Yookyung Jung

Distinct bone marrow blood vessels differentially regulate haematopoiesis.

Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.

Comprehensive Evaluation of Peripheral Nerve Regeneration in the Acute Healing Phase Using Tissue Clearing and Optical Microscopy in a Rodent Model

Peripheral nerve injury (PNI), a common injury in both the civilian and military arenas, is usually associated with high healthcare costs and with patients enduring slow recovery times, diminished quality of life, and potential long-term disability. Patients with PNI typically undergo complex interventions but the factors that govern optimal response are not fully characterized. A fundamental understanding of the cellular and tissue-level events in the immediate postoperative period is essential for improving treatment and optimizing repair. Here, we demonstrate a comprehensive imaging approach to evaluate peripheral nerve axonal regeneration in a rodent PNI model using a tissue clearing method to improve depth penetration while preserving neural architecture. Sciatic nerve transaction and end-to-end repair were performed in both wild type and thy-1 GFP rats. The nerves were harvested at time points after repair before undergoing whole mount immunofluorescence staining and tissue clearing. By increasing the optic depth penetration, tissue clearing allowed the visualization and evaluation of Wallerian degeneration and nerve regrowth throughout entire sciatic nerves with subcellular resolution. The tissue clearing protocol did not affect immunofluorescence labeling and no observable decrease in the fluorescence signal was observed. Large-area, high-resolution tissue volumes could be quantified to provide structural and connectivity information not available from current gold-standard approaches for evaluating axonal regeneration following PNI. The results are suggestive of observed behavioral recovery in vivo after neurorrhaphy, providing a method of evaluating axonal regeneration following repair that can serve as an adjunct to current standard outcomes measurements. This study demonstrates that tissue clearing following whole mount immunofluorescence staining enables the complete visualization and quantitative evaluation of axons throughout nerves in a PNI model. The methods developed in this study could advance PNI research allowing both researchers and clinicians to further understand the individual events of axonal degeneration and regeneration on a multifaceted level.

Longitudinal, 3D In Vivo Imaging of Sebaceous Glands by Coherent Anti-Stokes Raman Scattering Microscopy: Normal Function and Response to Cryotherapy

Sebaceous glands perform complex functions, and are centrally involved in the pathogenesis of acne vulgaris. Current techniques for studying sebaceous glands are mostly static in nature, whereas the gland’s main function – excretion of sebum via the holocrine mechanism – can only be evaluated over time. We present a longitudinal, real-time alternative – the in vivo, label-free imaging of sebaceous glands using Coherent Anti-Stokes Raman Scattering (CARS) microscopy, which is used to selectively visualize lipids. In mouse ears, CARS microscopy revealed dynamic changes in sebaceous glands during the holocrine secretion process, as well as in response to damage to the glands caused by cooling. Detailed gland structure, plus the active migration of individual sebocytes and cohorts of sebocytes were measured. Cooling produced characteristic changes in sebocyte structure and migration. This study demonstrates that CARS microscopy is a promising tool for studying the sebaceous gland and its associated disorders in three-dimensions in vivo.

Staged development of long lived TCRαβ Th17 resident memory T cell population to Candida albicans after skin infection

Background: Candida albicans is a dimorphic fungus to which human subjects are exposed early in life, and by adulthood, it is part of the mycobiome of skin and other tissues. Neonatal skin lacks resident memory T (TRM) cells, but in adults the C albicans skin test is a surrogate for immunocompetence. Young adult mice raised under specific pathogen‐free conditions are naive to C albicans and have been shown recently to have an immune system resembling that of neonatal human subjects. Objective: We studied the evolution of the adaptive cutaneous immune response to Candida species. Methods: We examined both human skin T cells and the de novo and memory immune responses in a mouse model of C albicans skin infection. Results: In mice the initial IL‐17–producing cells after C albicans infection were dermal &ggr;&dgr; T cells, but by day 7, &agr;&bgr; TH17 effector T cells were predominant. By day 30, the majority of C albicans–reactive IL‐17–producing T cells were CD4 TRM cells. Intravital microscopy showed that CD4 effector T cells were recruited to the site of primary infection and were highly motile 10 days after infection. Between 30 and 90 days after infection, these CD4 T cells became increasingly sessile, acquired expression of CD69 and CD103, and localized to the papillary dermis. These established TRM cells produced IL‐17 on challenge, whereas motile migratory memory T cells did not. TRM cells rapidly clear an infectious challenge with C albicans more effectively than recirculating T cells, although both populations participate. We found that in normal human skin IL‐17–producing CD4+ TRM cells that responded to C albicans in an MHC class II–restricted fashion could be identified readily. Conclusions: These studies demonstrate that C albicans infection of skin preferentially generates CD4+ IL‐17–producing TRM cells, which mediate durable protective immunity.

Longitudinal, quantitative monitoring of therapeutic response in 3D in vitro tumor models with OCT for high-content therapeutic screening

In vitro three-dimensional models of cancer have the ability to recapitulate many features of tumors found in vivo, including cell-cell and cell-matrix interactions, microenvironments that become hypoxic and acidic, and other barriers to effective therapy. These model tumors can be large, highly complex, heterogeneous, and undergo time-dependent growth and treatment response processes that are difficult to track and quantify using standard imaging tools. Optical coherence tomography is an optical ranging technique that is ideally suited for visualizing, monitoring, and quantifying the growth and treatment response dynamics occurring in these informative model systems. By optimizing both optical coherence tomography and 3D culture systems, it is possible to continuously and non-perturbatively monitor advanced in vitro models without the use of labels over the course of hours and days. In this chapter, we describe approaches and methods for creating and carrying out quantitative therapeutic screens with in vitro 3D cultures using optical coherence tomography to gain insights into therapeutic mechanisms and build more effective treatment regimens.

Transient Alterations of Cutaneous Sensory Nerve Function by Noninvasive Cryolipolysis

Cryolipolysis is a non-invasive, skin cooling treatment for local fat reduction that causes prolonged hypoesthesia over the treated area. We tested the hypothesis that cryolipolysis can attenuate nociception of a range of sensory stimuli, including stimuli that evoke itch. The effects of cryolipolysis on sensory phenomena were evaluated by quantitative sensory testing (QST) in 11 healthy subjects over a period of 56 days. Mechanical and thermal pain thresholds were measured on treated and contralateral untreated (control) flanks. Itch duration was evaluated following histamine iontophoresis. Unmyelinated epidermal nerve fiber and myelinated dermal nerve fiber densities were quantified in skin biopsies from six subjects. Cryolipolysis produced a marked decrease in mechanical and thermal pain sensitivity. Hyposensitivity started between two to seven days after cryolipolysis and persisted for at least thirty-five days post-treatment. Skin biopsies revealed that cryolipolysis decreased epidermal nerve fiber density as well as dermal myelinated nerve fiber density, which persisted throughout the study. In conclusion, cryolipolysis causes significant and prolonged decreases in cutaneous sensitivity. Our data suggest that controlled skin cooling to specifically target cutaneous nerve fibers has the potential to be useful for prolonged relief of cutaneous pain and might have a use as a research tool to isolate and study cutaneous itch-sensing nerves in human skin.

Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT

Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation.

Intravital Imaging of Mouse Bone Marrow: Hemodynamics and Vascular Permeability

The bone marrow is a unique microenvironment where blood cells are produced and released into the circulation. At the top of the blood cell lineage are the hematopoietic stem cells (HSC), which are thought to reside in close association with the bone marrow vascular endothelial cells (Morrison and Scadden, Nature 505:327-334, 2014). Recent efforts at characterizing the HSC niche have prompted us to make close examinations of two distinct types of blood vessel in the bone marrow, the arteriolar vessels originating from arteries and sinusoidal vessels connected to veins. We found the two vessel types to exhibit different vascular permeabilites, hemodynamics, cell trafficking behaviors, and oxygen content (Itkin et al., Nature 532:323-328, 2016; Spencer et al., Nature 508:269-273, 2014). Here, we describe a method to quantitatively measure the permeability and hemodynamics of arterioles and sinusoids in murine calvarial bone marrow using intravital microscopy.

In vivo flow cytometry for blood cell analysis using differential epi-detection of forward scattered light

The present standard of blood cell analysis is an invasive procedure requiring the extraction of patient’s blood, followed by ex-vivo analysis using a flow cytometer or a hemocytometer. We are developing a noninvasive optical technique that alleviates the need for blood extraction. For in-vivo blood analysis we need a high speed, high resolution and high contrast label-free imaging technique. In this proceeding report, we reported a label-free method based on differential epi-detection of forward scattered light, a method inspired by Jerome Mertzs oblique back-illumination microscopy (OBM) (Ford et al, Nat. Meth. 9(12) 2012). The differential epi-detection of forward light gives phase contrast image at diffraction-limited resolution. Unlike reflection confocal microscopy (RCM), which detects only sharp refractive index variation and suffers from speckle noise, this technique is suitable for detection of subtle variation of refractive index in biological tissue and it provides the shape and the size of cells. A custom built high speed electronic detection circuit board produces a real-time differential signal which yields image contrast based on phase gradient in the sample. We recorded blood flow in-vivo at 17.2k lines per second in line scan mode, or 30 frames per second (full frame), or 120 frame per second (quarter frame) in frame scan mode. The image contrast and speed of line scan data recording show the potential of the system for noninvasive blood cell analysis.

Abstract B44: Mapping, targeting, and eliminating therapeutically unresponsive ovarian cancer with high content imaging and photodynamic therapy

Ovarian cancer is the 5th most common cancer among women, in which epithelial ovarian cancer (EOC) is the most common type, accounting for 90% of cases. Currently, the standard treatment for ovarian cancer is surgical debulking followed by chemotherapy. Although initially EOC patients are highly responsive to standard platinum-derived chemotherapy, the majority of cancer patients will eventually relapse and experience chemoresistance. In spite of numerous efforts to improve EOC treatment, the five-year overall survival rate is still disappointing at 30%. It is therefore necessary to examine the fundamental causes of treatment resistance and use this information to design new therapeutic strategies. Recently, the concept of tumor-initiating cells and their supporting tumor microenvironment has been proposed as potential targets for cancer treatment due to their observed chemoresistance and ability to repopulate tumors. These cells are proposed to reside deep within tumors, potentially in oxygen-deprived environments. We recently observed that a light-based treatment modality known as photodynamic therapy (PDT) may be uniquely suited to targeting and eliminating this cellular population. PDT applies a light activated drug (photosensitizer) to selectively target and kill cancer cells through generation of reactive oxygen species (ROS), making it an attractive alternative cancer treatment modality with the potential to overcome adaptive therapeutic resistance. Characteristics of PDT, including the direct destruction of cellular organelles, selective tumor accumulation, and photochemistry-driven tumor killing mechanism give PDT advantages over traditional chemotherapy and radiotherapy. We have found that a lysosome targeted photosensitizer known as EtNBS specifically localizes and accumulates into the acidic and hypoxic tumor microenvironment in 3D cultures to successfully kill otherwise unresponsive cellular populations. EtNBS-mediated PDT is thus a potential modality in treating tumor initiating cells. In addition, the quantitative and label-free optical coherence tomography (OCT) imaging modality has recently been demonstrated to provide real time information regarding tumor tissue structure and therapeutic dynamics, making it a potential tool to study tumors and their associated change during the course of tumor development and treatment. Here, we utilized a 3D in vitro metastatic ovarian cancer culture model, with an oxygen sensor and OCT imaging as a combinational platform to study tumor initiating cells and the effects of their associated tumor microenvironment on therapeutic response under standard and EtNBS-PDT treatment. By mapping tumor response with this integrated platform, this work represents a mechanism- based targeted approach that will hopefully enable the identification of cellular targets to overcome disease relapse and adaptive chemoresistance. Citation Format: Hsin-I Hung, Oliver J. Klein, Yookyung Jung, Kashmira S. Kulkarni, Bo R. Rueda, Rosemary Foster, Conor L. Evans. Mapping, targeting, and eliminating therapeutically unresponsive ovarian cancer with high content imaging and photodynamic therapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B44.