Evaluation of reverse phase protein array (RPPA)-based pathway-activation profiling in 84 non-small cell lung cancer (NSCLC) cell lines as platform for cancer proteomics and biomarker discovery

AbstractThe reverse phase protein array (RPPA) approach was employed for a quantitative analysis of 71 cancer-relevant proteins and phosphoproteins in 84 non-small cell lung cancer (NSCLC) cell lines and by monitoring the activation state of selected receptor tyrosine kinases, PI3K/AKT and MEK/ERK1/2 signaling, cell cycle control, apoptosis, and DNA damage. Additional information on NSCLC cell lines such as that of transcriptomic data, genomic aberrations, and drug sensitivity was analyzed in the context of proteomic data using supervised and non-supervised approaches for data analysis. First, the unsupervised analysis of proteomic data indicated that proteins clustering closely together reflect well-known signaling modules, e.g. PI3K/AKT- and RAS/RAF/ERK-signaling, cell cycle regulation, and apoptosis. However, mutations of EGFR, ERBB2, RAF, RAS, TP53, and PI3K were found dispersed across different signaling pathway clusters. Merely cell lines with an amplification of EGFR and/or ERBB2 clustered closely together on the proteomic, but not on the transcriptomic level. Secondly, supervised data analysis revealed that sensitivity towards anti-EGFR drugs generally correlated better with high level EGFR phosphorylation than with EGFR abundance itself. High level phosphorylation of RB and high abundance of AURKA were identified as candidates that can potentially predict sensitivity towards the aurora kinase inhibitor VX680. Examples shown demonstrate that the RPPA approach presents a useful platform for targeted proteomics with high potential for biomarker discovery. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge

A genome-wide library of MADM mice for single-cell genetic mosaic analysis

Mosaic analysis with double markers (MADM) offers one approach to visualize and concomitantly manipulate genetically defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage, single-cell morphology and physiology, genomic imprinting phenotypes, and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM can only be applied to 96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond a proof of principle, we apply our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We find striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division

Multidisciplinary Ophthalmic Imaging In Vivo Imaging of a New Indocyanine Green Micelle Formulation in an Animal Model of Laser-Induced Choroidal Neovascularization

METHODS. The ICG was formulated with the nonionic solubilizer and emulsifying agent Kolliphor HS 15 to create ICG/HS 15 to improve the chemical stability and fluorescence efficacy. In vivo imaging was performed in rats that had undergone laser photocoagulation. Retinal uptake and fluorescence intensity of ICG and ICG/HS 15 were compared following intravenous injection of 3 dosages (0.05, 0.1, and 0.15 mg/kg body weight) at 7, 14, and 21 days following laser treatment. Postmortem analysis included histology with frozen sections and flat mounts. RESULTS. Immediately following injection of ICG or ICG/HS 15, a strong fluorescence was visible in the retinal vasculature and at the site of laser lesions. Pixel intensity was higher for ICG/HS 15 compared to conventional ICG at 8 minutes after injection for all different injection days and dosages. Over time, a continuous decrease of the fluorescent signal was observed for up to 60 minutes to baseline level. Flow cytometry data showed an increased uptake of micellar dye of macrophages and endothelial cells. Histology revealed an accumulation of the micellar dye within the laser lesion. CONCLUSIONS. Micelle formulated ICG can be visualized in the retinal vasculature and laserinduced CNV in vivo and ex vivo. Micellar ICG/HS 15 showed in vivo stronger signal intensity when compared to ICG for all tested dosages. Following further investigations, ICG/HS 15 may be evaluated in patients with retinal and choroidal diseases for more refined diagnosis

ESPEN Guideline: Clinical Nutrition in inflammatory bowel disease

Introduction: The ESPEN guideline presents a multidisciplinary focus on clinical nutrition in inflammatory bowel disease (IBD). Methodology: The guideline is based on extensive systematic review of the literature, but relies on expert opinion when objective data were lacking or inconclusive. The conclusions and 64 recommendations have been subject to full peer review and a Delphi process in which uniformly positive responses (agree or strongly agree) were required. Results: IBD is increasingly common and potential dietary factors in its aetiology are briefly reviewed. Malnutrition is highly prevalent in IBD – especially in Crohn's disease. Increased energy and protein requirements are observed in some patients. The management of malnu-trition in IBD is considered within the general context of support for malnourished patients. Treatment of iron deficiency (parenterally if necessary) is strongly recommended. Routine provision of a special diet in IBD is not however supported. Parenteral nutrition is indicated only when enteral nutrition has failed or is impossible. The recommended perioperative man-agement of patients with IBD undergoing surgery accords with general ESPEN guidance for patients having abdominal surgery. Probiotics may be helpful in UC but not Crohn's disease. Primary therapy using nutrition to treat IBD is not supported in ulcerative colitis, but is mod-erately well supported in Crohn's disease, especially in children where the adverse conse-quences of steroid therapy are proportionally greater. However, exclusion diets are generally not recommended and there is little evidence to support any particular formula feed when nutritional regimens are constructed. Conclusions: Available objective data to guide nutritional support and primary nutritional therapy in IBD are presented as 64 recommendations, of which 9 are very strong recom-mendations (grade A), 22 are strong recommendations (grade B) and 12 are based only on sparse evidence (grade 0); 21 recommendations are good practice points (GPP)

Biomarkeridentifikation und Analyse von Signalweg-Aktivitätsmustern bei Brustkrebs mittels Protein Microarrays

Breast cancer, the most frequent cancer entity among women, is nowadays widely recognized as a heterogeneous disease in terms of histopathology as well as on the molecular level. Over the last few years, gene expression profiling studies have improved our understanding of the underlying molecular mechanisms associated with the very heterogeneous outcomes of breast cancer patients. The existence of intrinsic molecular subtypes, which are linked to unique biological and prognostic features, was repeatedly demonstrated and points to the need of tailored therapy options. However on the functional level, breast cancer is not only a genomic but mainly a proteomic disease and gene expression profiling might provide only limited insights. Following the hypothesis that intrinsic biologic features of breast tumors affect prognosis and also therapy response, the general aim of this thesis was to further explore breast cancer heterogeneity with protein microarrays on the functional proteomics level. Around 70 – 80% of all breast cancer patients belong to the luminal intrinsic molecular subtype, characterized as a surrogate marker by overexpression of hormone receptors. An improved classification of this subtype is crucial for therapy decision as part of the patients are at higher risk of recurrence requiring chemo-endocrine treatment, whereas the other part is at lower risk and does not benefit from chemotherapy. However, accurate definition of low and high risk hormone receptor-positive breast cancer has remained a challenge so far. Thus, the first objective of this thesis was the identification of a robust and quantitative protein biomarker signature to facilitate risk classification of hormone receptor-positive breast cancer. To approach this aim reverse phase protein arrays were used to screen across over 120 breast cancer relevant proteins and a novel bioinformatics workflow for biomarker hit selection was applied. Using this approach, a biomarker signature consisting of caveolin-1, NDKA, RPS6, and Ki-67, was identified as most promising to distinguish between low and high risk hormone receptor-positive breast cancer. Since genomic and transcriptomic profiling alone cannot sufficiently predict protein pathway activation, it is important to explore and define the heterogeneity of hormone receptor-positive breast cancer on the proteome level. Especially as protein signaling pathways present the direct targets of new classes of therapeutics. Thus, the second thesis objective addressed the question of whether hormone receptor-positive breast cancer can be further categorized according to similar signaling pathway activation patterns and whether these patterns reflect common molecular mechanisms. Therefore, comprehensive protein pathway activation profiles of breast cancer specimens were obtained using reverse phase protein arrays. To complement this analysis, a microspot immunoassay was developed, which enabled the simultaneous quantification of eight different growth factors in tumor lysate as well as blood plasma of matching patient samples. Four subgroups were identified, based on differential expression of 90 cancer-relevant signaling proteins. Each subgroup showed unique characteristics which were also related to established clinicopathological features as well as growth factor expression. One subgroup, for example, was characterized by high expression levels of almost all analyzed proteins. In addition, VEGF tumor lysate levels were significantly higher in this subgroup and an enrichment of poorly differentiated tumors was observed underlining the aggressive phenotype. In contrast, another subgroup was characterized by weak signaling activity. Interestingly, this subgroup was mostly associated with invasive lobular carcinoma, the second most common histologic type of breast cancer, reflecting a link between histopathology and underlying molecular mechanisms. In summary, the reverse phase protein array based pathway activation profiling of hormone receptor-positive breast cancer, presented in this thesis, provides a comprehensive snapshot of the heterogeneity of this subtype on the proteomic level. Insights obtained can serve as basis to refine the concept of clinically relevant subtypes towards an improved definition of patient-tailored therapy options.Brustkrebs ist die häufigste Krebsart bei Frauen und wird mittlerweile als heterogene Erkrankung wahrgenommen. Diese Heterogenität spiegelt sich in der Histopathologie wie auch auf molekularer Ebene wieder. In den letzten Jahren haben vor allem Genexpressionsstudien zum besseren Verständnis der molekularen Mechanismen, die für die unterschiedlichen Krankheitsverläufe der Patientinnen verantwortlich sind, beigetragen. Die Existenz von intrinsischen molekularen Subtypen konnte wiederholt bestätigt werden. Die unterschiedlichen biologischen und prognostische Eigenschaften dieser molekularen Subtypen deuten auf die Notwendigkeit von maßgeschneiderten Therapien hin. Jedoch ist Brustkrebs auf funktionaler Ebene vor allem eine proteomische Erkrankung. Genexpressionsstudien können daher wahrscheinlich nur limitierte Einblicke bieten. Basierend auf der Hypothese, dass intrinsische biologische Eigenschaften der Tumore die Prognose sowie das Therapieansprechen beeinflussen, war das allgemeine Ziel dieser Dissertation, die Heterogenität von Brustkrebs auf funktionaler Proteome Ebene mittels Protein-Microarrays, besser zu verstehen. Die Merheit aller Brustkrebs Patientinnen wird mit einem Tumor des luminalen Subtyps diagnostiziert, welcher hauptsächlich durch eine Überexpression von Hormonrezeptoren charakerisiert ist. Eine weitere Klassifizierung dieses Subtyps ist äußerst wichtig, um die Therapieentscheidung zu unterstützen. Bei Patientinnen mit einem hohen Rückfallrisiko ist zusätzlich zu einer antihormonellen Behandlung eine Chemotherapie indiziert, wohingegen Patientinnen mit einem niedrigen Rückfallriskio nicht von einer Chemotherapie profitieren. Jedoch stellt die präzise Unterscheidung zwischen niedrigem und hohem Rückfallrisiko zur Zeit noch eine Herausforderung dar. Der Fokus dieser Dissertation lag daher zunächst auf der Identifizierung einer robusten und quantitativen Biomarker Signatur, die zur verbesserten Risikoklassifizierung bei Hormonrezeptor-positivem Brustkrebs beitragen kann. Reverse Phase Protein-Arrays wurden verwendet, um in Kombination mit einem neu entwickelten Biomarker-Identifikationsprozess die optimale Biomarker Signatur aus über 120 brustkrebsrelevanten Proteinen zu bestimmen. Mit diesem Vorgehen konnte eine Biomarker Signatur, bestehend aus Caveolin-1, RPS6, NDKA und Ki-67, als am vielversprechendsten identifiziert werden, um zwischen einem niedrigen und hohen Rückfallrisiko zu unterscheiden. Der zweite Teil dieser Dissertation befasste sich mit der Fragestellung, ob Hormonrezeptor-positiver Brustkrebs anhand von ähnlichen Signaltransduktionsaktivitätsmustern weiter stratifiziert werden kann und ob diese Aktivitätsmuster gemeinsame molekulare Mechanismen wiederspiegeln. Es wurden umfassende Proteinaktivitätsprofile von Tumoren mittels Reverse Phase Protein-Arrays erstellt. Um die Analyse zu erweitern, wurde zusätzlich ein Microspot-Immunoassay entwickelt, welcher die parallele Quantifizierung von acht unterschiedlichen Wachstumsfaktoren in Tumor- und Blutplasmaproben ermöglichte. Anhand der Analyse von 90 verschiedenen krebsrelevanten Signalproteinen konnten vier unterschiedliche Gruppen identifiziert werden. Jede Gruppe zeigte charakteristische Signaltransduktionsaktivitätsmuster. Desweiteren konnten diese vier Gruppen mit etablierten pathologischen Faktoren sowie mit der Expression von Wachstumsfaktoren in Verbindung gebracht werden. Eine Gruppe zeigte zum Beispiel eine stark erhöhte Signalaktivität bei einer Vielzahl der analysierten Proteine. In dieser Gruppe konnte zusätzlich eine signifikant erhöhte VEGF Konzentration sowie eine Anreicherung von schlecht differenzierten Tumoren beobachtet werden, wodurch der aggressive Phänotyp noch weiter bestärkt wurde. Im Gegensatz dazu, zeigte eine andere Gruppe eher schwache Signalaktivität für eine Vielzahl der analysierten Proteine. Interessanterweise wurde in dieser Gruppe ein hoher Anteil an Tumoren vom Typ des invasiven lobulären Karzinoms beobachet. Diese Beobachtung macht die Verbindung von histophatologischen und molekularen Eigenschaften deutlich. Die durchgeführte Analyse der Signaltransduktions-aktivitätsmuster bei Hormonrezeptor-positivem Brustkrebs zeigt die Heterogenität dieses Subtyps auf Proteome Ebene. Die neu gewonnenen Einblicke können als Grundlage für die Verbesserung der Definition von klinisch relevanten Subtypen dienen und zur Entwicklung von personalisierten Therapieansätzen beitragen