Intact Lung Research Articles

Induction of Human Lung Mast Cell Apoptosis by Granule Permeabilization: A Novel Approach for Targeting Mast Cells.

Mast cells are implicated as detrimental players in inflammatory lung diseases, particularly asthma. Mast cells respond to activating stimuli by releasing a wide panel of pro-inflammatory compounds that can contribute profoundly to the pathology, and there is currently an unmet need for strategies that efficiently ameliorate harmful effects of mast cells under such conditions. Here, we sought to evaluate a novel concept for targeting human lung mast cells, by assessing the possibility of selectively depleting the lung mast cells by induction of apoptosis. For this purpose, we used lysosomotropic agents, i.e., compounds that are known to permeabilize the secretory granules of mast cells, thereby releasing the contents of the granules into the cytosol. Either intact human lung tissue, purified human lung mast cells or mixed populations of human lung cells were incubated with the lysosomotropic agents mefloquine or siramesine, followed by measurement of apoptosis, reactive oxygen species (ROS) production, and release of cytokines. We show that human lung mast cells were highly susceptible to apoptosis induced by this strategy, whereas other cell populations of the lung were largely refractory. Moreover, we demonstrate that apoptosis induced by this mode is dependent on the production of ROS and that the treatment of lung tissue with lysosomotropic agents causes a decrease in the release of pathogenic cytokines. We conclude that selective apoptosis of human lung mast cells can be accomplished by administration of lysosomotropic agents, thus introducing the possibility of using such drugs as novel therapeutics in the treatment of inflammatory lung disorders such as asthma.

High dose irradiation after pleurectomy/decortication or biopsy for pleural mesothelioma treatment

PurposeThe role played by radiation therapy after pleurectomy/decortication or surgical biopsy in malignant pleural mesothelioma is uncertain. We treated patients with accelerated hypofractionated radiotherapy using helical tomotherapy and intensity-modulated arc therapy in an attempt to keep lung toxicity to a minimum. The present study reports the feasibility and toxicity of this approach. Material and MethodsBetween 2008 and 2012, 36 patients with malignant pleural mesothelioma underwent accelerated hypofractionated radiotherapy to the hemithorax after pleurectomy/decortication (19 patients) or biopsy (17 patients). The prescription dose was 25Gy in five fractions over 5 consecutive days. ResultsWe observed three patients with G3 pneumonitis, five cases of grade 2 dyspnea and six cases of grade 2 cough. The median follow-up was 37 months (range: 3–54 months). The median overall survival for patients who underwent pleurectomy/decortication followed by radiotherapy was 21.6 months [95% confidence interval (95% CI): 15.5–24.1] compared to 19.4 months for patients not submitted to surgery. ConclusionTreatment of intact lung with pleural intensity-modulated arc irradiation in malignant pleural mesothelioma patients with malignant pleural mesothelioma proved safe and feasible, with an acceptable rate of pneumonitis. Survival rates were encouraging for both biopsy-only and pleurectomy/decortication groups. We are currently conducting a phase II dose escalation trial in a similar patient setting to prospectively evaluate the impact of radiotherapy on toxicity, disease-free survival and overall survival.

P3.09-001 The Dosimetric Advantages of Intensity Modulated Proton Therapy (IMPT) for Mesothelioma after Pleurectomy/Decortication

Radiotherapy (RT) after surgery for locoregionally advanced mesothelioma is particularly challenging. Recent surgical advances with pleurectomy and decortication techniques are clinically promising, however, applying comprehensive ipsilateral pleural irradiation is technically difficult with two RT-sensitive, intact lungs. With state-of-the-art IMPT, we have successfully treated a 77-year-old woman who has now been disease-free and alive for 7 months after RT (and 18 months since diagnosis), with minimal therapy-related toxicities so far.

42 - Critical Role of Peroxiredoxin 6 in the Repair of Peroxidized Lung Membrane Phospholipids

Peroxidation of membrane lipids represents a severe threat to cellular integrity and its repair is crucial to prevent cell death. Peroxiredoxin 6 (Prdx6), a protein with both GSH peroxidase and phospholipase A2 (PLA2) activities, plays a major role in antioxidant defense of the lung, an organ with very low expression of GPx4.We investigated the role of Prdx6 in the repair of peroxidized cell membranes in intact mouse lungs and isolated pulmonary microvascular endothelial cells (PMVEC) treated with tert-butylhydroperoxide. Lipid peroxidation was evaluated by measurements of: a) thiobarbituric acid reactive substances (TBARS); b) oxidation of diphenyl-1-pyrenylphosphine (DPPP); and c) ferrous- xylenol orange (FOX). The exposure dose in the different models was varied to give a similar increase of about 4-fold over control in lipid peroxidation measurements at end-exposure. Values for lipid peroxidation returned to control levels within 2 h after oxidant removal in wild type (WT) lungs and PMVEC; however,there was essentially no repair in Pxdx6 null lungs or PMVEC. There also was no recovery in lungs and PMVEC that express H26A-Prdx6; this mutation abolishes the phospholipid hydroperoxidase function of Prdx6 but not its ability to reduce H2O2. An intermediate degree of repair was observed with lungs and PMVEC that express either C47S- or D140A-Prdx6; the former mutantion abolishes the peroxidase activity of Prdx6 while the latter abolishes PLA2 activity. Since repair was not seen with Prdx6 knock-out, GPx4 has no role in recovery from peroxidative stress by lung cells while Prdx6 plays a critical role; both the peroxidase and the PLA2 activities of Prdx6 contribute to the recovery process.

MS 06.05 The Use of Adjuvant IMRT after Pleurectomy/Decortication

MS 06.05 The Use of Adjuvant IMRT after Pleurectomy/Decortication

ROCK regulates the intermittent mode of interstitial T cell migration in inflamed lungs

Effector T cell migration through tissues can enable control of infection or mediate inflammatory damage. Nevertheless, the molecular mechanisms that regulate migration of effector T cells within the interstitial space of inflamed lungs are incompletely understood. Here, we show T cell migration in a mouse model of acute lung injury with two-photon imaging of intact lung tissue. Computational analysis indicates that T cells migrate with an intermittent mode, switching between confined and almost straight migration, guided by lung-associated vasculature. Rho-associated protein kinase (ROCK) is required for both high-speed migration and straight motion. By contrast, inhibition of Gαi signaling with pertussis toxin affects speed but not the intermittent migration of lung-infiltrating T cells. Computational modeling shows that an intermittent migration pattern balances both search area and the duration of contacts between T cells and target cells. These data identify that ROCK-dependent intermittent T cell migration regulates tissue-sampling during acute lung injury.

Early Posttreatment Assessment of MRI Perfusion Biomarkers Can Predict Long-Term Response of NSCLC Brain Metastases to SRS: A Longitudinal Analysis

Optimizing patient selection and identification of patients who may not respond to brain metastasis treatment is critical in personalizing therapy. MRI-based imaging biomarkers using perfusion MRI (pMRI) may satisfy this crucial unmet need. The aim of this study is to identify early post-treatment perfusion MRI changes and correlate to long-term outcomes after treatment of lung cancer brain metastases (BM) with stereotactic radiosurgery (SRS). Pre-treatment and post-treatment pMRI within 8 weeks was obtained in patients treated with SRS for an intact non-small cell lung cancer (NSCLC) brain metastasis. Time-dependent leakage (Ktrans), blood plasma volume (Vp), and extravascular extracellular volume (Ve) were calculated on regions of interest delineated on pMRI. Patients were followed longitudinally with serial MRI until death, progression, or intervention (whole brain radiation or surgery). Genotyping was performed on a biopsy of each patient’s primary or metastatic lesion with the Sequenom MassARRAY system. We hypothesize that early post SRS quantitative analysis of perfusion imaging can predict whether or not lung cancer BMs will ultimately respond or fail treatment Fifty-three lesions were included from 41 total patients. All lesions were treated with SRS with median dose 21 Gy (range = 18-22 Gy). Median follow-up after treatment was 11 months. Actuarial local control at 1 year was 85%. Univariate analysis showed that there is a significant difference in post-RT Ktrans standard deviation (STD) between patients with progressive disease (PD) (mean = 0.0317) and without progressive disease (mean = 0.0219) (P = 0.032). We additionally determined a cutoff value for predicting PD and non-PD using Ktrans STD. ROC curve analysis resulted in the area under the curve (AUC) = 0.73, sensitivity = 89%, and specificity = 67%, with a cutoff value 0.017. Pre-treatment Ve was significantly lower in KRAS mutants vs nonmutants (P = 0.0428). Using a median value of 0.1334 as a cutoff, Ve was able to significantly predict KRAS mutant status compared to the Sequenom reference standard (AUC = 0.71, sensitivity = 81.8%, specificity = 61.9%). Post-RT Ktrans STD may be used as an early post treatment (within 8 weeks) imaging biomarker to help predict long-term response of lung BMs to SRS. This can help identify patients who will ultimately fail SRS in a timelier manner and allow for early adjustment in treatment approach. We determine that Ve from pMRI can identify KRAS mutation status with >80% sensitivity. This study demonstrates promising evidence of the utility of early assessment of post-treatment imaging biomarkers. These data are to be validated in another cohort of patients and also in other histologies.

Automated computer-assisted quantitative analysis of intact murine lungs at the alveolar scale.

Using state-of-the-art X-ray tomographic microscopy we can image lung tissue in three dimensions in intact animals down to a micrometer precision. The structural complexity and hierarchical branching scheme of the lung at this level of details, however, renders the extraction of biologically relevant quantities particularly challenging. We have developed a methodology for a detailed description of lung inflation patterns by measuring the size and the local curvature of the parenchymal airspaces. These quantitative tools for morphological and topological analyses were applied to high-resolution murine 3D lung image data, inflated at different pressure levels under immediate post mortem conditions. We show for the first time direct indications of heterogeneous intra-lobar and inter-lobar distension patterns at the alveolar level. Furthermore, we did not find any indication that a cyclic opening-and-collapse (recruitment) of a large number of alveoli takes place.

Biochemical and histological alterations induced by the smoke of allethrin based mosquito coil on mice model

BackgroundMosquito coil (MC) emits insecticide upon burning which provides limited protection against lethal mosquito borne diseases. However, apart from killing the insect, toxicities associated with the inhalation of these insecticides poses severe health hazards. However, the use of MC is increasing day by day in third world countries in particular but, yet to receive enough attention of both policy maker and general public. The current study was aimed to assess the MC smoke induced damage of pulmonary and hepatic tissues along with observing the alterations of several blood biochemical parameters in mice model.MethodsA total of twenty four Swiss albino mice were allowed to inhale the smoke of allethrin based MC at different duration per day for 120 days. By the end of treatment period, blood sample was drawn from each mouse and blood biochemical parameters including alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen(BUN), serum total protein, cholesterol, low density lipoprotein (LDL) and triglyceride (TG) were analyzed. Intact lung and liver were collected for histological analysis using standard protocol.ResultsBiochemical study indicates elevated activity of two hepatic enzymes: ALT (89%), AST (85%), in comparison with the respective control. Increased level of some parameters of lipid profile including cholesterol (36%), LDL (48%) and triglyceride (30%) in smoke inhaled mice is the new finding of this study. On the contrary, the activity of serum total protein and BUN was decreased by 20% and 24%, respectively in inhaled mice. Pulmonary tissue of treated mice shows severe forms of emphysema and hyperplasia, especially in the peripheral region of lung, which is the hallmark of chronic obstructive pulmonary disease (COPD). Histological study of hepatic tissue shows apoptosis mediated damage of hepatocytes along with severe form of necrosis. Infiltration of Inflammatory cells was also observed in both of the organs.ConclusionResults from the present studies suggest that chronic exposure of allethrin based MC is responsible factor for severe health complications such as COPD due to the alterations of the key biochemical parameters of blood and histo-organization of lung and liver.

Early posttreatment assessment of MRI perfusion biomarkers can predict long-term response of lung cancer brain metastases to stereotactic radiosurgery.

Imaging criteria to evaluate the response of brain metastases to stereotactic radiosurgery (SRS) in the early posttreatment period remains a crucial unmet need. The aim of this study is to correlate early (within 12 wk) posttreatment perfusion MRI changes with long-term outcomes after treatment of lung cancer brain metastases with SRS. Pre- and posttreatment perfusion MRI scans were obtained in patients treated with SRS for intact non-small cell lung cancer brain metastases. Time-dependent leakage (Ktrans), blood plasma volume (Vp), and extracellular extravascular volume (Ve) were calculated for each lesion. Patients were followed longitudinally with serial MRI until death, progression, or intervention (whole brain radiation or surgery). We included 53 lesions treated with SRS from 41 total patients. Median follow-up after treatment was 11 months. Actuarial local control at one year was 85%. Univariate analysis demonstrated a significant difference (P = 0.032) in posttreatment Ktrans SD between patients with progressive disease (mean = 0.0317) and without progressive disease (mean = 0.0219). A posttreatment Ktrans SD cutoff value of 0.017 was highly sensitive (89%) for predicting progressive disease and no progressive disease. Early posttreatment volume change was not associated with outcome (P = 0.941). Posttreatment Ktrans SD may be used as an early posttreatment imaging biomarker to help predict long-term response of lung cancer brain metastases to SRS. This can help identify patients who will ultimately fail SRS and allow for timelier adjustment in treatment approach. These data should be prospectively validated in larger patient cohorts and other histologies.

In vivo neutralization of α4 and β7 integrins inhibits eosinophil trafficking and prevents lung injury during tropical pulmonary eosinophilia in mice.

Integrins regulate leukocyte trafficking during homeostasis and inflammatory conditions. However, the role of α4 and β7 integrins in guiding eosinophil transmigration into the lungs during filarial manifestation of Tropical Pulmonary Eosinophilia (TPE) has not been explored. In this study, mice exhibiting TPE manifestations were administered with in vivo neutralizing antibodies against integrins α4 and β7 or their combination and immuno-pathological parameters were evaluated. Results show an intact lung barrier, significantly lower lung inflammation and reduced eosinophil counts in the Bronchoalveolar lavage fluid and lungs of mice receiving anti-α4+ β7 treatment. Reduced eosinophil peroxidase and β-hexosaminidase activity, downregulation of inflammatory genes, lower production of inflammatory lipid intermediates like prostaglandins E2 and D2, leukotriene B4 and cysteinyl leukotrienes were also noted in anti-α4+ β7 treated mice. Reduced accumulation of central memory, effector memory, regulatory T cells and lower production of IL-4, IL-5, and TGF-β were other cardinal features of anti-α4+ β7 treated mice lungs. Flow cytometry-sorted lung eosinophils from anti-α4+ β7 treated mice showed higher apoptotic potential, downregulated anti-apoptotic gene Bcl-2, and exhibited reduced F-actin polymerization and calcium influx as compared to IgG controls. In summary, neutralization of α4+ β7 integrins impairs the transmigration, activation and survival of eosinophils and reduces TPE induced pathology in mice lungs.

The Differential Absorption of a Series of P-Glycoprotein Substrates in Isolated Perfused Lungs from Mdr1a/1b Genetic Knockout Mice can be Attributed to Distinct Physico-Chemical Properties: an Insight into Predicting Transporter-Mediated, Pulmonary Specific Disposition

PurposeTo examine if pulmonary P-glycoprotein (P-gp) is functional in an intact lung; impeding the pulmonary absorption and increasing lung retention of P-gp substrates administered into the airways. Using calculated physico-chemical properties alone build a predictive Quantitative Structure-Activity Relationship (QSAR) model distinguishing whether a substrate’s pulmonary absorption would be limited by P-gp or not.MethodsA panel of 18 P-gp substrates were administered into the airways of an isolated perfused mouse lung (IPML) model derived from Mdr1a/Mdr1b knockout mice. Parallel intestinal absorption studies were performed. Substrate physico-chemical profiling was undertaken. Using multivariate analysis a QSAR model was established.ResultsA subset of P-gp substrates (10/18) displayed pulmonary kinetics influenced by lung P-gp. These substrates possessed distinct physico-chemical properties to those P-gp substrates unaffected by P-gp (8/18). Differential outcomes were not related to different intrinsic P-gp transporter kinetics. In the lung, in contrast to intestine, a higher degree of non-polar character is required of a P-gp substrate before the net effects of efflux become evident. The QSAR predictive model was applied to 129 substrates including eight marketed inhaled drugs, all these inhaled drugs were predicted to display P-gp dependent pulmonary disposition.ConclusionsLung P-gp can affect the pulmonary kinetics of a subset of P-gp substrates. Physico-chemical relationships determining the significance of P-gp to absorption in the lung are different to those operative in the intestine. Our QSAR framework may assist profiling of inhaled drug discovery candidates that are also P-gp substrates. The potential for P-gp mediated pulmonary disposition exists in the clinic.

Endocytic Uptake, Transport and Macromolecular Interactions of Anionic PAMAM Dendrimers within Lung Tissue

PurposePolyamidoamine (PAMAM) dendrimers are a promising class of nanocarrier with applications in both small and large molecule drug delivery. Here we report a comprehensive evaluation of the uptake and transport pathways that contribute to the lung disposition of dendrimers.MethodsAnionic PAMAM dendrimers and control dextran probes were applied to an isolated perfused rat lung (IPRL) model and lung epithelial monolayers. Endocytosis pathways were examined in primary alveolar epithelial cultures by confocal microscopy. Molecular interactions of dendrimers with protein and lipid lung fluid components were studied using small angle neutron scattering (SANS).ResultsDendrimers were absorbed across the intact lung via a passive, size-dependent transport pathway at rates slower than dextrans of similar molecular sizes. SANS investigations of concentration-dependent PAMAM transport in the IPRL confirmed no aggregation of PAMAMs with either albumin or dipalmitoylphosphatidylcholine lung lining fluid components. Distinct endocytic compartments were identified within primary alveolar epithelial cells and their functionality in the rapid uptake of fluorescent dendrimers and model macromolecular probes was confirmed by co-localisation studies.ConclusionsPAMAM dendrimers display favourable lung biocompatibility but modest lung to blood absorption kinetics. These data support the investigation of dendrimer-based carriers for controlled-release drug delivery to the deep lung.

ИНТРАОПЕРАЦИОННАЯ ПРОТЕКТИВНАЯ ИСКУССТВЕННАЯ ВЕНТИЛЯЦИЯ ЛЕГКИХ В АБДОМИНАЛЬНОЙ ХИРУРГИИ

Artificial pulmonary ventilation (APV) is one of the main methods of intensive care, but it differs greatly from natural external respiration and can promote post-operative pulmonary complications (POPC). The objective of the review is to evaluate the efficiency of protective APV in general and its certain components (small respiratory volume, high positive end-expiratory pressure (PEEP), and alveolar mobilization maneuver) in the reduction of risk of POPC in the patients with intact lungs undergoing abdomen surgery. Conclusion. The protective APV in the peri-operative period of abdomen surgery reduces the frequency of post-operative pulmonary complications but provides no impact on mortality. The main component of protective APV is small respiratory volume while using high PEEP and alveolar mobilization maneuver in the open surgery in the non-obese patients is still to be discussed. In laparoscopic surgery and obese patients, it is feasible to combine small respiratory volume, high PEEP and alveolar mobilization maneuver.

Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells.

Inhalation of allergens and pathogens elicits multiple changes in a variety of immune cell types in the lung. Flow cytometry is a powerful technique for quantitative analysis of cell surface proteins on immune cells, but it provides no information on the localization and migration patterns of these cells within the lung. Similarly,chemotaxis assays can be performed to study the potential of cells to respond to chemotactic factors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these aforementioned techniques, the location of individual cell types within the lung can be readily visualized by generating Precision-cut Lung Slices (PCLS), staining them with commercially available, fluorescently tagged antibodies, and visualizing the sections by confocal microscopy. PCLS can be used for both live and fixed lung tissue, and the slices can encompass areas as large as a cross section of an entire lobe. We have used this protocol to successfully visualize the location of a wide variety of cell types in the lung, including distinct types of dendritic cells, macrophages, neutrophils, T cells and B cells, as well as structural cells such as lymphatic, endothelial, and epithelial cells. The ability to visualize cellular interactions, such as those between dendritic cells and T cells, in live, three-dimensional lung tissue, can reveal how cells move within the lung and interact with one another at steady state and during inflammation. Thus, when used in combination with other procedures, such as flow cytometry and quantitative PCR, PCLS can contribute to a comprehensive understanding of cellular events that underlie allergic and inflammatory diseases of the lung.

The Vagus Nerve in Development of Lung Tumor

ObjectiveLung cancer is the leading cause of cancer death worldwide. Its treatment remains largely unsatisfactory, because our knowledge of its underlying mechanisms is limited. In addition to genetic factors, the initiation, development and progression of lung tumors result from a network of interaction among mediators, cytokines, and growth factors derived from various cells. The vagus nerve is closely related to each of these network components, thus must influence the outcome. Here we tested the hypothesis that the vagus nerve protectes the lung from tumor formation and explored its underlying mechanism.MethodsTumors were assessed microscopically by HE staining and macroscopically by directly counting tumor numbers and measuring their sizes in the two lungs in unilaterally vagotomized AJ mice following urethane treatment. Tumors were also dissected from the lung tissue and grinded for FACS analysis to assess myeloid‐derived suppressor cells.ResultsMorphologically, hyperplasia lesions and nodules were similar in the two lungs. However, tumor numbers and sizes (normalized with lung surface area) 20 weeks after urethane treatment were greater in vagotomized (0.068 /mm2 and 21.6 area%) than intact lungs (0.052 /mm2 and 16.0 area%) (n = 13 and n = 8; p < 0.05 and p < 0.01). Similar results were observed at 10 weeks after urethane treatment. In addition, CD11b+Ly6− C++Ly6G− suppressor cells were more in the vagotomized lung than in the intact lung.ConclusionOur data support that the vagus nerve may protect the lung from tumor formation by modulating immune cell expression.Support or Funding InformationFunded by the Pulmonary Fibrosis Foundation

Decellularization of Intact Lung Tissue Through Vasculature and Airways Using Negative and Positive Pressure.

Decellularization allows the production of extracellular matrix (ECM) scaffolds. Here we describe the use of combined positive pressure and negative pressure to drive decellularization reagents into the vasculature and airways, respectively, of structurally intact lungs in order to remove cells and cellular material leaving an intact ECM scaffold.

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell's arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

Real-time imaging of inflation-induced ATP release in the ex vivo rat lung.

Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that regulate diverse processes critical for lung function, including mucociliary clearance, surfactant secretion, and local blood flow. Cellular ATP release is mechanosensitive; however, the impact of physical stimuli on ATP release during breathing has never been tested in intact lungs in real time and remains elusive. In this pilot study, we investigated inflation-induced ATP release in rat lungs ex vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled with simultaneous infrared tissue imaging to identify ATP-releasing sites. With LL solution introduced into air spaces, brief inflation of such edematous lung (1 s, ∼20 cmH2O) induced transient (<30 s) ATP release in a limited number of air-inflated alveolar sacs during their recruitment/opening. Released ATP reached concentrations of ∼10-6 M, relevant for autocrine/paracrine signaling, but it remained spatially restricted to single alveolar sacs or their clusters. ATP release was stimulus dependent: prolonged (100 s) inflation evoked long-lasting ATP release that terminated upon alveoli deflation/derecruitment while cyclic inflation/suction produced cyclic ATP release. With LL introduced into blood vessels, inflation induced transient ATP release in many small patchlike areas the size of alveolar sacs. Findings suggest that inflation induces ATP release in both alveoli and the surrounding blood capillary network; the functional units of ATP release presumably consist of alveolar sacs or their clusters. Our study demonstrates the feasibility of real-time ATP release imaging in ex vivo lungs and provides the first direct evidence of inflation-induced ATP release in lung air spaces and in pulmonary blood capillaries, highlighting the importance of purinergic signaling in lung function.

Abstract 4104: Characterization and validation of an implantable chronic lung imaging window

Abstract Metastatic disease is the major cause of cancer mortality and is responsible for over 1/2 million deaths each year in the U.S. alone. Of the three most common sites of metastasis observed clinically (bone, lung and liver), the most difficult to study using intravital microscopy is the lung; a delicate organ in perpetual motion. Recent advances in intravital microscopy have enabled visualization of the live intact lung, but are limited in the duration over which they can be utilized (hours) and require major invasive surgeries. Both of these limitations reduce their usefulness in the study of single cell events in the lung and are susceptible to introducing artefacts. We report the development of a novel, minimally invasive surgical protocol for the implantation of a long-lasting lung imaging window. Once implanted, this window allows for the repeated, non-invasive visualization of living, breathing lung tissue over a period of two weeks without the need for ventilation, vacuum plates, or gated imaging equipment. Here we characterize and validate the use of this window in living mice. The window is well tolerated, does not cause infection or inflammation, and offers a 4mm view of the left lung. We utilize it to visualize, with single cell resolution, the progression of experimental metastasis from the arrival of a single cell to the formation of micro-metastases. The development of this novel optical window will allow visualization of, with single cell resolution, tumor microenvironments and metastatic progression within the lung over extended periods of time. This approach to imaging live lung tissue has the potential to reveal the mechanisms underlying many diseases affecting the lung as well as the means to directly evaluate responses to therapeutics in in real time and within a single animal. Citation Format: David Entenberg, Allison Harney, Sonia E. Voiculescu, Yarong Wang, Maja Oktay, John Condeelis. Characterization and validation of an implantable chronic lung imaging window. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4104.