Jochen Kieninger

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies.

Cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs. There are, however, two different aspects of the problem caused by tumor hypoxia when cancer therapy is concerned: One is due to the chemical reactions that molecular oxygen enters into therapeutically targeted cells. This results in a direct chemical protection against therapy by the hypoxic microenvironment, which has little to do with cellular biological regulatory processes. This part of the protective effect of hypoxia has been known for more than half a century and has been studied extensively. However, in recent years there has been more focus on the other aspect of hypoxia, namely the effect of this microenvironmental condition on selecting cells with certain genetic prerequisites that are negative with respect to patient prognosis. There are adaptive mechanisms, where hypoxia induces regulatory cascades in cells resulting in a changed metabolism or changes in extracellular signaling. These processes may lead to changes in cellular intrinsic sensitivity to treatment irrespective of oxygenation and, furthermore, may also have consequences for tissue organization. Thus, the adaptive mechanisms induced by hypoxia itself may have a selective effect on cells, with a fine-tuned protection against damage and stress of many kinds. It therefore could be that the adaptive mechanisms may take advantage of for new tumor labeling/imaging and treatment strategies. One of the Achilles’ heels of hypoxia research has always been the exact measurements of tissue oxygenation as well as the control of oxygenation in biological tumor models. Thus, development of technology that can ease this control is vital in order to study mechanisms and perform drug development under relevant conditions. An integrated EU Framework project 2004–2009, termed EUROXY, demonstrates several pathways involved in transcription and translation control of the hypoxic cell phenotype and evidence of cross-talk with responses to pH and redox changes. The carbonic anhydrase isoenzyme CA IX was selected for further studies due to its expression on the surface of many types of hypoxic tumors. The effort has led to marketable culture flasks with sensors and incubation equipment, and the synthesis of new drug candidates against new molecular targets. New labeling/imaging methods for cancer diagnosing and imaging of hypoxic cancer tissue are now being tested in xenograft models and are also in early clinical testing, while new potential anti-cancer drugs are undergoing tests using xenografted tumor cancers. The present article describes the above results in individual consortium partner presentations.

Targeting tumour hypoxia to prevent cancer metastasis: from biology, biosensing and technology to drug development : the METOXIA consortium

Abstract The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation–deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009–2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [18F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O2), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

Polymer-based, flexible glutamate and lactate microsensors for in vivo applications

We present a flexible microsensor, based on a polymer substrate, for multiparametric, electrochemical in vivo monitoring. The sensor strip with a microelectrode array at the tip was designed for insertion into tissue, for fast and localized online monitoring of physiological parameters. The microsystem fabrication on a wafer-level is based on a polyimide substrate and includes the patterning of platinum microelectrodes as well as epoxy and dry-film-resist insulation in a cost-effective thin-film and laminate process. A stable, electrodeposited silver/silver chloride reference electrode on-chip and a perm-selective membrane as an efficient interference rejection scheme are integrated on a wafer-level. Amperometric, electrochemical, enzyme-based biosensors for the neurotransmitter L-glutamate and the energy metabolite L-lactate have been developed. Hydrogel membranes or direct cross-linking as stable concepts for the enzyme immobilization are shown. Sensor performance including high selectivity, tailoring of sensitivity and long-term stability is discussed. For glutamate, a high sensitivity of 2.16 nAmm(-2) µM(-1) was found. For lactate, a variation in sensitivity between 2.6 and 32 nAmm(-2)mM(-1) was achieved by different membrane compositions. The in vivo application in an animal model is demonstrated by glutamate measurements in the brain of rats. Local glutamate alterations in the micromolar range and in nanoliter-range volumes can be detected and quantified with high reproducibility and temporal resolution. A novel, versatile platform for the integration of various electrochemical sensors on a small, flexible sensor strip for a variety of in vivo applications is presented.

Microfabricated, amperometric, enzyme-based biosensors for in vivo applications

AbstractMiniaturized electrochemical in vivo biosensors allow the measurement of fast extracellular dynamics of neurotransmitter and energy metabolism directly in the tissue. Enzyme-based amperometric biosensing is characterized by high specificity and precision as well as high spatial and temporal resolution. Aside from glucose monitoring, many systems have been introduced mainly for application in the central nervous system in animal models. We compare the microsensor principle with other methods applied in biomedical research to show advantages and drawbacks. Electrochemical sensor systems are easily miniaturized and fabricated by microtechnology processes. We review different microfabrication approaches for in vivo sensor platforms, ranging from simple modified wires and fibres to fully microfabricated systems on silicon, ceramic or polymer substrates. The various immobilization methods for the enzyme such as chemical cross-linking and entrapment in polymer membranes are discussed. The resulting sensor performance is compared in detail. We also examine different concepts to reject interfering substances by additional membranes, aspects of instrumentation and biocompatibility. Practical considerations are elaborated, and conclusions for future developments are presented. Graphical Abstractᅟ

Multiparametric, Flexible Microsensor Platform for Metabolic Monitoring \(In~Vivo\)

In this paper, we present a multiparametric microsensor platform for metabolic monitoring in vivo. In contrast to silicon or ceramic-based systems, the flexible, implantable polymer-based sensor strip is manufactured in a cost-effective hybrid of thin-film and laminate technology in a wafer-level process. Flexibility allows easy handling and placement in soft tissue. It comprises a microelectrode array for up to four electrochemical, amperometric micro-(bio)sensors, and an integrated reference electrode. The energy metabolism parameters glucose, lactate, and oxygen as well as the neurotransmitter glutamate were measured. The sensors allow dynamic, highly sensitive, localized, long-term, online measurement of up to four metabolic parameters with a single device. The reliable analytical performance of the sensors, stability of the reference electrode, and the multiparametric measurement are shown. The sensor can be inserted directly into the tissue for in vivo applications.

Pericellular oxygen monitoring with integrated sensor chips for reproducible cell culture experiments

Here we present an application, in two tumour cell lines, based on the Sensing Cell Culture Flask system as a cell culture monitoring tool for pericellular oxygen sensing.

Superoxide microsensor integrated into a Sensing Cell Culture Flask microsystem using direct oxidation for cell culture application.

A new electrochemical sensor system for reliable and continuous detection of superoxide radical release from cell culture was developed utilizing direct oxidation of superoxide on polymer covered gold microelectrodes. Direct superoxide oxidation was demonstrated to provide robust measurement principle for sensitive and selective reactive oxygen species (ROS) quantification without the need for biocomponent supported conversion. Sensor performance was investigated by using artificial enzymatic superoxide production revealing a sensitivity of 2235AM(-1)m(-2). An electrode protection layer with molecular weight cut-off property from adsorbed linear branched polyethylenimine was successfully introduced for long term and selectivity improvement. Thin-film based sensor chip fabrication with implemented three-electrode setup and full integration into the technological platform Sensing Cell Culture Flask was described. Cell culturing directly on-chip and free radical release by phorbol-12-myristate-13-acetate (PMA) stimulation was demonstrated using T-47D human breast cancer carcinoma cell model. Transient extracellular superoxide production upon stimulation was successfully observed from amperometric monitoring. Signal inhibition from scavenging of extracellular superoxide by specific superoxide dismutase (SOD) showed the applicability for selective in vitro ROS determination. The results confirm the possibility of direct superoxide oxidation, with exclusion of the main interfering substances uric acid and hydrogen peroxide. This offers new insights into the development of reliable and robust ROS sensors.

Accessing 3D microtissue metabolism: Lactate and oxygen monitoring in hepatocyte spheroids.

3D hepatic microtissues, unlike 2D cell cultures, retain many of the in-vivo-like functionalities even after long-term cultivation. Such 3D cultures are increasingly applied to investigate liver damage due to drug exposure in toxicology. However, there is a need for thorough metabolic characterization of these microtissues for mechanistic understanding of effects on culture behaviour. We measured metabolic parameters from single human HepaRG hepatocyte spheroids online and continuously with electrochemical microsensors. A microsensor platform for lactate and oxygen was integrated in a standard 96-well plate. Electrochemical microsensors for lactate and oxygen allow fast, precise and continuous long-term measurement of metabolic parameters directly in the microwell. The demonstrated capability to precisely detect small concentration changes by single spheroids is the key to access their metabolism. Lactate levels in the culture medium starting from 50µM with production rates of 5µMh-1 were monitored and precisely quantified over three days. Parallel long-term oxygen measurements showed no oxygen depletion or hypoxic conditions in the microwell. Increased lactate production by spheroids upon suppression of the aerobic metabolism was observed. The dose-dependent decrease in lactate production caused by the addition of the hepatotoxic drug Bosentan was determined. We showed that in a toxicological application, metabolic monitoring yields quantitative, online information on cell viability, which complements and supports other methods such as microscopy. The demonstrated continuous access to 3D cell culture metabolism within a standard setup improves in vitro toxicology models in replacement strategies of animal experiments. Controlling the microenvironment of such organotypic cultures has impact in tissue engineering, cancer therapy and personalized medicine.

Lift-Off Free Fabrication Approach for Periodic Structures with Tunable Nano Gaps for Interdigitated Electrode Arrays.

We report a simple, low-cost and lift-off free fabrication approach for periodic structures with adjustable nanometer gaps for interdigitated electrode arrays (IDAs). It combines an initial structure and two deposition process steps; first a dielectric layer is deposited, followed by a metal evaporation. The initial structure can be realized by lithography or any other structuring technique (e.g., nano imprint, hot embossing or injection molding). This method allows the fabrication of nanometer sized gaps and completely eliminates the need for a lift-off process. Different substrate materials like silicon, Pyrex or polymers can be used. The electrode gap is controlled primarily by sputter deposition of the initial structure, and thus, adjustable gaps in the nanometer range can be realized independently of the mask or stamp pattern. Electrochemical characterizations using redox cycling in ferrocenemethanol (FcMeOH) demonstrate signal amplification factors of more than 110 together with collection factors higher than 99%. Furthermore, the correlation between the gap width and the amplification factor was studied to obtain an electrochemical performance assessment of the nano gap electrodes. The results demonstrate an exponential relationship between amplification factor and gap width.

Designed miniaturization of microfluidic biosensor platforms using the stop-flow technique

Here, we present a novel approach to increase the degree of miniaturization as well as the sensitivity of biosensor platforms by the optimization of microfluidic stop-flow techniques independent of the applied detection technique (e.g. electrochemical or optical). The readout of the labeled bioassays, immobilized in a microfluidic channel, under stop-flow conditions leads to a rectangular shaped peak signal. Data evaluation using the peak height allows for a high level miniaturization of the channel geometries. To study the main advantages and limitations of this method by numerical simulations, a universally applicable model system is introduced for the first time. Consequently, proof-of-principle experiments were successfully performed with standard and miniaturized versions of an electrochemical biosensor platform utilizing a repressor protein-based assay for tetracycline antibiotics. Herein, the measured current peak heights are the same despite the sextuple reduction of the channel dimensions. Thus, this results in a 22-fold signal amplification compared to the constant flow measurements in the case of the miniaturized version.