Charles P. Lin, Ph.D. - US grants

We propose to develop a method for selective RPE photocoagulation with minimal damage to the overlying photoreceptors. The technique will be applied to address a long standing question of how much retinal tissue one needs to photocoagulate in order to elicit a useful therapeutic outcome. While the answer will depend on the specific disorder being treated, our hypothesis is that selective killing of diseased RPE cells will stimulate surrounding RPE cells to proliferate and form a new, functional RPE layer. The proposed technique may be useful for treating diseases thought to be associated with the RPE, including central serous retinopathy, diabetic macular edema, and drusen. Two major technical challenges need to be overcome in order to implement the selective RPE targeting scheme. First, a suitable laser source has to be developed, with the desired treatment parameters which confine laser damage to the RPE. Second, a feedback system needs to be implemented to ensure that reproducible lesions are being created, since RPE damage alone without neuroretina coagulation is not visible ophthalmoscopically. This proposal aims to address both of these issues, as well as to understand the mechanism for RPE cell killing.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] The overall long-term goal of this multidisciplinary research program is to apply new optical technology to investigate early changes associated with cellular immune response and to monitor response to therapy over time in vivo and at the cellular level. Specifically, we proposed to develop in vivo immunofluorescence microscopy for imaging specific cell surface markers expressed by vascular and lymphatic endothelial cells, circulating leukocytes, and tissue dendritic cells. The technology that enables noninvasive, real-time cellular imaging is a video rate confocal and multiphoton fluorescence microscope that we recently developed in our laboratory. We will apply this technology to study important steps involved in the regulation of cellular immune response in vivo, including endothelial cell activation, leukocyteendothelial interaction, and dendritic cell recruitment and migration. In addition, we proposed to develop an in vivo flow cytometer, a novel technology for real-time, noninvasive detection and quantification of circulating cells that are tagged with fluorescent antibodies or antibody fragments. We will use this system to monitor changes in specific T cell population in response to immunomodulation, to detect circulating tumor cells and to investigate the correlation between tumor cell shedding and metastatic potential. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This is a multidisciplinary, collaborative research program bringing together investigators from several institutions to work across technology and disciplinary boundaries. The group shares a common interest in vascular biology, particularly in the eye, and specifically in the application of modern optical technology to answer critical questions related to vascular biology. The technology platform will be based on the scanning laser ophthalmoscope and the real-time in vivo confocal microscope previously developed at Schepens Eye Research Institute and at the Wellman Laboratories of Photomedicine. The existing technology will be enhanced with new development to improve image resolution, contrast, sensitivity, methods for quantification, and flexibility of imaging in living animals. Specific questions to be addressed include: 1. What are the cellular processes governing normal vascular development and stabilization? 2. What are the factors governing angiogenesis, Iymphangiogenesis, and immune cell trafficking? 3. What are the cellular mechanisms for the development of sickle cell and diabetic retinopathy? 4. Can we visualize early changes in the retinal pigment epithelium noninvasively in vivo? 5. Can we detect circulating cells in vivo without drawing blood? Is the number of circulating tumor cells a good predictor for tumor burden and response to therapy? Imaging at the cellular level will enable biologists to study problems in living animals over time, gaining physiological insights beyond what can be obtained by classic static measurement (histology, immunocytochemistry, etc.), substantially reducing the number of animals required to answer these critical questions.

DESCRIPTION (provided by applicant): The 2002 Gordon Research Conference on Lasers in Medicine and biology will be held from July 14-19, 2002 at Kimball Union Academy in Meriden, New Hampshire. The intent of the conference is to gather investigators from academia, governmental agencies, national laboratories and industry to examine future applications of lasers and optical sciences in medicine and surgery. Particular emphasis is placed on technological advances that are close to clinical application and/or commercial development. Sessions include: Optical Coherence Tomography; Nanotechnologies for Optical Sensing, Imaging, and Manipulation; Biological Applications of Micro-optical Devices; Combined Imaging Methodologies; Fluorescence Methods for Biomedical Diagnostics; Advances in Optical Microscopy; Intra-vital Imaging and Microscopy; Wavefront Sensing and Adaptive Optics in Vision Correction; and Photodynamic Therapy. Seven of the nine sessions include at least one talk with substantial applications in the area of cancer diagnostics. An important focus of the Gordon Conference is to bring students and post-doctoral fellows in biomedical engineering together with academic engineers and scientists, clinicians and members of industry to discuss and define the most important questions and problems to be solved in this field. Ample time between sessions has been schedule to foster informal discussion of new ideas emerging from the conference. In addition to traditional scientific presentations, we have organized two special symposia to facilitate this discussion process. The first symposium, "Career Paths in Biomedical Engineering", will feature short presentations from clinicians and engineers in academia, industry and government to provide career advice to graduate students and post-doctoral fellows. The second symposium, a special industrial forum, will highlight new technologic developments on the cusp of clinical viability. Industrial participants will each make a brief presentation followed by questions and answers. Both symposia will conclude with a general discussion followed by a reception. Finally, a special issue of the Journal of Biomedical Optics will be devoted to Optical Detection: Clinical and Industrial Advances. Conference attendees presenting talks or posters will be invited to submit their work for this special issue.

DESCRIPTION (provided by applicant): Hematopoiesis is the process of blood cell formation from hematopoietic stem cells (HSCs). Adult HSCs reside in a special microenvironment within the bone marrow (BM) called the HSC niche whose anatomical and functional properties are just beginning to be uncovered. The importance of the microenvironment is illustrated by the fact that HSCs taken out of their niche have yet to be successfully expanded ex vivo. While multiple cellular components and soluble factors have been shown to contribute to the establishment of the HSC niche, the central role of oxygen as a critical physiological factor governing cell function cannot be overlooked in considering the biology of the HSC in vivo. The BM is generally considered to be hypoxic (low oxygen tension), but the relationship between local oxygen distribution and the microanatomy of the HSC niche remain poorly defined, in part because of our inability to probe oxygen tension in vivo with cellular resolution. Here we propose to develop molecular imaging technology to enable high-resolution 3D visualization and quantification of local oxygen tension in the BM of live mice. We will use the technology to study if HSC homing and retention in the BM is regulated by local oxygen tension and by hypoxia-inducible factor-1 (HIF-1) dependent cellular response. We will further investigate if anaerobic metabolism is essential for HSC function in vivo. The proposed research requires close collaboration of a multidisciplinary team of investigators with diverse expertise in molecular imaging, optical technology, and stem cell biology. PUBLIC HEALTH RELEVANCE: Narrative Hematopoiesis is the process of blood cell formation from hematopoietic stem cells that reside in the bone marrow. We propose to examine how stem cells respond to oxygen distribution in the bone marrow.

The overarching goal of this project is to forward and backward track cancer cell fates and histories using new imaging approaches to redefine cancer evolution and therapeutic escape on the cellular and molecular level. The project will focus on clonal selection, in order to develop a single unifying hypothesis, which can explain a variety of clinical observations including that of primary tumor cell heterogeneity, the propensity for metastases to develop at specific sites, and the differences in therapeutic responses. Current methodologies for studying in vivo clonal expansion and tumor growth have been hampered by the overwhelming challenge of being able to follow the development of distinct cellular lineages from a single common progenitor. By adopting vanguard technologies such as combinatorial expression of multiple fluorescent proteins (Livet et al., 2007, Nature, 450, 56-62), we will be able to perform fluorescent-based lineage tracing of cancer cells in vivo. The overarching goal for this project is to better understand the clonal fates of both tumor cells and their associated stromal cells. The specific aims are thus: 1) to develop, test and validate multicolor tools and reagents to track multiple clonally derived cellular lineages; 2) to determine whether tumor formation, progression and resistance to therapy are characterized by dominant subclones; and 3) to examine whether tumor cells recruit unique subpopulations of host stromal cells to the growing tumor.

? DESCRIPTION (provided by applicant): This proposal aims to address the provocative question PQC- 2: What molecular or cellular events establish tumor dormancy after treatment and what leads to recurrence? We will address this question by examining the bone marrow (BM), a location known to provide a favorable microenvironment not only for hematologic malignancies, but also for solid tumor cells that frequently seed and persist in this location. These cells can reemerge after prolonged periods of apparent inactivity and become the source of new cancer growth. Understanding how dormancy is regulated is important for developing therapeutic strategies that aim to prevent cancer relapse. Our central hypothesis is that localization is an important determinant of dormancy. Localization of normal hematopoietic stem cells (HSCs) to proper niches in the BM is critical for maintaining quiescence and long-term function of the HSC. Here we propose to investigate the relationship between dormancy and localization of malignant cells in the BM following chemotherapy. We hypothesize that dormancy is maintained by localization of malignant cells in quiescent niches, and subsequent events that alter their localization can trigger the malignant cells to exit their quiescent state. By tracking individual cancer cells expressing a novel cell cycle fluorescent indicator in the mouse BM using intravital microscopy, we will identify the location of quiescent niches where cells are maintained in the G0 state. We will examine the link between quiescence and hypoxia by mapping local oxygen concentration in the BM with high spatial resolution using two-photon phosphorescence lifetime microscopy (2PLM), and identify niche-specific molecular expression by laser microdissection and capturing of niche cells for RNA-seq. Finally, we will examine if genetic or pharmacologic manipulations of the niche components can result in altered cellular localization, changes in cell cycle status, and susceptibility to chemotherapy. If successful, this work can lead to a new way of sensitizing cancer cells to chemotherapy by targeting their localization.

ABSTRACT The balance between hematopoietic stem cell (HSC) self-renewal and differentiation directly impacts hematopoietic homeostasis. We hypothesize that signals from the bone marrow microenvironment (or ?niche?), together with cues from cell-intrinsic networks, contribute to fine-tuning this balance. However, our understanding of the niche has been limited by the current approach relying on sequential deletion of individual regulatory factors from candidate cells in available mouse models, and analysis of individual HSCs and their in vivo interactions with the niche has also been hindered by the heterogeneity of available HSC-enriched fractions and the technical challenges of imaging HSC fate in vivo. To illuminate the behavior of individual HSCs in vivo, we have established a new technical regimen which includes prospective isolation of HSCs with high purity based on Tie2 positivity, a local transplantation technique which delivers a single HSC under multiphoton microscopy guidance into the bone marrow of a live mouse, and micropipette aspiration to extract single cells after division directly from the marrow for transcriptomic assay. Our project will utilize these advances to describe the molecular basis of HSC fate choice in the niche. This in turn will facilitate novel therapeutic strategies for cell- fate manipulation which could accelerate hematopoietic recovery after transplantation, and possibly contribute to improved transplantation efficiency for non-malignant blood diseases. Thus, the goals of this proposal are three-fold: (1) to identify molecular mechanisms which enhance symmetric self-renewing division of HSCs, (2) to understand the niche factors governing HSC division balance, and (3) to assess the HSC niche under non- genotoxic conditioning. If successful, the proposed research will positively impact the HSC field by identifying molecular targets that will improve hematopoietic recovery after transplantation, and enable improvements in the ex vivo engineering of niche models.

? DESCRIPTION (provided by applicant): This proposal aims to address the provocative question PQC- 2: What molecular or cellular events establish tumor dormancy after treatment and what leads to recurrence? We will address this question by examining the bone marrow (BM), a location known to provide a favorable microenvironment not only for hematologic malignancies, but also for solid tumor cells that frequently seed and persist in this location. These cells can reemerge after prolonged periods of apparent inactivity and become the source of new cancer growth. Understanding how dormancy is regulated is important for developing therapeutic strategies that aim to prevent cancer relapse. Our central hypothesis is that localization is an important determinant of dormancy. Localization of normal hematopoietic stem cells (HSCs) to proper niches in the BM is critical for maintaining quiescence and long-term function of the HSC. Here we propose to investigate the relationship between dormancy and localization of malignant cells in the BM following chemotherapy. We hypothesize that dormancy is maintained by localization of malignant cells in quiescent niches, and subsequent events that alter their localization can trigger the malignant cells to exit their quiescent state. By tracking individual cancer cells expressing a novel cell cycle fluorescent indicator in the mouse BM using intravital microscopy, we will identify the location of quiescent niches where cells are maintained in the G0 state. We will examine the link between quiescence and hypoxia by mapping local oxygen concentration in the BM with high spatial resolution using two-photon phosphorescence lifetime microscopy (2PLM), and identify niche-specific molecular expression by laser microdissection and capturing of niche cells for RNA-seq. Finally, we will examine if genetic or pharmacologic manipulations of the niche components can result in altered cellular localization, changes in cell cycle status, and susceptibility to chemotherapy. If successful, this work can lead to a new way of sensitizing cancer cells to chemotherapy by targeting their localization.

The integrity of the lining of the airways relies on multiple functions of its epithelial stem cells. We hypothesize that the airway stem cell behavior is regulated by the cells and the forces that surrounds them, including other epithelial cells, subepithelial mesenchymal cells, and external mechanical forces exerted on the epithelium. How these varied components regulate the behavior of airway progenitor cells is largely unknown. Furthermore, basal stem cell ? niche interactions may explain the increasingly recognized heterogeneity within this progenitor cell population, including regional and tissue-level heterogeneity in basal stem cell behavior. Although progenitor cell ? niche interactions have been described with live imaging in other systems, the constant motion and challenging access of the respiratory system has hampered the development of platforms for live imaging of the airways at high resolution. We combined emerging stem cell biology and live imaging technologies to develop a novel airway explant live imaging platform. We imaged the maintenance and regeneration of the airway epithelium in a mouse airway explant, surrounded by all of the major components of its 3D multicomponent microenvironment, including the ECM and the mesenchyme. We present preliminary data on tracking individual stem cells during regeneration with two photon laser scanning microscopy, including visualization of stem cell differentiation with fluorescent reporters and analysis of ciliated cell function with novel video-rate imaging modalities. To define the airway stem cell microenvironment and to test its role in progenitor cells function, we visualize and putative cellular niche components and test their role by laser and toxin-mediated ablation. We propose to use cellular engraftment followed by application of mechanical forces to check the role of the microenvironment in establishing planar polarity, and to study the relationship of proliferation, migration and differentiation in collective cell migration in the airway epithelium.

The concept of the stem cell niche is central both to the fundamental understanding of how stem cells are regulated by their microenvironment, and to clinical translation that targets the microenvironment for improving therapeutic outcome. The bone marrow (BM), where hematopoietic stem cells (HSCs) reside, is a crowded space packed with a diversity of cell types derived from both hematopoietic and nonhematopoietic precursors. A major challenge in studying the HSC niche has been the difficulty in identifying the rare HSCs and their neighboring cells in the native BM microenvironment. Elegant cell type-specific deletion of molecules critical for HSC maintenance has led to the identification of vascular endothelial cells (ECs) and CXCL12-abundant reticular (CAR) cells as two major cell types of the HSC niche. However, deletion of such factors impacts all ECs and CAR cells that are present throughout the BM, and are therefore not specific in terms of their local impact in the HSC niche. Direct imaging has the potential to uncover which cell types are in close contact with the HSCs, provided that specific markers are available for all cell types involved. As markers for HSCs are now just beginning to emerge, and visualization of minor cell types remains a challenge, the direct imaging approach has not progressed beyond resolving whether HSCs are in proximity to ECs, CAR cells, or bone-lining osteoblasts. Imaging on its own also does not provide the molecular information essential for understanding how the signals from the niche are communicated to the HSCs. We propose that two things are needed for the field to move forward. First, development of an HSC-specific reporter mouse will enable the identification of endogenous stem cells in their native microenvironment without transplantation. Second, development of a method to selectively isolate the cells in close proximity to the HSCs will enable unbiased profiling of cell types and their molecular signatures (for example, by single-cell RNA sequencing) involved in HSC maintenance. We have now taken steps to address both of these needs. First, we have developed (Camargo Lab) a dual genetic strategy in mice that restricts reporter labeling near exclusively to the most quiescent long-term subset of the HSC compartment (LT-HSCs). This reporter line is fully compatible with current intravital imaging approaches in the calvarial BM and enables live animal tracking of native HSCs (Lin Lab) based on the expression of the green fluorescent protein (GFP) alone, without the need for additional markers and without transplantation. In addition, we have developed a technique for micropipette aspiration of single cells and cell clusters directly from the BM under two-photon image guidance, enabling single cell analysis with high spatial definition. Here, we propose to bring the two teams together to work on an integrated approach for marking, isolating and profiling the native HSCs together with their neighboring ?niche cells?, whose cell types will be identified retrospectively from the transcriptome profiles.

The mission of the cellular diagnostics/imaging core is two fold. First, we will provide state-of-the art imaging technology and cell analysis tools to meet the scientific needs of the four projects in this PPG. We will work closely with project investigators to perform quantitative measurements and data analysis, customizing instrumentation and optimizing experimental protocols as necessary. Second, we will pursue new technical innovations that will lead to future scientific advances beyond what can be accomplished with existing technologies. The Core is a unique resource with deep expertise in optical technology, intravital microscopy, instrumentation design and fabrication, as well as image processing and data analysis. We also have a record of productive collaborations with multiple investigators in this PPG, including Dr. Scadden (Project 1), Dr. Nahrendorf (Project 2), and Dr. Swirski (Project 3). Our laboratories are all physically co-located in the same building, making the Core a common meeting place where team members from various laboratories converge, not just to use the facility but also to interact and exchange ideas. To carry out the mission of the Core, we propose the following Specific Aims. In Aim 1, we will work with Projects 1 and 4 to perform clonal analysis of hematopoietic cells based on their expression of multi-color fluorescent proteins. We will expand the multi-color capability of our in vivo flow cytometer, a technology developed in our laboratory for real-time detection and quantification of fluorescent cells in the circulation of live animals without the need to draw blood samples, to enable noninvasive monitoring of the clonal dynamics in the peripheral circulating leukocyte population. In Aim 2, we will work with Project 2 to assess how the bone marrow (BM) vasculature is altered by cardiovascular diseases (CVD). We will measure functional parameters such as blood flow, vascular permeability, vascular reactivity, and trans-endothelial migration, by performing multiphoton imaging of the BM vasculature together with second-harmonic imaging of the extracellular matrix component. We will also work with Project 3 to characterize hematopoietic stem cell (HSC) localization and dynamics in the BM in the settings of MI and atherosclerosis using gene-edited HSCs provided by the Genome Engineering Core. In Aim 3 we propose to develop a new method to enable image-guided laser microdissection and extraction of live cells from the BM for single cell molecular profiling. We will initially focus on capturing BM vascular endothelial cells, as they form a critical component of the HSC niche and also regulate leukocyte trafficking and macromolecular transport. The technique will bridge the existing divide between single-cell analysis on the one hand, which provides molecular but no spatial information, and live imaging on the other, which supplies the 3D spatial context lacking in the molecular analysis. The technique can be extended in future studies to the analysis of other cell types and in other tissues.