Christian Kugler

Downregulation of the TGFβ Pseudoreceptor BAMBI in Non-Small Cell Lung Cancer Enhances TGFβ Signaling and Invasion.

Non-small cell lung cancer (NSCLC) is characterized by early metastasis and has the highest mortality rate among all solid tumors, with the majority of patients diagnosed at an advanced stage where curative therapeutic options are lacking. In this study, we identify a targetable mechanism involving TGFβ elevation that orchestrates tumor progression in this disease. Substantial activation of this pathway was detected in human lung cancer tissues with concomitant downregulation of BAMBI, a negative regulator of the TGFβ signaling pathway. Alterations of epithelial-to-mesenchymal transition (EMT) marker expression were observed in lung cancer samples compared with tumor-free tissues. Distinct alterations in the DNA methylation of the gene regions encoding TGFβ pathway components were detected in NSCLC samples compared with tumor-free lung tissues. In particular, epigenetic silencing of BAMBI was identified as a hallmark of NSCLC. Reconstitution of BAMBI expression in NSCLC cells resulted in a marked reduction of TGFβ-induced EMT, migration, and invasion in vitro, along with reduced tumor burden and tumor growth in vivo In conclusion, our results demonstrate how BAMBI downregulation drives the invasiveness of NSCLC, highlighting TGFβ signaling as a candidate therapeutic target in this setting. Cancer Res; 76(13); 3785-801. ©2016 AACR.

Mechanisms of Cilia-Driven Transport in the Airways in the Absence of Mucus

Airway mucus is thought to be required for the clearance of inhaled particles by mucociliary transport, but this view has recently been challenged. To test if mucus is necessary for cilia-driven particle transport, we removed mucus from murine and human ex vivo airway preparations by thorough rinsing with buffer with or without additional dithiothreitol washing. The transport of particles with diameters of 4.5 μm, 200 nm, and 40 nm and of bacteria was analyzed by video microscopy. Complete removal of mucus was verified by wheat germ agglutinin staining and by scanning electron microscopy. In the absence of mucus, we observed efficient transport of particles and bacteria by direct cilia-mediated propulsion or via fluid flow generated by ciliary beating. Virus-sized particles had the tendency to attach to cilia. Because direct contact of particles with ciliated cells occurs in the absence of mucus, we examined if this direct interaction changes epithelial function. Neither bacteria- nor LPS-induced nuclear translocation of NF-κB p65 in ciliated cells occurred, indicating that mere contact between ciliated cells and bacteria during transport does not activate the epithelium. Attachment of virus-sized particles to cilia could induce mucus release and/or increase the ciliary beat frequency. Our results indicate that cilia-driven transport of particles with various sizes is possible in murine and human airways without the presence of mucus. If mucus-free transport fails, the epithelium can react by releasing mucus or increasing the ciliary beat frequency to maintain particle transport.

Pulmonary Haptoglobin and CD163 Are Functional Immunoregulatory Elements in the Human Lung

Background: The acute-phase protein haptoglobin (Hp) and its receptor CD163 serve as immunomodulators and possess anti-inflammatory besides antioxidant functions. Objectives: To further understand the role of the recently described pulmonary Hp (pHp) and its receptor CD163 in case of inflammation and infection, pHp and CD163 were investigated on mRNA and protein level to gain insight into the cellular events taking place upon stimulation with the inflammatory mediators LPS, Pam3, cytokine IL-6 and dexamethasone, and upon infection with respiratory pathogens (Haemophilus influenzae, Streptococcuspneumoniae and Chlamydia pneumoniae) by use of a human ex vivo tissue culture model and cell cultures of A549 and alveolar epithelial cells type II. In addition, pHp and CD163 expression in COPD and sarcoidosis was assessed. Methods: We conducted experiments using 942 ex vivo cultured lung samples applying immunohistochemistry, immunocytochemistry, in situ hybridization, immunofluorescence, real-time PCR, RT-PCR, slot and Western immunoblot analyses with tissue lysates and culture supernatants as well as ELISA and cytometric bead array analyses. Results: This study describes for the first time the expression, regulation and secretion of pHp and its receptor CD163 in the human lung. The release of soluble mediators from A549 cell line and human monocyte-derived macrophages was observed indicating that Hp differentially activates the release of soluble mediators and major chemoattractants. Conclusions: The findings indicate a native function of pHp and CD163 as functional pulmonary defense elements due to local expression, regulation and secretion during lung infection and as part of the inflammatory immune response of the respiratory system.

Correlation of EGFR expression, gene copy number and clinicopathological status in NSCLC

BackgroundEpidermal Growth Factor Receptor (EGFR) targeting therapies are currently of great relevance for the treatment of lung cancer. For this reason, in addition to mutational analysis immunohistochemistry (IHC) of EGFR in lung cancer has been discussed for the decision making of according therapeutic strategies. The aim of this study was to obtain standardization of EGFR-expression methods for the selection of patients who might benefit of EGFR targeting therapies.MethodsAs a starting point of a broad investigation, aimed at elucidating the expression of EGFR on different biological levels, four EGFR specific antibodies were analyzed concerning potential differences in expression levels by Immunohistochemistry (IHC) and correlated with fluorescence in situ hybridization (FISH) analysis and clinicopathological data. 206 tumor tissues were analyzed in a tissue microarray format employing immunohistochemistry with four different antibodies including Dako PharmDx kit (clone 2-18C9), clone 31G7, clone 2.1E1 and clone SP84 using three different scoring methods. Protein expression was compared to FISH utilizing two different probes.ResultsEGFR protein expression determined by IHC with Dako PharmDx kit, clone 31G7 and clone 2.1E1 (p ≤ 0.05) correlated significantly with both FISH probes independently of the three scoring methods; best correlation is shown for 31G7 using the scoring method that defined EGFR positivity when ≥ 10% of the tumor cells show membranous staining of moderate and severe intensity (p = 0.001).ConclusionOverall, our data show differences in EGFR expression determined by IHC, due to the applied antibody. Highest concordance with FISH is shown for antibody clone 31G7, evaluated with score B (p = 0.001). On this account, this antibody clone might by utilized for standard evaluation of EGFR expression by IHC.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_165

Surgical Technique of Lower Lobe Lung Transplantation

Among patients with end-stage lung disease awaiting lung transplantation, pediatric and small adult patients have a significantly lower chance of getting size-matched pulmonary grafts in time because of the severe scarcity of small donors. It is our strategy to perform lobar lung transplantations in small recipients with restrictive pulmonary disease once their clinical status demands urgent transplantation. Here we describe our surgical technique and discuss the benefits and risks of this procedure.

The tissue is the issue: improved methylome analysis from paraffin-embedded tissues by application of the HOPE technique

Alterations in the DNA methylome are characteristic for numerous diseases and a typical hallmark of cancer. Therefore, DNA methylation is currently under investigation in research labs and has also entered diagnostics. Recently, protocols like the BeadChip technology have become commercially available to study DNA methylation in an array format and semiquantitative fashion. However, it is known that fixation of the sample material with formalin prior to BeadChip analysis can affect the results. In this study we compared the influence of fixation on the outcome of BeadChip analysis. From six patients each a lung cancer tissue sample and a corresponding tumor-free lung tissue sample were collected. The samples were separated into three pieces. One piece of each sample was fixed with formalin, another one by the non-cross-linking HOPE technique (Hepes-glutamic acid buffer mediated Organic solvent Protection Effect). Subsequently, both became paraffin embedded. As a reference, the remaining third piece was cryopreserved. In addition we used three adenocarcinoma cell lines (H838, A549, and H1650) to validate the results from patient tissues. We show that using the HOPE technique instead of formalin largely prevents the introduction of formalin-fixation related artifacts. An ANOVA analysis significantly separated HOPE- and cryopreserved from formalin-fixed samples (FDR<0.05), while differences in the methylation data obtained from HOPE-fixed and cryopreserved material were minor. Consequently, HOPE fixation is superior to formalin fixation if a subsequent BeadChip analysis of paraffin-embedded sample material is intended.

HOPE-Fixation of Lung Tissue Allows Retrospective Proteome and Phosphoproteome Studies

Hepes-glutamic acid buffer-mediated organic solvent protection effect (HOPE)-fixation has been introduced as an alternative to formalin fixation of clinical samples. Beyond preservation of morphological structures for histology, HOPE-fixation was demonstrated to be compatible with recent methods for RNA and DNA sequencing. However, the suitability of HOPE-fixed materials for the inspection of proteomes by mass spectrometry so far remained undefined. This is of particular interest, since proteins constitute a prime resource for drug research and can give valuable insights into the activity status of signaling pathways. In this study, we extracted proteins from human lung tissue and tested HOPE-treated and snap-frozen tissues comparatively by proteome and phosphoproteome analyses. High confident data from accurate mass spectrometry allowed the identification of 2603 proteins and 3036 phosphorylation sites. HOPE-fixation did not hinder the representative extraction of proteins, and investigating their biochemical properties, covered subcellular localizations, and cellular processes revealed no bias caused by the type of fixation. In conclusion, proteome as well as phosphoproteome data of HOPE lung samples were qualitatively equivalent to results obtained from snap-frozen tissues. Thus, HOPE-treated tissues match clinical demands in both histology and retrospective proteome analyses of patient samples by proteomics.

Human alveolar epithelial cells type II are capable of TGFβ-dependent epithelial-mesenchymal-transition and collagen-synthesis

BackgroundThe origin of collagen-producing cells in lung fibrosis is unclear. The involvement of embryonic signaling pathways has been acknowledged and trans-differentiation of epithelial cells is discussed critically. The work presented here investigates the role of TGFB in cytoskeleton remodeling and the expression of Epithelial-Mesenchymal-Transition markers by Alveolar Epithelial Cells Type II and tests the hypothesis if human alveolar epithelial cells are capable of trans-differentiation and production of pro-fibrotic collagen.MethodsPrimary human alveolar epithelial cells type II were extracted from donor tissues and stimulated with TGFβ and a TGFβ-inhibitor. Transcriptome and pathway analyses as well as validation of results on protein level were conducted.ResultsA TGFβ-responsive fingerprint was found and investigated for mutual interactions. Interaction modules exhibited enrichment of genes that favor actin cytoskeleton remodeling, differentiation processes and collagen metabolism. Cross-validation of the TGFβ-responsive fingerprint in an independent IPF dataset revealed overlap of genes and supported the direction of regulated genes and TGFβ-specificity.ConclusionsPrimary human alveolar epithelial cells type II seem undergo a TGFβ-dependent phenotypic change, exhibit differential expression of EMT markers in vitro and acquire the potential to produce collagen.

Lipidomes of lung cancer and tumour-free lung tissues reveal distinct molecular signatures for cancer differentiation, age, inflammation, and pulmonary emphysema

Little is known about the human lung lipidome, its variability in different physiological states, its alterations during carcinogenesis and the development of pulmonary emphysema. We investigated how health status might be mirrored in the lung lipidome. Tissues were sampled for both lipidomic and histological analysis. Using a screening approach, we characterised lipidomes of lung cancer tissues and corresponding tumour-free alveolar tissues. We quantified 311 lipids from 11 classes in 43 tissue samples from 26 patients. Tumour tissues exhibited elevated levels of triacylglycerols and cholesteryl esters, as well as a significantly lower abundance of phosphatidylglycerols, which are typical lung surfactant components. Adenocarcinomas and squamous cell carcinomas were distinguished with high specificity based on lipid panels. Lipidomes of tumour biopsy samples showed clear changes depending on their histology and, in particular, their proportion of active tumour cells and stroma. Partial least squares regression showed correlations between lipid profiles of tumour-free alveolar tissues and the degree of emphysema, inflammation status, and the age of patients. Unsaturated long-chain phosphatidylserines and phosphatidylinositols showed a positive correlation with a worsened emphysema status and ageing. This work provides a resource for the human lung lipidome and a systematic data analysis strategy to link clinical characteristics and histology.

Acute Pulmonary Artery Obstruction as the Primary Manifestation of a Rapidly Growing Intimal Sarcoma in a 54-Year-Old Patient.

Pulmonary artery sarcoma is a rare malignant neoplasm that is often misdiagnosed and most often only recognized postmortem during the autopsy. We present the case of a male patient with a rapidly progressive pulmonary tumor who underwent urgent pneumonectomy for increasing symptoms of chest pain and septic clinical picture. Histological analysis revealed the diagnosis of a pulmonary artery sarcoma. Despite an R1-resection and adjuvant chemotherapy, the patient is in good clinical health and free of tumor relapse 1 year after the surgery.