no overlap with the currently proposed project. PENDING: R01-GM104287 Hammer (PI) 09/01/12 - 08/31/17 0.6 Calendar NIH Chen Lab Annual Directs: $100,045 Using micropost arrays to measure traction forces during dendritic cell motility The goal of this project is to develop novel tools to understand and quantify the behavior of dendritic cells. Dendritic cells are key cellular regulators of the immune response, and they coordinate the activity of many other immune cells. Overlap:This is the proposal currently under consideration. 2 R01 HL089315-06 Woo (PI) 03/01/13 - 02/28/18 0.3 Calendar NIH Chen Lab Annual Directs: $10,000 Angiogenic tissue engineering to limit post-infarction ventricular remodeling This project seeks to study in further depth the specific interactive mechanisms underlying SDF-mediated EPC neovasculogenesis, develop novel, clinically translatable delivery platforms that enhance EPC function and survival, and refine these for potential human clinical investigation. Overlap: There is no overlap with the currently proposed project. R01-HL115553 Assoian & Chen (MPI) 04/01/13 - 03/31/18 1.8 Calendar NIH Chen Lab Annual Directs: $207,272 Stiffness, cadherins, and Integrins in Mechanochemical Signaling This project focuses on the mechanisms by which cadherins, integrins and the cytoskeleton cooperate to regulate proliferation in vascular smooth muscle cells (VSMCs). Overlap: There is no overlap with the currently proposed project. R21 EB016802 Reich (PI) 04/01/13 - 03/31/15 0.6 Calendar NIH via Rutgers University Chen Lab Annual Directs: $46,405 Magnetic Microdevice to Actuate Arrays of Engineered Microtissues Our role in this project is the development of the TUG design to optimize performance with the integrated actuation system. Overlap: There is no overlap with the currently proposed project. For New and Renewal Applications (PHS 398) - DO NOT SUBMIT UNLESS REQUESTED For Non-competing Progress Reports (PHS 2590) - Submit only Active Support for Key Personnel PHS 398/2590 OTHER SUPPORT BURKHARDT, J.K. ACTIVE R01 AI065644 Burkhardt (PI) 1/1/11 - 12/31/13 (2nd NCE)* 1.8 calendar NIH/NIAID $240,370 "Cytoskeletal Remodeling During T Cell Activation" The goals of this project are to define HS1's function as a component of the T cell actin regulatory network, to identify the distinct roles of HS1, WIP and WASP during T cell activation, and to conduct structure-function analysis of HS1 during T cell activation. Role: PI Overlap: There is no overlap with the currently proposed project. This project is under a no cost extension and will end shortly after the proposal under evaluation is funded. 2P01 CA093615 Koretzky (PI) 9/1/11 - 8/31/13 (NCE)* 1.2 calendar NIH/NCI $241,452 (ann. project direct costs) "Temporal and Spatial organization of signaling complexes in T and B cells" Project 2: "Organization and Function of the Distal Pole Complex During T cell activation" The goals of this project are to define the unique role of ezrin in T cell activation, to assess the role of the DPC in T cell signaling, and to assess the role of the DPC in determining T cell polarity. Role: Project PI. Overlap: There is no overlap with the currently proposed project. This project is under a no cost extension and will end shortly after the proposal under evaluation is funded. Renewal for this P01 will not be sought. R21 AI088376 Burkhardt and Argon (co-PIs) 9/1/11 - 8/31/13 (NCE)* 0.72 calendar NIH/NIAID $150,000 "HS1 Function in Dendritic Cells" The goals of this project are to identify the HS1-dependent steps in antigen presentation and to determine the functional significance of binding partners. Role: co-PI. Overlap: Although this proposal is about dendritic cells, there is no direct overlap with the aims of the currently proposed project. This project is under a no cost extension and will end shortly after the proposal under evaluation is funded. Institutional Pilot Project 07/01/12 - 6/30/13 1.2 calendar Foerderer Foundation $50,000 "Fascin Function in Dendritic Cells" The goals of this pilot project are to obtain newly-available fascin-/- mice, and to initiate analysis of fascin function in DC migration and T cell priming in vitro. This grant does not allow PI salary recovery. Role: PI Overlap: This project will end before the proposal under evaluation is funded. PENDING: 1R01 AI108645 Huang (PI) 10/1/2013 - 9/30/2018 0.6 calendar NIH/NIAID $250,000 "The function of Crk adaptor proteins in T cell migration and activation" The goals of this grant are to analyze the role of Crk and CrkL in T cell migration and T cell activation, and to define the molecular mechanisms that underlie Crk protein signaling in T cells. Role: Co-Investigator. Overlap: There is no overlap with the currently proposed project. R01 AI106641 Bergelson (PI) 8/1/2013 - 7/31/16 0.24 calendar NIH/NIAID $358,209 "EV71 Pathogenesis: cytokine responses and virus-immune cell interactions" The goals of this grant are to define the consequences of enterovirus 71 (EV71) interactions with PSGL-1 on leukocytes, and to define the mechanisms responsible for EV71 entry into leukocytes. Role: Co-Investigator Overlap: There is no overlap with the currently proposed project. R01 GM1071731 Burkhardt (PI) 7/1/2013 - 6/30/2018 3.0 calendar NIH/NIGMS $327,821 "Fascin Function in Dendritic Cells" The goals of this project are to test the role of the actin bundling protein fascin in organizing DC shape and migration, to test the idea that fascin promotes T cell activation by bundling actin filaments at the DC-IS, and to characterize the role of fascin in promoting T cell priming in vivo. Role: PI. Overlap: There is no direct overlap with the aims of the currently proposed project. Though this grant was assigned to General Medicine because of its cell biological nature, it is ultimately aimed at designing better DC-based cancer vaccines. It received a 19% on the first submission. FCCC-UPENN SPORE Burkhardt (PI) 3/1/2013 - 2/28/2014 0.6 calendar Pilot Program in Ovarian Cancer $49,995 "Characterization of Cytoskeletal Regulation in Dendritic Cells Cultured for Tumor Vaccines" The goals of this project are to characterize the migratory properties of clinically relevant human DCs, and to analyze cytoskeletal protein expression at the mRNA and protein levels. Role: PI. Overlap: There is no overlap with the currently proposed project. This grant represents a new, more translational initiative in our lab. It is directly cancer related. R01 GM104867 Burkhardt (PI) 11/1/2012 - 10/31/2017 3.0 calendar NIAID/NIGMS $287,359 "Costimulatory ligand mobility effects on T cell activation" In this project, we will test the hypothesis that constrained mobility of ICAM-1 and CD80 by specific actin binding proteins is essential for efficient T cell activation by DCs. Overlap: There is no direct overlap with the currently proposed project. R01A1 GM104287 Hammer (PI) 07/01/2013 - 06/30/2018 1.2 calendar NIH/NIGMS $149,201 (ann. subcontract direct costs) "Using micropost arrays to measure traction forces during dendritic cell motility" The goals of this project are to use a novel micropost force detector to measure DC motility in well defined chemokine gradients, to measure the effects of individual actin regulatory proteins on DC migration, and to measure the forces of DC migration in chemokine countergradients. Role: Co-Investigator Overlap: This is the proposal currently under investigation. OVERLAP There is no overlap between active or pending projects and the current proposal.