High-resolution Intravital Imaging Research Articles

Abstract 1463: Intravital discovery of miRNA drivers of human cancer cell directional invasion

Abstract Metastatic cancer cells often use directional ECM cues such as blood vessels or collagen fibers when invading through live tissue. Oncogenic miRNAs have been implicated as key regulators of cancer progression yet the systemic discovery of miRNAs that drive directional cancer cell invasion has not been achieved. Here we describe the first in vivo quantitative whole human miRNAome screen for miRNA drivers of directional cancer cell invasion using an ex ovo avian embryo model of human cancer cell metastasis combined with high resolution intravital imaging. We identified more than twenty novel miRNAs that promote cancer cell invasion during the key rate-limiting step of cancer metastasis, the initiation and expansion of overt metastatic lesions. In silico miRNA target analysis (DIANA, Targetscan) showed significant enrichment in gene targets/pathways that control cancer cell-ECM adhesion and cancer cell contractility. These findings were further confirmed using Affymetrix gene expression analysis that showed significant changes in the expression of genes that regulate actin cytoskeleton rearrangement, cancer cell-ECM interaction and cell surface-receptor signaling. Public cancer gene expression database analysis (Oncoprint) showed that these miRNA-mRNA networks deregulated in several deadly cancers (pancreas, prostate, breast) and negatively correlate with the cancer patient survival. To elucidate potential mechanisms of these pro-metastatic miRNAs, we utilized an intravital imaging approach. In vivo 4D cancer cell tracking revealed that these pro-metastatic miRNAs are required for successful invasion into collagen-rich tissue and for attachment to the outer surface of the vascular wall. Deregulation of these miRNAs led to formation of loose contacts with the vasculature and chaotic, non-directional cancer cell invasion patterns in living tissue. Intravital second-harmonic microscopy analysis showed that inhibition of the expression of these miRNAs blocked the ability of cancer cells to rearrange the disorganized fine collagen fiber network into thicker collagen bundles that guide directional tumor cell invasion. In contrast to parental tumor cells that persistently moved along the collagen bundles, miRNA-deficient tumor cells were observed to transiently associate with multiple collagen fibers without establishing persistent collagen fiber-tumor cell protrusion contacts. Moreover, miRNA-deficient tumor cells failed to attach and protrude along preexisting perivascular collagen bundles, resulting in the complete abrogation of cancer cell/blood vessel co-option. In summary, we identified a novel panel of human miRNAs that are functionally involved in the regulation of directional invasion and metastasis. This work establishes these miRNAs as promising therapeutic targets to block the metastatic spread of lethal cancers. Citation Format: Konstantin V. Stoletov, Lian Willetts, Juan Jovel, Emma Woolner, John D. Lewis. Intravital discovery of miRNA drivers of human cancer cell directional invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1463. doi:10.1158/1538-7445.AM2017-1463

Abstract 3963: Neoadjuvant chemotherapy promotes prometastatic changes in the primary breast tumor microenvironment in mice and humans

Abstract Chemotherapy induces influx of bone marrow-derived proangiogenic Tie2hi monocytes in primary tumors. Tie2hi perivascular macrophages specifically induce the prometastatic Mena isoforms in tumor cells and can assemble specialized microanatomical sites called “tumor microenvironment of metastasis” (TMEM), structures that may serve as doorways for intravasation of tumor cells in mammary tumors. Both TMEM and MenaINV are required for tumor cell intravasation and dissemination. Thus, we hypothesized that chemotherapy may increase the density of TMEM sites and MenaINV-expressing, intravasation-competent tumor cells, resulting in increased tumor cell invasion and metastasis. We studied these potential pro-metastatic effects of chemotherapy in a neoadjuvant setting (NAC) by either administering paclitaxel or a combination of doxorubicin and cyclophosphamide in several mammary carcinoma mouse and human breast cancer models. As expected, chemotherapy delayed tumor growth, yet it significantly increased the recruitment of TMEM-forming, perivascular Tie2hi/Vegfhi macrophages and TMEM density. Using high-resolution multiphoton intravital imaging in live tumor-bearing mice, we observed that paclitaxel also increased the activity of TMEM sites, visualized as endothelial cell tight-junction disruption around TMEM and subsequent intravasation of the migratory cancer cell subpopulation. Indeed, paclitaxel-treated mice have higher numbers of circulating tumor cells, single cell seeding in lungs and incidence and number of micrometastatic foci, all associated with increased TMEM activity, as demonstrated by high-resolution imaging techniques. Tie2 inhibitors reversed paclitaxel-induced pro-metastatic phenotypes without affecting the assembly of TMEM, indicating that Tie2-mediated signaling is required for paclitaxel-mediated cancer cell dissemination via TMEM. Paclitaxel also caused a significant increase in the expression of MenaINV at both the gene and protein levels. Furthermore, paclitaxel treatment in Mena-/- breast tumor-bearing mice resulted in failure to assemble TMEM and to increase circulating-tumor cells and cancer cell metastasis despite the fact that Tie2hi macrophages are attracted to perivascular niches as a result of paclitaxel treatment. This indicated that Mena is involved in the paclitaxel-mediated increase in cancer cell dissemination but not required for Tie2hi macrophage recruitment. These pre-clinical data are further supported by findings from a cohort (N=20) of breast cancer patients, who received pre-operative paclitaxel-based chemotherapy and demonstrated significant increases in TMEM density and MenaINV expression. Together, our data provide solid evidence that NAC leads to metastasis in rodents via TMEM/ MenaINV-mediated mechanisms, and to cancer cell dissemination in certain clinical scenarios in humans. Citation Format: George S. Karagiannis, Jessica Pastoriza, Jeanine Pignatelli, Yarong Wang, Allison S. Harney, David Entenberg, Ved P. Sharma, Emily Xue, Esther Cheng, Timothy M. D'Alfonso, Joan G. Jones, Jesus Anampa, Thomas E. Rohan, Joseph A. Sparano, John S. Condeelis, Maja H. Oktay. Neoadjuvant chemotherapy promotes prometastatic changes in the primary breast tumor microenvironment in mice and humans [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3963. doi:10.1158/1538-7445.AM2017-3963

Radiation therapy primes tumors for nanotherapeutic delivery via macrophage-mediated vascular bursts.

Efficient delivery of therapeutic nanoparticles (TNPs) to tumors is critical in improving efficacy, yet strategies that universally maximize tumoral targeting by TNP modification have been difficult to achieve in the clinic. Instead of focusing on TNP optimization, we show that the tumor microenvironment itself can be therapeutically primed to facilitate accumulation of multiple clinically relevant TNPs. Building on the recent finding that tumor-associated macrophages (TAM) can serve as nanoparticle drug depots, we demonstrate that local tumor irradiation substantially increases TAM relative to tumor cells and, thus, TNP delivery. High-resolution intravital imaging reveals that after radiation, TAM primarily accumulate adjacent to microvasculature, elicit dynamic bursts of extravasation, and subsequently enhance drug uptake in neighboring tumor cells. TAM depletion eliminates otherwise beneficial radiation effects on TNP accumulation and efficacy, and controls with unencapsulated drug show that radiation effects are more pronounced with TNPs. Priming with combined radiation and cyclophosphamide enhances vascular bursting and tumoral TNP concentration, in some cases leading to a sixfold increase of TNP accumulation in the tumor, reaching 6% of the injected dose per gram of tissue. Radiation therapy alters tumors for enhanced TNP delivery in a TAM-dependent fashion, and these observations have implications for the design of next-generation tumor-targeted nanomaterials and clinical trials for adjuvant strategies.

Abstract A069: Quantification of lymph node transit times reveals differences in the trafficking behavior of regulatory versus conventional CD4+ T cells

Abstract Regulatory T cells (Tregs) express the canonical marker Foxp3 and are critical for suppressing the immune response in homeostatic and inflammatory conditions. Despite how widely studied CD4+Foxp3+ Tregs are in mice, their physiologic trafficking and scanning behavior of antigen-presenting cells in secondary lymphoid organs (SLOs) have yet to be quantitatively analyzed in the intravital setting. Our laboratory has previously described the dynamics of T cell trafficking through SLOs and scanning of dendritic cells (DCs), revealing that CD4+ T cells and CD8+ T cells exhibit different transit kinetics through lymph nodes (LNs). On average, CD4+ T cells spend less time scanning for self-peptide/MHC (self-pMHC), dwell shorter in LNs, and egress faster compared to CD8+ T cells. Here we use flow cytometry and 2-photon laser scanning microscopy (2-PLSM) to quantitate the homeostatic trafficking behavior and surveillance strategy of Tregs in LNs and to compare their behavior to conventional T cells (Tconvs). Our data reveal surprising differences in Treg versus Tconv trafficking in mesenteric versus other peripheral LNs. We find that Treg LN transit time is heavily dependent on recognition of self-pMHC in peripheral but not mesenteric LN. Furthermore, we describe a novel imaging platform which enables high resolution intravital imaging of gut SLOs, especially mesenteric LN, for up to 10 hours. Finally, we use intravital 2-PLSM to quantify the contribution of self-pMHC recognition to Treg kinetics and scanning of DCs in peripheral and mesenteric LNs. These results add quantitative data to the emerging paradigm that self-pMHC interactions dynamically control homeostatic Treg retainment and localized suppressive function in peripheral LNs. Citation Format: Alexander Tong. Quantification of lymph node transit times reveals differences in the trafficking behavior of regulatory versus conventional CD4+ T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A069.

Abstract 352: Real-time Intravital Optical Imaging Reflects the Dynamic Changes of Oxidative Stress Induced by Cigarette Smoking in Vasculatures

Background: Reactive oxygen species (ROS) play a central role in cigarette smoking-induced atherogenesis. The present study aims to assess the smoking-induced acute oxidative stress within vasculatures, and evaluates whether the resveratrol, a natural polyphenol antioxidant, can counteract this ROS production, using a customized, high resolution intravital optical imaging in real-time. Methods and Results: 20-week-old male C57BL/6 mice were divided into four groups according to the preceding administration of resveratrol (R) (25mg/kg via gavage, for 7 days) and exposure to cigarette smoke (CS). To in vivo assess acute oxidative stress in blood vessels, dihydroethidium, which forms a red fluorescence (ethidium, excitation/emission: 520nm/610nm) upon reaction with ROS, was injected intraperitoneally. During CS exposure, temporal changes of fluorescence signals from the mouse cremaster muscle including vasculatures were assessed by intravital optical imaging for 15 minutes. Fluorescence signals were much more pronounced in CS exposed mice than controls (p<0.001). Resveratrol p.o. significantly reduced the CS-induced ROS signals compared to the non-treated group (fluorescence signal to noise ratio, SNR, 2.51±0.09 vs. 12.52±2.116, p=0.0002) (Figure A). Without CS exposure, fluorescence signals in targeted vasculatures were very low showing no difference between groups (SNR, 1.65±0.19 vs. 1.53±0.07, p=0.80) (Figure A). Lipid peroxidation was increased in CS group and significantly attenuated in resveratrol-treated mice (Figure B). Fluorescence microscopy and immunostainings corroborated the in vivo findings. Conclusions: The intravital optical imaging was able to in vivo estimate the dynamic changes of ROS production by CS exposure. Our data demonstrated that even a brief exposure to CS increased oxidative stress in vasculatures promptly, and the resveratrol exerts protective effects against the CS-induced acute oxidative stress.

Abstract 17815: The Effects of Nanoparticle-mediated PPARγ Agonist Delivery on Plaque Stabilization: A Multichannel High-resolution Intravital Imaging Analysis

Introduction: Plaque inflammation is associated with fibrous cap destabilization and rupture. Hypothesis: We hypothesized that the specific delivery of a novel PPARγ agonist, lobeglitazone, to inflamed plaques via nanoparticle (NP) targeting mannose receptors on macrophages could reduce inflammation in atheromata and thus, tested it using serial optical intravital molecular imaging. Methods and Results: A novel near-infrared fluorescence (NIRF) probe was fabricated by chemically conjugating thiolated glycol chitosan with mannose-PEG, and NIRF dye Cy5.5 (ext/emi 675/694 nm) for imaging. Lobeglitazone was incorporated into NIRF-NP targeting macrophage mannose receptors (MMR). In vitro, MMR-NIRF-NP was selectively uptaken by RAW264.7 cells, and NIRF signals were estimated to be 10-fold over control group (p<0.01). In ELISA and immunoblot analysis, lobeglitazone-incorporated MMR-NIRF-NP significantly decreased inflammatory cytokines. To assess the therapeutic effects in vivo, macrophage activity in the carotid plaques of apoE-/- mice were serially imaged using a customized multichannel intravital fluorescence microscope (IVFM) under mechanical carotid stabilization. After baseline imaging, lobeglitazone-loaded MMR-NIRF-NPs were intravenously injected (7 mg/kg, twice weekly) and plaque NIRF signals were compared with p.o. treated group and controls. Four weeks later, IVFM at the same plaques revealed that macrophage signals much more decreased in lobeglitazone-loaded NP treated mice compared to those at baseline, and controls (P<0.05). FM and immunostainings corroborated the in vivo findings. Conclusions: Lobeglitazone-incorporated MMR-NIRF-NP was able to selectively target and image atheroma macrophages, and effectively reduce plaque inflammation as assessed by intravital optical imaging. NP-mediated lobeglitazone delivery could be a promising therapeutic strategy for high-risk plaque stabilization.

High-resolution live imaging reveals axon-glia interactions during peripheral nerve injury and repair in zebrafish.

ABSTRACTNeural damage is a devastating outcome of physical trauma. The glia are one of the main effectors of neuronal repair in the nervous system, but the dynamic interactions between peripheral neurons and Schwann cells during injury and regeneration remain incompletely characterized. Here, we combine laser microsurgery, genetic analysis, high-resolution intravital imaging and lattice light-sheet microscopy to study the interaction between Schwann cells and sensory neurons in a zebrafish model of neurotrauma. We found that chronic denervation by neuronal ablation leads to Schwann-cell death, whereas acute denervation by axonal severing does not affect the overall complexity and architecture of the glia. Neuronal-circuit regeneration begins when Schwann cells extend bridging processes to close the injury gap. Regenerating axons grow faster and directionally after the physiological clearing of distal debris by the Schwann cells. This might facilitate circuit repair by ensuring that axons are guided through unoccupied spaces within bands of Büngner towards their original peripheral target. Accordingly, in the absence of Schwann cells, regenerating axons are misrouted, impairing the re-innervation of sensory organs. Our results indicate that regenerating axons use haptotaxis as a directional cue during the reconstitution of a neural circuit. These findings have implications for therapies aimed at neurorepair, which will benefit from preserving the architecture of the peripheral glia during periods of denervation.

High-Resolution Intravital Imaging for Monitoring the Transplanted Islets in Mice

Intravital imaging techniques will be a valuable tool to monitor the post-transplantation dynamics of the cells/tissues in regenerative medicine research. Among the conventional live imaging techniques, the cranial window model has various advantages regarding resolution, longevity, and easy manipulability. We describe the use of the cranial window model to visualize the post-transplantation processes of primary pancreatic islets in the living mouse. Macroscopic or microscopic analyses were performed to evaluate the post-transplantation dynamics of primary murine islets, including the revascularization process inside the cranium. Consistent with earlier literature on clinical outcomes of islet transplantation, marked loss of transplanted islets was observed within 7 days. Intravital confocal microscope analysis revealed that functional revascularization seldom occurred in the central regions of the transplants. Our results suggest that the cranial window model offers an ideal platform for understanding cellular dynamics, through the possibility of long-term imaging studies over time scales. This platform is possibly applied not only for transplant studies of pancreatic islets, but also for other endodermal cell/tissue types in vivo.

High-resolution intravital imaging reveals that blood-derived macrophages but not resident microglia facilitate secondary axonal dieback in traumatic spinal cord injury

After traumatic spinal cord injury, functional deficits increase as axons die back from the center of the lesion and the glial scar forms. Axonal dieback occurs in two phases: an initial axon intrinsic stage that occurs over the first several hours and a secondary phase which takes place over the first few weeks after injury. Here, we examine the secondary phase, which is marked by infiltration of macrophages. Using powerful time-lapse multi-photon imaging, we captured images of interactions between Cx3cr1+/GFP macrophages and microglia and Thy-1YFP axons in a mouse dorsal column crush spinal cord injury model. Over the first few weeks after injury, axonal retraction bulbs within the lesion are static except when axonal fragments are lost by a blebbing mechanism in response to physical contact followed by phagocytosis by mobile Cx3Cr1+/GFP cells. Utilizing a radiation chimera model to distinguish marrow-derived cells from radio-resistant CNS-resident microglia, we determined that the vast majority of accumulated cells in the lesion are derived from the blood and only these are associated with axonal damage. Interestingly, CNS-resident Cx3Cr1+/GFP microglia did not increasingly accumulate nor participate in neuronal destruction in the lesion during this time period. Additionally, we found that the blood-derived cells consisted mainly of singly labeled Ccr2+/RFP macrophages, singly labeled Cx3Cr1+/GFP macrophages and a small population of double-labeled cells. Since all axon destructive events were seen in contact with a Cx3Cr1+/GFP cell, we infer that the CCR2 single positive subset is likely not robustly involved in axonal dieback. Finally, in our model, deletion of CCR2, a chemokine receptor, did not alter the position of axons after dieback. Understanding the in vivo cellular interactions involved in secondary axonal injury may lead to clinical treatment candidates involving modulation of destructive infiltrating blood monocytes.

Intravital FRET Imaging of Tumor Cell Viability and Mitosis during Chemotherapy

Taxanes, such as docetaxel, are microtubule-targeting chemotherapeutics that have been successfully used in the treatment of cancer. Based on data obtained from cell cultures, it is believed that taxanes induce tumor cell death by specifically perturbing mitotic progression. Here, we report on data that suggest that this generally accepted view may be too simplified. We describe a high-resolution intravital imaging method to simultaneously visualize mitotic progression and the onset of apoptosis. To directly compare in vitro and in vivo data, we have visualized the effect of docetaxel on mitotic progression in mouse and human colorectal tumor cell lines both in vitro and in isogenic tumors in mice. We show that docetaxel-induced apoptosis in vitro occurs via mitotic cell death, whereas the vast majority of tumor cells in their natural environment die independent of mitotic defects. This demonstrates that docetaxel exerts its anti-tumor effects in vivo through means other than mitotic perturbation. The differences between in vitro and in vivo mechanisms of action of chemotherapeutics may explain the limited response to many of the anti-mitotic agents that are currently validated in clinical trials. Our data illustrate the requirement and power of our intravital imaging technique to study and validate the mode of action of chemotherapeutic agents in vivo, which will be essential to understand and improve their clinical efficacy.

Abstract 1534: CD163-expressing macrophages constitute a distinct malignancy-promoting phenotype in melanoma facilitating tumor development, progression, and metastasis: Implications for novel anticancer therapy.

Abstract Tumor-infiltrating macrophages are recruited to the tumor microenvironment and believed to play a key role in tumor development, progression, and metastasis by producing growth factors and promoting neoangiogenesis. Here, we show that CD163-expressing macrophages express a distinct gene expression signature capable of facilitating tumor development and progression. This supports our earlier findings showing significant correlation between CD163+ macrophage density, tumor grade, and poor prognosis in patients with melanoma. Selective eradication of CD163-expressing tumor-associated macrophages through receptor-mediated delivery of nanoparticles encapsulating the cytotoxic agent doxorubicin significantly inhibited tumor formation and prolonged survival in an immune-competent conditional mouse model of BrafV600E-induced, Pten-deficient metastatic melanoma. High-resolution dynamic intravital imaging showed specific targeting of a subpopulation of tumor-infiltrating macrophages expressing the scavenger receptor CD163, including Tie2-expressing macrophages recently identified as being highly proangiogenic. In conclusion, our findings identify CD163-expressing macrophages as a distinct malignancy-promoting phenotype and indicate a novel therapeutic target in cancer. Citation Format: Maciej B. Maniecki, Anders Etzerodt, William E. Damsky Jr., Aisha Rafique, David Gonzalez, Lars Dyrskjøt, Holger J. Møller, Marcus W. Bosenberg, Søren K. Moestrup. CD163-expressing macrophages constitute a distinct malignancy-promoting phenotype in melanoma facilitating tumor development, progression, and metastasis: Implications for novel anticancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1534. doi:10.1158/1538-7445.AM2013-1534

Intravital imaging of hair-cell development and regeneration in the zebrafish

Direct videomicroscopic visualization of organ formation and regeneration in toto is a powerful strategy to study cellular processes that often cannot be replicated in vitro. Intravital imaging aims at quantifying changes in tissue architecture or subcellular organization over time during organ development, regeneration or degeneration. A general feature of this approach is its reliance on the optical isolation of defined cell types in the whole animals by transgenic expression of fluorescent markers. Here we describe a simple and robust method to analyze sensory hair-cell development and regeneration in the zebrafish lateral line by high-resolution intravital imaging using laser-scanning confocal microscopy (LSCM) and selective plane illumination microscopy (SPIM). The main advantage of studying hair-cell regeneration in the lateral line is that it occurs throughout the life of the animal, which allows its study in the most natural context. We detail protocols to achieve continuous videomicroscopy for up to 68 hours, enabling direct observation of cellular behavior, which can provide a sensitive assay for the quantitative classification of cellular phenotypes and cell-lineage reconstruction. Modifications to this protocol should facilitate pharmacogenetic assays to identify or validate otoprotective or reparative drugs for future clinical strategies aimed at preserving aural function in humans.

Abstract 1695: Imaging of the interaction of pancreatic cancer and stellate cells during liver metastasis

Abstract Pancreatic stellate cells are involved in fibrosis of pancreatic cancer. An understanding of pancreatic cancer-cell interactions with stellate cells is critical to our ability to develop effective anti-tumor therapeutics for pancreatic cancer. We report here imaging of the interaction of pancreatic cancer cells and pancreatic stellate cells in liver metastasis. Human pancreatic cancer cell lines (XPA1 and MiaPaCa-2) were engineered to express green fluorescent protein (GFP) in the nucleus and red fluorescent protein (RFP) in the cytoplasm. Pancreatic stellate cells, engineered to express RFP, were co-injected with the cancer cells into the spleen of transgenic cyan fluorescent protein (CFP) nude mice. Three hours after splenic injection dual-color pancreatic cancer cells and pancreatic stellate cells were found distributed in the host liver. Seven days after cancer cell-stellate cell co-injection, most pancreatic cancer cells and stellate cells were dead. However, by 28 days after injection, liver metastases were observed in the host CFP nude mice. With the high-resolution intravital imaging afforded by the Olympus FV1000 confocal microscope, the interaction of the dual-colored pancreatic cancer cells and the RFP-expressing pancreatic stellate cells could be clearly imaged in the liver metastasis, suggesting that stellate cells participate in metastasis formation. Our hypothesis is that pancreatic stellate cells form a niche for liver metastasis of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1695. doi:1538-7445.AM2012-1695

High Resolution Intravital Imaging of Subcellular Structures of Mouse Abdominal Organs Using a Microstage Device

Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated ‘microstage’ that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in real-time as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox.

CSM Murray Award Lecture — Functional studies of the Lyme disease spirochete — from molecules to mice1This article is based on a presentation by Dr. George Chaconas at the 61st Annual Meeting of the Canadian Society of Microbiologists in St. John’s, Newfoundland, on 20 June 2011. Dr. Chaconas was the recipient of the CSM Murray Award for Career Achievement.

Lyme borreliosis, also known as Lyme disease, is now the most common vector transmitted disease in the northern hemisphere. It is caused by the spirochete Borrelia burgdorferi and related species. In addition to their clinical importance, these organisms are fascinating to study because of the wide variety of unusual features they possess. Ongoing work in the laboratory in several areas will be described. (1) The segmented genomes contain up to two dozen genetic elements, the majority of which are linear with covalently closed hairpin ends. These linear DNAs also display a very high degree of ongoing genetic rearrangement. Mechanisms for these processes will be described. (2) Persistent infection by Borrelia species requires antigenic variation through a complex DNA rearrangement process at the vlsE locus on the linear plasmid lp28-1. Novel features of this recombination process will be presented. (3) Evidence for a new global regulatory pathway of B. burgdorferi gene expression that is required for pathogenicity will be described. The DEAH box RNA helicase HrpA is involved in this pathway, which may be relevant in other bacteria. (4) The mechanism of B. burgdorferi to effectively disseminate throughout its host is being studied in real time by high resolution intravital imaging in live mice. Recent work will be presented.

Real-time intravital imaging of cancer models

High-resolution intravital imaging (IVM) has proven to be a powerful technique to visualise dynamic processes that are important for tumour progression, such as the interplay between tumour cells and cellular components of the tumour microenvironment. The development of IVM tools, including imaging windows and photo-marking of individual cells, has led to the visualisation of dynamic processes and tracking of individual cells over a time span of days. In order to visualise these dynamic processes, several strategies have been described to develop fluorescent IVM tumour models. Genetic tools to engineer fluorescent tumour cell lines have advanced the applications of cell line-based tumour models to study, for example, changes in behaviour or transcriptional and differentiation state of individual cells in a tumour. In order to study tumour progression, fluorescent genetic mouse models have been engineered that better recapitulate human tumours. These technically challenging tumour models are key in visualising dynamic processes during cancer progression and in the translational aspects of IVM experiments.

Accurate measurement of pancreatic islet β-cell mass using a second-generation fluorescent exendin-4 analog

The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing β-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial β-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in β-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent β-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K(12) position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4(×12)-VT750) had a high binding affinity (~3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to β-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and β-cell mass in mice treated with streptozotocin, a β-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4(×12)-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of β-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts.

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice

The near-infrared photoluminescence intrinsic to semiconducting single-walled carbon nanotubes is ideal for biological imaging owing to the low autofluorescence and deep tissue penetration in the near-infrared region beyond 1 microm. However, biocompatible single-walled carbon nanotubes with high quantum yield have been elusive. Here, we show that sonicating single-walled carbon nanotubes with sodium cholate, followed by surfactant exchange to form phospholipid-polyethylene glycol coated nanotubes, produces in vivo imaging agents that are both bright and biocompatible. The exchange procedure is better than directly sonicating the tubes with the phospholipid-polyethylene glycol, because it results in less damage to the nanotubes and improves the quantum yield. We show whole-animal in vivo imaging using an InGaAs camera in the 1-1.7 microm spectral range by detecting the intrinsic near-infrared photoluminescence of the 'exchange' single-walled carbon nanotubes at a low dose (17 mg l(-1) injected dose). The deep tissue penetration and low autofluorescence background allowed high-resolution intravital microscopy imaging of tumour vessels beneath thick skin.