Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.     

In Vivo Imaging in Cancer

Imaging has become an indispensable tool in the study of cancer biology and in clinical prognosis and treatment. The rapid advances in high resolution fluorescent imaging at single cell level and MR/PET/CT image registration, combined with new molecular probes of cell types and metabolic states, will allow the physical scales imaged by each to be bridged. This holds the promise of translation of basic science insights at the single cell level to clinical application. In this article, we describe the recent advances in imaging at the macro- and micro-scale and how these advances are synergistic with new imaging agents, reporters, and labeling schemes. Examples of new insights derived from the different scales of imaging and relevant probes are discussed in the context of cancer progression and metastasis.Imaging has become an indispensable tool in cancer research, clinical trials and medical practice. The last three decades have seen an explosive growth in the number and applications of different imaging technologies (Fig. 1). Imaging systems can be grouped by the energy used to derive visual information (X-rays, positrons, photons, sound waves), the spatial resolution attained (macro-, meso-, microscopic), or the type of information obtained (anatomic, physiological, molecular/cellular). Macroscopic imaging systems providing anatomic and physiological information are now in widespread clinical and preclinical use (computed tomography, CT; magnetic resonance imaging, MRI; ultrasound, US), while molecular imaging systems are either in clinical (positron emission tomography, PET; single-photon emission computed tomography, SPECT) or experimental use (fluorescence reflectance imaging, FRI; fluorescence-mediated tomography, FMT; bioluminescence imaging, BLI; laser-scanning confocal microscopy, LSCM; multiphoton microscopy, MPM). Ultimately, it is hoped that some of the molecular imaging systems will allow clinicians to not only see where a tumor is located in the body, but also to visualize the expression and activity of specific molecules (e.g., receptors, protein kinases, proteases), cells (e.g., T cells, macrophages, stem cells), and biological processes (e.g., apoptosis, angiogenesis, metastasis) that influence tumor behavior and/or responsiveness to therapeutic drugs.Open in a separate windowFigure 1.Imaging technologies used in oncology. Several macroscopic imaging technologies (above date line) are in routine clinical use and have advanced tremendously in their capabilities to obtain anatomic and functional information. Microscopic and other intravital optical techniques (below date line) have evolved over the last decade and now allow experimental studies of genetic, molecular, and cellular events in vivo (reproduced with permission from Nature).Perhaps the biggest growth area is fluorescence imaging, with different microscopic and macroscopic technologies being adapted to in vivo use. Indeed, we are on the verge of being able to address some big questions in molecular oncology: How does the molecular machinery of signaling pathways interact in real time; what are the kinetics and flux rates of such networks; what are the differences between networks in malignant cells and normal tissues; can we exploit differences to make less toxic and more efficacious drugs; what are the “hubs” that will translate into most efficient read-outs of cancer development and therapeutic efficacy; and what is the spatial and temporal extent of tumor microenvironments that cause metastasis? In this article, we highlight applications of imaging technologies for breast cancer. Some recent review articles provide more in-depth information on clinical imaging technologies (Neves and Brindle 2006; Torigian et al. 2007) and cellular nanoimaging (Deisseroth et al. 2006; Soon et al. 2007).     

On In Vivo Imaging in Cancer

At any one moment, >500,000 proteins are present within a human cell. Imaging techniques can capture complex molecular mechanisms, including those involved in cancer, in their normal physiological context.There are ∼23,500 genes in every human cell, but it is estimated that >500,000 proteins are present within the cell at any one moment, and 80% of these reside in protein heterocomplexes. Many proteins are altered by posttranslational modifications that impact subcellullar location, protein activity, protein-binding partners, and organellar trafficking. All of this complexity impacts gene expression and cell function. Importantly, many protein interactions arise following cell-to-cell signaling in a tissue-restricted manner and we now understand that protein–protein interactions, signal transduction, and gene expression are context-specific. For example, the functional consequences of a given gene being expressed during development can be quite different from those when the same gene is expressed in the adult, as seen with embryonic genes that are reexpressed in cancer cells (Monk and Holding 2001). Indeed, it can be stated with confidence that cell autonomous genetic changes within an incipient cancer cell in collaboration with alterations in the microenvironment contribute to neoplastic progression. This concept was not always appreciated, but is now widely accepted following the pioneering work of Mina Bissell and others (Novak 2005). The importance of the microenvironment in neoplastic progression is underscored by studies demonstrating that fibroblasts isolated from a tumor stimulate growth of preneoplastic and neoplastic cells in xenograft models. Gene expression patterns in cancer-associated fibroblasts are reminiscent of that found within a wound and include expression of numerous growth factors, chemokines, and angiogenic factors (Bissell and Radisky 2001), which suggests that the inflammatory response plays a key role in tumorigenesis. Similarly, senescent fibroblasts promote preneoplastic cell growth in vitro and in vivo (Pazolli et al. 2009), and the stromal compartment also undergoes age-related changes in mutational load.Thus, there is increasing need for studies of the genetic and molecular basis of cancer to migrate to the whole organism to correctly capture relevant molecular mechanisms in the proper context. Molecular imaging provides one such platform for noninvasive analysis of cancer biology in vivo. This new set of molecular probes, detection technologies, and imaging strategies, collectively termed molecular imaging, now provides researchers and clinicians alike with new opportunities to visualize gene expression, biochemical reactions, signal transduction, protein–protein interactions, regulatory pathways, cell trafficking, and drug action noninvasively and repetitively in their normal physiological context within living organisms in vivo (Singer et al. 2005; Villalobos et al. 2007; Weissleder and Pittet 2008; Dothager et al. 2009).In particular, integration of genetically encoded imaging reporters into live cells and small animal models of cancer has provided powerful tools to monitor cancer-associated molecular, biochemical, and cellular pathways in vivo (Gross and Piwnica-Worms 2005). New animal models combined with imaging techniques (nuclear, fluorescence, and bioluminescence) at both macroscopic and microscopic scales will make it possible to explore the consequences of the interactions between tumor cells and microenvironment during tumor progression and between stromal cells and normal epithelial cells during normal morphogenesis in vivo in real time. Novel injectable agents under development that target key activities may someday enable investigators and clinicians to visualize these processes in patients.Condeelis and Weissleder (2011) review many of the principles and strategies for molecular imaging that will introduce the general reader to this exciting area of context-specific visualization of cancer biology in vivo.     

活体动物体内光学成像技术的研究进展及其应用

活体动物体内光学成像是利用基因改构进行内源性成像试剂或外源性成像试剂标记细胞、蛋白或DNA，从而非侵入性地报告小动物体内的特定生物学事件的技术。活体成像可以直观灵敏地监测基因的表达模式、标记和示踪细胞、探讨蛋白间的相互作用，因而这一技术被广泛地用于分析基因的表达模式、评价基因治疗效果、评估肿瘤的发生和转移、监测移植器官等。简要综述了现有活体动物体内光学成像技术的基本原理、技术进展和相关应用。     

In Vivo Imaging of Human Cerebral Acetylcholinesterase

Abstract: We report here the first positron emission tomography (PET) images showing the in vivo regional distribution of acetylcholinesterase (AChE) in human brain. The study was carried out in eight healthy human volunteers using as a tracer [¹¹C]physostigmine ([¹¹C]PHY), an inhibitor of AChE. After intravenous injection of [¹¹C]PHY, radioactivity was rapidly taken up in brain tissue and reached maximal uptake within a few minutes, following a regional pattern mostly related to cerebral perfusion. After the peak, the cerebral radioactivity gradually decreased with a half-life varying from 20 to 35 min, depending on the brain structure. [¹¹C]PHY retention was higher in regions rich in AChE, such as the striatum (half-life, 35 min), than in regions poor in AChE, such as the cerebral cortex (half-life, 20 min). At later times (25–35 min postinjection), the cerebral distribution of [¹¹C]PHY was typical of AChE activity: putamen-caudate > cerebellum > brainstem > thalamus > cerebral cortex, with a striatal to cortex ratio of 2. These results suggest that PET studies with [¹¹C]PHY can provide in vivo brain mapping of human AChE and are promising for the study of changes in AChE levels associated with neurodegenerative diseases.     

小动物活体成像技术的应用

小动物活体成像技术在国内外得到越来越多的普及应用,极大地促进了生命科学特别是肿瘤研究的发展。本文就小动物活体成像技术的原理、标记方法和实际应用做简单介绍。     

In Vivo Detection of Extrapancreatic Insulin Gene Expression in Diabetic Mice by Bioluminescence Imaging

Background

Extrapancreatic tissues such as liver may serve as potential sources of tissue for generating insulin-producing cells. The dynamics of insulin gene promoter activity in extrapancreatic tissues may be monitored in vivo by bioluminescence-imaging (BLI) of transgenic mice Tg(RIP-luc) expressing the firefly luciferase (luc) under a rat-insulin gene promoter (RIP).

Methods

The Tg(RIP-luc) mice were made diabetic by a single injection of the pancreatic β-cell toxin streptozotocin. Control mice were treated with saline. Mice were subject to serum glucose measurement and bioluminescence imaging daily. On day eight of the treatment, mice were sacrificed and tissues harvested for quantitative luciferase activity measurement, luciferase protein cellular localization, and insulin gene expression analysis.

Results

Streptozotocin-induced diabetic Tg(RIP-luc) mice demonstrated a dramatic decline in the BLI signal intensity in the pancreas and a concomitant progressive increase in the signal intensity in the liver. An average of 5.7 fold increase in the liver signal intensity was detected in the mice that were exposed to hyperglycemia for 8 days. Ex vivo quantitative assays demonstrated a 34-fold induction of the enzyme activity in the liver of streptozotocin-treated mice compared to that of the buffer-treated controls. Luciferase-positive cells with oval-cell-like morphology were detected by immunohistochemistry in the liver samples of diabetic mice, but not in that of non-treated control transgenic mice. Gene expression analyses of liver RNA confirmed an elevated expression of insulin genes in the liver tissue exposed to hyperglycemia.

Conclusions

BLI is a sensitive method for monitoring insulin gene expression in extrapancreatic tissues in vivo. The BLI system may be used for in vivo screening of biological events or pharmacologic activators that have the potential of stimulating the generation of extrapancreatic insulin-producing cells.     

在体生物发光成像技术在生物医学中的应用

在体生物发光成像技术采用萤光素酶基因标记细胞及DNA,利用灵敏的光学检测仪器,实时直观地监测活体小动物体内感染性疾病的发生发展、肿瘤的生长及转移及引发的免疫反应、特定基因的表达等诸多生物过程。通过对同一组实验对象在不同时间点进行记录,跟踪同一观察目标(标记细胞、病原微生物或基因)的移动及变化,与传统的动物实验方法相比,在体生物发光成像得到的数据具有更高的可信度。近年来因其灵敏度较高、不涉及放射性物质、所得结果直观等优势,该技术已普遍应用于生物医学、药物开发等研究领域。     

Mechanical Stress Is a Pro-Inflammatory Stimulus in the Gut: In Vitro,In Vivo and Ex Vivo Evidence

Aims

Inflammatory infiltrates and pro-inflammatory mediators are found increased in obstructive and functional bowel disorders, in which lumen distention is present. However, what caused the low level inflammation is not well known. We tested the hypothesis that lumen distention- associated mechanical stress may induce expression of specific inflammatory mediators in gut smooth muscle.

Methods

Static mechanical stretch (18% elongation) was applied in vitro in primary culture of rat colonic circular smooth muscle cells (RCCSMCs) with a Flexercell FX-4000 Tension Plus System. Mechanical distention in vivo was induced in rats with an obstruction band placed in the distal colon.

Results

In the primary culture of RCCSMCs, we found that static stretch significantly induced mRNA expression of iNOS, IL-6, and MCP-1 in 3 hours by 6.0(±1.4), 2.5(±0.5), and 2.2(±0.5) fold (n = 6∼8, p<0.05), respectively. However, gene expression of TNF-α, IL-1β, and IL-8 was not significantly affected by mechanical stretch. In the in vivo model of colon obstruction, we found that gene expression of iNOS, IL-6, and MCP-1 is also significantly increased in a time-dependent manner in the mechanically distended proximal segment, but not in the sham controls or distal segments. The conditioned medium from the muscle strips of the stretched proximal segment, but not the distal segment or control, significantly induced translocation and phosphorylation of NF-κB p65. This treatment further increased mRNA expression of inflammatory mediators in the naïve cells. However, treatment of the conditioned medium from the proximal segment with neutralizing antibody against rat IL-6 significantly attenuated the activation of NF-κB and gene expression of inflammatory mediators.

Conclusions

Our studies demonstrate that mechanical stress induces gene expression of inflammatory mediators i.e. iNOS, IL-6, and MCP-1 in colonic SMC. Further ex vivo study showed that mechanical stress functions as a pro-inflammatory stimulus in the gut.     

Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo

Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200–250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.     

In Vivo Imaging and Characterization of Actin Microridges

Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo.