Evaluation of protein microarray technology for tumor autoantibody screening in colon cancer

Dickdarmkrebs ist die dritthäufigste Krebserkrankung weltweit, mit zunehmender Häufigkeit an Neuerkrankungen in Industrieländern. Die Progression der Erkrankung erstreckt sich über mehrere Jahre, Früherkennung und Diagnose innerhalb eines bevölkerungsweiten Screenings haben die Überlebensrate beträchtlich gesteigert. Da die üblichen klinischen Screening-Verfahren wie fäkaler okkulter Bluttest (FOBT) geringe Sensitivität aufweisen bzw. Koloskopie eine invasive und für den Patient unangenehme Methode darstellen, ist die Entwicklung von serumbasierten minimal invasiven Methoden von hohem Interesse. Das Konzept von spezifischen molekularen Signaturen in unterschiedlichen Phasen der Tumorgenese und die sich daraus ergebenden tumorassoziierten Antigenen können voraussichtlich als Biomarker in der Krebsdiagnose fungieren. Tumorassoziierte Antigene wie z.B.: CEA und CA 19.9 finden schon klinische Anwendung, zeigen jedoch niedrige Sensitivität und Spezifität. Außerdem zeigen Studien, dass die Detektion von Krebserkrankungen mit Hilfe eines Tumorbiomarkerpanels höhere Sensitivität und Selektivität zeigt als bei Verwendung einzelner individueller Biomarker. Es ist bekannt, dass abnorme Expression von Proteinen innerhalb von Tumoren antigenische Eigenschaften aufweist. Diese werden durch das Immunsystem erkannt und draus folgend Tumorautoantikörper (TAA) gebildet. In Kombination mit Protein-Microarray Technologie sind TAAs ein vielversprechender Ansatz für die Entdeckung von Tumorbiomarkern. Jedoch auf Grund der komplexen Natur von Proteinen, zeigen Experimente mit Protein-Microarrays eine niedrige Reproduzierbarkeit, verglichen mit DNA-Microoarrays, und bedürfen daher sorgfältiger Optimierung. Diese Arbeit präsentiert ein Tumor-Autoantibody Screening von Dickdarmkrebsproben (gesunder Kontrollgruppe, Darmpolypen mit niedrigem Risiko Darmpolypen, Darmpolypen mit hohem Risiko und Dickdarmkarzinom) mittels Protein-Microarrays bestehend aus mehreren tausend Proteinen. Ziel ist es spezifische TAAs zu identifizieren, um ein Kandidatenmarker-Array herstellen und dieses mit einem größeren Probenset zu validieren. Aufgrund der niedrigen Effizienz des konventionellen Assay-Protokolls wurden einige Optimierungen vorgenommen, um so eine standardisierte Arbeitsvorschrift für den Umgang mit den vorliegenden Protein-Microarrays zu erstellen. Wichtige Aspekte bei der Prozessierung der Protein-Microarrays wurden berücksichtigt und auf mögliche Alternativen getestet, wie beispielsweise Protein-Microarray Oberflächen, Blockier- und Puffer chemikalien und experimentelle Bedingungen. Am wichtigsten war es, die Verwendbarkeit von aufgereinigtem IgG für das Tumorautiantikörper Screening zu testen. Letztendlich wurden einige Veränderungen des Protokolls vorgenommen: Probentestung in rotierenden Kammern bevorzugt gegenüber statischer Feuchtigkeitskammer, Verlängerung der Probentestzeit von zwei auf vier Stunden, Zusatz von Milchpulver zu den Proben, Optimierung der Verdünnung des Detektions-Antikörpers, Ersetzen des Detergenz Tween20 mit Triton X-100 im Puffer und Verwendung von aufgereinigtem IgG besser als Serum. Die Leistung des Tumorantikörper-Kandidatscreenings konnte signifikant, im Bezug auf Sensitivitäten und Spezifizitäten, gegenüber früheren Screenings verbessert werden. Eine Steigerung von ca. 54% auf 97% für die Unterscheidung zwischen Patienten gegen Kontrollen wurde unter der Verwendung von 25 greedy-pairs gene selection für statistische Klassenvorhersage erreicht. Mit diesem verbesserten Protokoll wurden mittels class prediction analyses 632 Klassifikatorklone, welche 593 Gene repräsentieren, als die am besten vorhersagenden TAAs ausgewählt. Zusätzlich wurden weitere 100 Gene aus publizierten Tumor-Antikörper Screenings mittel Protein-Microarrays in Dickdarmkrebs herangezogen. Die insgesamt 732 Klone werden auf einem Kandidatenmarker-Array zusammengefasst und in der Zukunft für eine Leistungsvalidierung des Arrays mit 384 Proben (unterteilt in 4 Probengruppen) verwendet.Colorectal cancer is the third ranking cancer type worldwide with increasing incidences in developed countries. Progression of the disease takes several years and early detection and diagnosis following population based screenings has increased survival rate considerably. The screening methods in clinical application such as faecal occult blood test (FOBT) lack sensitivity and colonoscopy is unpleasant and invasive. Therefore, serum based minimal invasive methods are in great demand. The concepts of specific molecular signatures in the different stages of tumorigenesis, and from there generation of tumor-associated antigens are highly anticipated as biomarkers for applications in diagnostics. Tumor associated antigens such as CEA and CA 19.9 are in clinical applications, but exhibit low sensitivity and specificity. Moreover, many studies have presented that panel of tumor biomarkers show higher sensitivity and specificity in detecting cancer than individual biomarkers. It is recognized that abnormal expression of proteins in tumors exhibits antigenic ability and are recognized by the immune system, consequently producing tumor autoantibodies (TAA). TAAs in combination with protein microarray technology are a promising approach for tumor biomarker discovery. However, due to the complex nature of proteins, protein microarray experiments have low reproducibility and require careful optimization. This thesis presents screening for tumor autoantibodies using colon cancer plasma samples of healthy controls, low risk polyps, high risk polyps and colon carcinoma groups utilizing protein microarrays containing several thousand proteins, aiming at identifying specific TAAs for generating a candidate marker array for subsequent validation in larger sample set. Due to the low performance of conventional assay protocols, several optimizations were carried out to establish a standard operating procedure for the particular type of protein microarray. Several aspects important for protein microarray processing were addressed and tested for the possible alternatives, such as protein microarray surfaces, blocking and buffer chemistries, as well as reaction conditions of the assays. Most importantly, the possibility of using purified IgG for tumor autoantibody marker screening was tested. As a result, several changes to the protocol were made: probing in rotating chambers rather than horizontal humidity chambers, extension of sample incubation from 2 hours to 4 hours, addition of milk powder to samples, and optimization of detection antibody dilutions, replacement of the detergent Tween20 with Triton X-100 in buffers and using purified IgG of samples rather than serum. Tumor autoantibody candidate marker screening performance could be significantly improved from previous screening, with respect to sensitivities and specificities, increasing from ~54% to 97% for distinguishing patients versus controls using 25 greedy-pairs gene selection criteria for class prediction. With this improved protocol, 632 classifier clones representing 593 genes were deduced from class prediction analyses as the most predictive TAAs. Additionally, 100 genes were selected from published literature on screening for tumor autoantibodies in colorectal cancer using protein microarray technology. This total of 732 clones will comprise the candidate marker array and will be applied in future studies for validation with 384 samples of 4 sample groups: healthy controls, low risk polyps, high risk polyps and colon carcinoma

Evaluation of auto-antibody serum biomarkers for breast cancer screening and in silico analysis of sero-reactive proteins

Aberrantly expressed proteins in tumours evoke an immunological response. These immunogenic proteins can serve as potential biomarkers for the early diagnosis of cancers. In this study, we performed a candidate marker screen on macroarrays containing 38,016 human proteins, derived from a human fetal-brain expression library, with the pools of sera from breast cancer patients (1 pool of benign samples, 3 pools of ductal carcinoma and 2 pools of lobular carcinoma) and 1 pool of sera from healthy women. A panel of 642 sero-reactive clones were deduced from these macroarray experiments which include 284 in-frame clones. Over-representation analyses of the sero-reactive in-frame clones enabled the identification of the sets of genes over-expressed in various pathways of the functional categories (KEGG, Transpath, Pfam and GO). Protein microarrays, generated using the His-tag proteins derived from the macroarray experiments, were used to evaluate the sera from breast cancer patients (24 malignant, 16 benign) and 20 control individuals. Using the PAM algorithm we elucidated a panel of 50 clones which enabled the correct classification prediction of 93% of the breast-nodule positive group (benign & malignant) sera from healthy individuals’ sera with 100% sensitivity and 85% specificity. This was followed by over-representation analysis of the significant clones derived from the class prediction

Reversible plasmonic biosensors based on aptamers and hydrogels

This thesis presents several interlinked projects aimed at sensitive detection of chemical and biological species by the use of surface plasmon resonance-based biosensors. This optical technique takes advantage of electromagnetic field confined at the nanoscale that is generated by the coupling light to the collective oscillation of charge density at the surface of metallic films and metallic nanoparticles. In particular, the thesis utilizes surface plasmon field-enhanced fluorescence spectroscopy (SPFS) and surface plasmon resonance (SPR) biosensors for sensitive readout of heterogeneous assays on gold sensor surface. It focuses on several important aspects that are essential to unlock the potential of optical biosensors in the emerging field of biomedical sciences that require detection of species serving as biomarkers or drugs, in close contact with the human body, outside of specialized laboratories. Specially,it aims at optical biosensor systems that hold potential for continuous monitoring of compounds in complex liquid samples and the following three projects have been pursued.Firstly, fluorescence assay with weak affinity recognition elements that reversibly bind target analyte has been employed. In order to compensate for low fluorescence signal associated with the affinity binding of target analyte at the sensor surface, the SPFS was implemented. A fluorophore-labeled hairpin aptamer structure was designed for reversible real-time biosensor, which was demonstrated for a model analyte –adenosine triphosphate. The sensing concept relies on resonant fluorescence energy transfer between the fluorophore label and metal that occurs at short distances. The aptamer hairpin is opened and closed by the specific capture of target analyte which is translated to strong variations in fluorescence signal intensity amplified by the intense surface plasmon field. A fully reversible sensor was achieved with up to 23-fold increased fluorescence signal when target analyte was captured. In addition to the aptamer hairpin, split sequences were implemented in a sandwich-type assay for the same analyte. The lack of interaction of the aptamer split sequences in the absence of the analyte allowed for a highly reduced background interference. The sensor demonstrated full reversibility that allowed multiple rounds of detection on the same sensor chip, with time resolution of several minutes for ligands with equilibrium affinity binding constants at around mM concentration. Secondly, hydrogel materials were employed for the construction of a biointerface that is resistant to fouling from blood serum. It was utilized in the form of thin surface-attached layers and free-standing membranes spanning above gold-coated surface plasmon resonance-based biosensors. The hydrophilic nature of the used hydrogels provided efficient means to repel the unspecific sorption from serum, as demonstrated by optical waveguide spectroscopy method. In addition, the interaction of biomolecules with the poly-(N-isopropyl)acrylamide – based hydrogel used was thoroughly investigated by fluorescence correlation spectroscopy. These materials were tailored for filtering applications as the permeability of hydrogels can be efficiently controlled by tuning the pore size. A thermo-responsive hydrogel was used for dynamic switching of the membrane with about micrometer thickness between its closed and permeable states. Thirdly, a thin hydrogel layer was employed as an efficient 3D affinity binding matrix that takes advantage of its large surface area. This material was employed in a fluorescence assay that relies on horse-radish peroxidase label and tyramide-based enzymatic fluorescence signal amplification. On 2D surface architectures, self-quenching occurs which limits the performance characteristics of this approach. The use of 3D hydrogel architecture offers means to overcome this limitation and is demonstrated to provide 2 orders of magnitude increase in the fluorescence signal intensity compared to conventional fluorophore-labeled detection scheme.Doctor of Philosoph

Free-standing hydrogel-particle composite membrane with dynamically controlled permeability

The preparation and investigation of a free-standing membrane made from a composite of thermoresponsive poly(N-isopropylacrylamide) (pNIPAAm) and polystyrene nanoparticles (PS NP) with temperature-controlled permeability is reported. The method exploits the light-induced crosslinking of the photo-reactive pNIPAAm-based polymer and mechanical reinforcement of the membrane structure by the polystyrene nanoparticles. About micrometer thick layers were either directly attached to a gold surface or prepared as free-standing layers spanning over arrays of microfluidic channels with a width of about hundred microns by using template stripping. Diffusion of liquid medium, low molecular weight molecules, and large molecular weight proteins contained in blood through the composite membrane was observed with combined surface plasmon resonance (SPR) and optical waveguide spectroscopy (OWS). The swelling ratio, permeability, and nonspecific sorption to these composite membranes were investigated by SPR and OWS as a function of molecular weight of analyte, loading of PS NP in the composite film, and temperature. The authors show successful preparation of a defect-free membrane structure that acts as a thermoresponsive filter with nanoscale pores spanning over an area of several square millimeters. This membrane can be reversibly switched to block or allow the diffusion of low mass molecules to the sensor surface by temperature-triggered swelling and collapsing of the hydrogel component. Blocking of diffusion and low unspecific sorption of proteins contained in blood serum is observed. These features make this platform interesting for potential future applications in continuous monitoring biosensors for the analysis of low molecular weight drug analytes or for advanced cell-on-chip microfluidic studies.Published versio

Reversible Immunosensor for the Continuous Monitoring of Cortisol in Blood Plasma Sampled with Microdialysis

Cortisol is a steroid hormone involved in a wide range of medical conditions. The level of the hormone fluctuates over time, but with traditional laboratory-based assays, such dynamics cannot be monitored in real time. Here, a reversible cortisol sensor is reported that allows continuous monitoring of cortisol in blood plasma using sampling by microdialysis. The sensor is based on measuring single-molecule binding and unbinding events of tethered particles. The particles are functionalized with antibodies and the substrate with cortisol-analogues, causing binding and unbinding events to occur between particles and substrate. The frequency of binding events is reduced when cortisol is present in the solution as it blocks the binding sites of the antibodies. The sensor responds to cortisol in the high nanomolar to low micromolar range and can monitor cortisol concentrations over multiple hours. Results are shown for cortisol monitoring in filtered and in microdialysis-sampled human blood plasma

Plasmon Field-Enhanced Fluorescence Energy Transfer for Hairpin Aptamer Assay Readout

Surface plasmon field-enhanced fluorescence energy transfer is employed for sensitive optical readout of a reversible hairpin aptamer assay that is suitable for continuous monitoring of low-molecular-weight chemical analytes. A hairpin aptamer specific to adenosine and adenosine triphosphate with Alexa Fluor 647 fluorophore attached to its 5′ end was anchored via 3′ end thiol to a gold thin film. Molecular spacers were used to control the distance of the fluorophore from the surface in the aptamer “off” and “on” states. The specific binding of the target analyte changes the aptamer conformation, which alters the distance of the fluorophore from the gold surface and translates to variations in the detected fluorescence intensity. The plasmonically mediated fluorescence signal increases the measured signal-to-noise ratio and allows for real-time observation of the analyte binding. Theoretical as well as experimental study of the optical signal dependence on fluorophore orientation, design of spacers, and angular distribution of collected light is presented for rational design of the assay. The detected sensor signal increased by a factor as large as 23 upon switching the aptamer from the “off” to “on” state due to the hairpin opening associated with the specific capture of target analyte

The Immunome of Colon Cancer: Functional In Silico Analysis of Antigenic Proteins Deduced from IgG Microarray Profiling

Characterization of the colon cancer immunome and its autoantibody signature from differentially-reactive antigens (DIRAGs) could provide insights into aberrant cellular mechanisms or enriched networks associated with diseases. The purpose of this study was to characterize the antibody profile of plasma samples from 32 colorectal cancer (CRC) patients and 32 controls using proteins isolated from 15,417 human cDNA expression clones on microarrays. 671 unique DIRAGs were identified and 632 were more highly reactive in CRC samples. Bioinformatics analyses reveal that compared to control samples, the immunoproteomic IgG profiling of CRC samples is mainly associated with cell death, survival, and proliferation pathways, especially proteins involved in EIF2 and mTOR signaling. Ribosomal proteins (e.g., RPL7, RPL22, and RPL27A) and CRC-related genes such as APC, AXIN1, E2F4, MSH2, PMS2, and TP53 were highly enriched. In addition, differential pathways were observed between the CRC and control samples. Furthermore, 103 DIRAGs were reported in the SEREX antigen database, demonstrating our ability to identify known and new reactive antigens. We also found an overlap of 7 antigens with 48 “CRC genes.” These data indicate that immunomics profiling on protein microarrays is able to reveal the complexity of immune responses in cancerous diseases and faithfully reflects the underlying pathology. Keywords: Autoantibody tumor biomarker, Cancer immunology, Colorectal cancer, Immunomics, Protein microarra

Continuous biomarker monitoring with single molecule resolution by measuring free particle motion

There is a need for sensing technologies that can continuously monitor concentration levels of critical biomolecules in applications such as patient care, fundamental biological research, biotechnology and food industry, as well as the environment. However, it is fundamentally difficult to develop measurement technologies that are not only sensitive and specific, but also allow monitoring over a broad concentration range and over long timespans. Here we describe a continuous biomolecular sensing methodology based on the free diffusion of biofunctionalized particles hovering over a sensor surface. The method records digital events due to single-molecule interactions and enables biomarker monitoring at picomolar to micromolar concentrations without consuming any reagents. We demonstrate the affinity-based sensing methodology for DNA-based sandwich and competition assays, and for an antibody-based cortisol assay. Additionally, the sensor can be dried, facilitating storage over weeks while maintaining its sensitivity. We foresee that this will enable the development of continuous monitoring sensors for applications in fundamental research, for studies on organs on a chip, for the monitoring of patients in critical care, and for the monitoring of industrial processes and bioreactors as well as ecological systems