Establishment of mouse lung tumor models and development of new therapeutic approaches

Two mouse models of lung cancer were used to investigate cancer progression, cancer treatment, and cancer imaging. One model was established by subcutaneous injection of human adenocarcinoma A549 cells and lewis lung carcinoma (LLC1) cells, the other by intratracheal instillation of LLC1 cells. In the first study, the role of HIF-1 in tumor progression was investigated. Overexpression of HIF-1alpha by genetic alteration of adenocarcinoma cells decreased tumor size, due to decreased proliferation and increased apoptosis, despite an augmented vascularization observed in these tumors. In a further study, tumor regression by immunological approaches was attempted. Hybrids were generated by fusing dendritic cells and syngeneic poorly immunogenic LLC1 cells of C57/BL6 origin. Hybrid immunization induced the expression of a variety of cytokines and the partial host protective immunity against LLC1 tumor challenge. Moreover, hybrid vaccination and adoptive immunotherapy resulted in notable tumor regression. For establishing small animal tumor imaging in our study, three different imaging modalities, micro computed tomography (müCT), multi slice computed tomography (MSCT), and flat panel volumetric computed tomography (fpvCT) were investigated. The müCT images of intrapulmonary tumors suggested that µCT is a reliable and non-destructive method for quantifying the volume of intrapulmonary tumors in the mouse model. In addition, müCT can be used to evaluate tumor angiogenesis. We exploited MSCT and fpvCT for the in vivo imaging and detection of lung nodules in a mouse LLC1 lung tumor model. FpvCT allowed easy monitoring of a lung tumor model with high resolution, facilitating follow-up investigations in cancer research. In addition, the superiority of fpvCT over MSCT was clearly demonstrated. Furthermore, both imaging modalities (müCT and fpvCT), along with fluorescent microspheres, were applied to delineate the relative contribution of blood supply via the pulmonary and the systemic arteries to LLC1 lung tumors. All three methods revealed the pulmonary artery to be the primary functional source for feeding vessels to the lung tumors. Moreover, both modalities demonstrated the microanatomy of the vessels and blood-supplying tissue. The development of experimental mouse lung tumor models is essential to the understanding of tumor pathophysiology and vascular microanatomy. Our findings can be used to identify novel targets for anticancer treatment and for site specific drug targeting. Additionally, the successful employment of various computed tomography systems for lung cancer imaging in rodents offers in vivo evaluation of such strategies.Zur Untersuchung von Tumorwachstum, Tumortherapie und Tumordarstellung wurden zwei verschiedene Lungentumor-Mausmodelle etabliert. Das eine Modell wurde durch die subkutane Injektion von menschlichen Adenokarzinom A549 Zellen oder Lewis-lung-karzinom zellen (LLC1), das andere durch die intratracheale Verabreichung von LLC1 Zellen ausgelöst. In der ersten Studie wurde die Rolle von HIF-1 auf das Tumorwachstum analysiert. Die genetisch verursachte Überexpression von HIF-1alpha in Adenokarzinom-Zellen führte, trotz erhöhter Gefäßbildung in den untersuchten Tumoren, über eine verminderte Proliferation und gesteigerte Apoptose zu einer Verminderung des Tumorwachstums. Eine weitere Untersuchung beschäftigte sich mit einem immunologischen Ansatz der Tumortherapie. Es wurden dendritische Zellen und syngene, schwach immunogene LLC1 Zellen, abgeleitet von C57/BL6 Mäusen, fusioniert. Immunisierung mit diesen Zellhybriden induzierte die Expression verschiedener Zytokine. Desweiteren wurde eine effektive Immunantwort gegen schwach immunogene Tumorzellen und eine gesteigerte T-lymphozytär vermittelte Zerstörung der Tumorzellen beobachtet. Dieser Effekt ließ sich sowohl durch Zellhybrid-Vakzinierung als auch durch die Übertragung von zuvor Zell hybrid-aktivierten T-Lymphozyten herbeiführen. Zur Darstellung der induzierten Maus-Tumore wurden die Methoden der Mikro-Computertomographie (müCT), der Mehrschicht- Computertomographie (MSCT) und der Flachwandvolumen- Computertomographie (fpvCT) angewendet. Die mü;CT Darstellung von intrapulmonalen Tumoren erwies sich als eine geeignete, nicht-destruktive Methode für die Quantifizierung des Tumorvolumens. Außerdem war es mit müCT möglich, die Gefäßversorgung der Tumore darzustellen. Weiterhin haben wir die in vivo Darstellung und Detektion von Lungentumorknoten im LLC1-Lungen Modell mittels MSCT und fpvCT, untersucht. Aufgrund der präzisen Bestimmung der Tumorgröße ermöglicht die fpvCT die Analyse der Tumorprogression in zeitlicher Abfolge. Zusätzlich konnte eine Überlegenheit der fpvCT gegenüber der MSCT nachgewiesen werden. In Ergänzung wurden die Methoden der müCT und fpvCT zur Detektion von Fluoreszenzmikropartikeln für die Differenzierung von pulmonalarteriellen und systemarteriellen Blutflusses hinsichtlich der Versorgung des Tumorgewebes eingesetzt. Der strenge Hinweis, dass die Pulmonalarterien als funktionelle Quelle der Lungentumorgefäßversorgung dienten, konnte von allen drei eingesetzten Methoden erbracht werden. Darüber hinaus war es möglich, die Mikroanatomie der Gefäße sowie blutversorgende Gewebsbrücken darzustellen

Association of Clonal Hematopoiesis of Indeterminate Potential with Inflammatory Gene Expression in Patients with COPD

Chronic obstructive pulmonary disease (COPD) is a disease with an inflammatory pheno type with increasing prevalence in the elderly. Expanded population of mutant blood cells carrying somatic mutations is termed clonal hematopoiesis of indeterminate potential (CHIP). The associ ation between CHIP and COPD and its relevant effects on DNA methylation in aging are mainly unknown. Analyzing the deep-targeted amplicon sequencing from 125 COPD patients, we found enhanced incidence of CHIP mutations (~20%) with a predominance of DNMT3A CHIP-mediated hypomethylation of Phospholipase D Family Member 5 (PLD5), which in turn is positively correlated with increased levels of glycerol phosphocholine, pro-inflammatory cytokines, and deteriorating lung function

Characterization of a murine model of monocrotaline pyrrole-induced acute lung injury

<p>Abstract</p> <p>Background</p> <p>New animal models of chronic pulmonary hypertension in mice are needed. The injection of monocrotaline is an established model of pulmonary hypertension in rats. The aim of this study was to establish a murine model of pulmonary hypertension by injection of the active metabolite, monocrotaline pyrrole.</p> <p>Methods</p> <p>Survival studies, computed tomographic scanning, histology, bronchoalveolar lavage were performed, and arterial blood gases and hemodynamics were measured in animals which received an intravenous injection of different doses of monocrotaline pyrrole.</p> <p>Results</p> <p>Monocrotaline pyrrole induced pulmonary hypertension in Sprague Dawley rats. When injected into mice, monocrotaline pyrrole induced dose-dependant mortality in C57Bl6/N and BALB/c mice (dose range 6–15 mg/kg bodyweight). At a dose of 10 mg/kg bodyweight, mice developed a typical early-phase acute lung injury, characterized by lung edema, neutrophil influx, hypoxemia and reduced lung compliance. In the late phase, monocrotaline pyrrole injection resulted in limited lung fibrosis and no obvious pulmonary hypertension.</p> <p>Conclusion</p> <p>Monocrotaline and monocrotaline pyrrole pneumotoxicity substantially differs between the animal species.</p

Repurposing Thioridazine (TDZ) as an anti-inflammatory agent

Nuclear factor-kB (NF-kB) is a crucial transcription factor in the signal transduction cascade of the inflammatory signaling. Activation of NF-κB depends on the phosphorylation of IκBα by IκB kinase (IKKβ) followed by subsequent ubiquitination and degradation. This leads to the nuclear translocation of the p50- p65 subunits of NF-κB, and further triggers pro-inflammatory cytokine gene expression. Thus, in the need of a more effective therapy for the treatment of inflammatory diseases, specific inhibition of IKKβ represents a rational alternative strategy to the current therapies. A computer-aided drug identification protocol was followed to identify novel IKKβ inhibitors from a database of over 1500 Food and Drug Administration (FDA) drugs. The best scoring compounds were compared with the already known high-potency IKKβ inhibitors for their ability to bind and inhibit IKKβ by evaluating their docking energy. Finally, Thioridazinehydrochloride (TDZ), a potent antipsychotic drug against Schizophrenia was selected and its efficiency in inhibiting IκBα protein degradation and NF-κB activation was experimentally validated. Our study has demonstrated that TDZ blocks IκBα protein degradation and subsequent NF-κB activation to inhibit inflammation. Thus, it is a potential repurposed drug against inflammation

Expression of B-RAF V600E in Type II Pneumocytes Causes Abnormalities in Alveolar Formation, Airspace Enlargement and Tumor Formation in Mice

Growth factor induced signaling cascades are key regulatory elements in tissue development, maintenance and regeneration. Perturbations of these cascades have severe consequences, leading to developmental disorders and neoplastic diseases. As a major function in signal transduction, activating mutations in RAF family kinases are the cause of human tumorigenesis, where B-RAF V600E has been identified as the prevalent mutant. In order to address the oncogenic function of B-RAF V600E, we have generated transgenic mice expressing the activated oncogene specifically in lung alveolar epithelial type II cells. Constitutive expression of B-RAF V600E caused abnormalities in alveolar epithelium formation that led to airspace enlargements. These lung lesions showed signs of tissue remodeling and were often associated with chronic inflammation and low incidence of lung tumors. The inflammatory cell infiltration did not precede the formation of the lung lesions but was rather accompanied with late tumor development. These data support a model where the continuous regenerative process initiated by oncogenic B-RAF-driven alveolar disruption provides a tumor-promoting environment associated with chronic inflammation

Exposomes to exosomes: exosomes as tools to study epigenetic adaptive mechanisms in high-altitude humans

Humans on earth inhabit a wide range of environmental conditions and some environments are more challenging for human survival than others. However, many living beings, including humans, have developed adaptive mechanisms to live in such inhospitable, harsh environments. Among different difficult environments, high-altitude living is especially demanding because of diminished partial pressure of oxygen and resulting chronic hypobaric hypoxia. This results in poor blood oxygenation and reduces aerobic oxidative respiration in the mitochondria, leading to increased reactive oxygen species generation and activation of hypoxia-inducible gene expression. Genetic mechanisms in the adaptation to high altitude is well-studied, but there are only limited studies regarding the role of epigenetic mechanisms. The purpose of this review is to understand the epigenetic mechanisms behind high-altitude adaptive and maladaptive phenotypes. Hypobaric hypoxia is a form of cellular hypoxia, which is similar to the one suffered by critically-ill hypoxemia patients. Thus, understanding the adaptive epigenetic signals operating in in high-altitude adjusted indigenous populations may help in therapeutically modulating signaling pathways in hypoxemia patients by copying the most successful epigenotype. In addition, we have summarized the current information about exosomes in hypoxia research and prospects to use them as diagnostic tools to study the epigenome of high-altitude adapted healthy or maladapted individuals

Macrophage and tumor cell cross-talk is fundamental for lung tumor progression: we need to talk

Regardless of the promising results of certain immune checkpoint blockers, current immunotherapeutics have met a bottleneck concerning response rate, toxicity, and resistance in lung cancer patients. Accumulating evidence forecasts that the crosstalk between tumor and immune cells takes center stage in cancer development by modulating tumor malignancy, immune cell infiltration, and immune evasion in the tumor microenvironment (TME). Cytokines and chemokines secreted by this crosstalk play a major role in cancer development, progression, and therapeutic management. An increased infiltration of Tumor-associated macrophages (TAMs) was observed in most of the human cancers, including lung cancer. In this review, we emphasize the role of cytokines and chemokines in TAM-tumor cell crosstalk in the lung TME. Given the role of cytokines and chemokines in immunomodulation, we propose that TAM-derived cytokines and chemokines govern the cancer-promoting immune responses in the TME and offer a new immunotherapeutic option for lung cancer treatment

Hidden treasures: macrophage long non-coding RNAs in lung cancer progression

Simple Summary: Cancer immunotherapy mainly targets immune system components, such as immune-suppressive networks generated by cancer cells in the tumor microenvironment (TME). Programmed cell death ligand 1, which is a secretory immune-suppressive factor, is released by tumor-associated macrophages (TAMs). The TME also disrupts production of tumor-specific T cells and generates immunosuppressive leukocytes, regulatory T cells, and myeloid-derived suppressor cells. Immune checkpoint inhibitors are effective in various cancers but only in a subset of patients. Non-coding RNAs, such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), are dysregulated in cancer through transcriptional, post-transcriptional, and epigenetic changes and have significant roles in cancer initiation and progression, which depends on deregulation of lncRNA expression. TAM function can be influenced by lncRNAs in various ways. However, our understanding of lncRNA dysregulation and function in cancer remains in the early stage. Abstract: Ever since RNA sequencing of whole genomes and transcriptomes became available, numerous RNA transcripts without having the classic function of encoding proteins have been discovered. Long non-coding RNAs (lncRNAs) with a length greater than 200 nucleotides were considered as “junk” in the beginning, but it has increasingly become clear that lncRNAs have crucial roles in regulating a variety of cellular mechanisms and are often deregulated in several diseases, such as cancer. Lung cancer is the leading cause of cancer-related deaths and has a survival rate of less than 10%. Immune cells infiltrating the tumor microenvironment (TME) have been shown to have a great effect on tumor development with macrophages being the major cell type within the TME. Macrophages can inherit an inflammatory M1 or an anti-inflammatory M2 phenotype. Tumor-associated macrophages, which are predominantly polarized to M2, favor tumor growth, angiogenesis, and metastasis. In this review, we aimed to describe the complex roles and functions of lncRNAs in macrophages and their influence on lung cancer development and progression through the TME

Evaluation of Angiogenesis Using Micro-Computed Tomography in a Xenograft Mouse Model of Lung Cancer

Quantitative evaluation of lung tumor angiogenesis using immunohistochemical techniques has been limited by difficulties in generating reproducible data. To analyze intrapulmonary tumor angiogenesis, we used high-resolution micro-computed tomography (micro-CT) of lung tumors of mice inoculated with mouse Lewis lung carcinoma (LLC1) or human adenocarcinoma (A549) cell lines. The lung vasculature was filled with the radiopaque silicone rubber, Microfil, through the jugular vein (in vivo application) or pulmonary artery (ex vivo application). In addition, human adenocarcinoma lung tumor-bearing mice treated site-specifically with humanized monoclonal antibody (bevacizumab) against vascular endothelial growth factor. Quantitative analysis of lung tumor microvessels imaged with micro-CT showed that more vessels (mainly small, <0.02 mm2) were filled using the in vivo (5.4%) compared with the ex vivo (2.1%) method. Furthermore, bevacizumab-treated lung tumor-bearing mice showed significantly reduced lung tumor volume and lung tumor angiogenesis compared with untreated mice as assessed by micro-CT. Interestingly, microvascularization of mainly the smaller vessels (<0.02 mm2) was reduced after bevacizumab treatment. This observation with micro-CT was nicely correlated with immunohistochemical measurement of microvessels. Therefore, micro-CT is a novel method for investigating lung tumor angiogenesis, and this might be considered as an additional complementary tool for precise quantification of angiogenesis