Postfabrication Tuning of Circular Bragg Resonators for Enhanced Emitter-Cavity Coupling.

Solid-state quantum emitters embedded in circular Bragg resonators are attractive due to their ability to emit quantum light with high brightness and low multiphoton probability. As for any emitter-microcavity system, fabrication imperfections limit the spatial and spectral overlap of the emitter with the cavity mode, thus limiting their coupling strength. Here, we show that an initial spectral mismatch can be corrected after device fabrication by repeated wet chemical etching steps. We demonstrate an ∼16 nm wavelength tuning for optical modes in AlGaAs resonators on oxide, leading to a 4-fold Purcell enhancement of the emission of single embedded GaAs quantum dots. Numerical calculations reproduce the observations and suggest that the achievable performance of the resonator is only marginally affected in the explored tuning range. We expect the method to be applicable also to circular Bragg resonators based on other material platforms, thus increasing the device yield of cavity-enhanced solid-state quantum emitters.

Hyperpolarized 13C Spectroscopy with Simple Slice-and-Frequency-Selective Excitation.

Hyperpolarized 13C nuclear magnetic resonance spectroscopy can characterize in vivo tissue metabolism, including preclinical models of cancer and inflammatory disease. Broad bandwidth radiofrequency excitation is often paired with free induction decay readout for spectral separation, but quantification of low-signal downstream metabolites using this method can be impeded by spectral peak overlap or when frequency separation of the detected peaks exceeds the excitation bandwidth. In this work, alternating frequency narrow bandwidth (250 Hz) slice-selective excitation was used for 13C spectroscopy at 7 T in a subcutaneous xenograft rat model of human pancreatic cancer (PSN1) to improve quantification while measuring the dynamics of injected hyperpolarized [1-13C]lactate and its metabolite [1-13C]pyruvate. This method does not require sophisticated pulse sequences or specialized radiofrequency and gradient pulses, but rather uses nominally spatially offset slices to produce alternating frequency excitation with simpler slice-selective radiofrequency pulses. Additionally, point-resolved spectroscopy was used to calibrate the 13C frequency from the thermal proton signal in the target region. This excitation scheme isolates the small [1-13C]pyruvate peak from the similar-magnitude tail of the much larger injected [1-13C]lactate peak, facilitates quantification of the [1-13C]pyruvate signal, simplifies data processing, and could be employed for other substrates and preclinical models.

Acquisition strategies for spatially resolved magnetic resonance detection of hyperpolarized nuclei.

Hyperpolarization is an emerging method in magnetic resonance imaging that allows nuclear spin polarization of gases or liquids to be temporarily enhanced by up to five or six orders of magnitude at clinically relevant field strengths and administered at high concentration to a subject at the time of measurement. This transient gain in signal has enabled the non-invasive detection and imaging of gas ventilation and diffusion in the lungs, perfusion in blood vessels and tissues, and metabolic conversion in cells, animals, and patients. The rapid development of this method is based on advances in polarizer technology, the availability of suitable probe isotopes and molecules, improved MRI hardware and pulse sequence development. Acquisition strategies for hyperpolarized nuclei are not yet standardized and are set up individually at most sites depending on the specific requirements of the probe, the object of interest, and the MRI hardware. This review provides a detailed introduction to spatially resolved detection of hyperpolarized nuclei and summarizes novel and previously established acquisition strategies for different key areas of application.

Biosensor for the characterisation of hMAO B inhibitors and the quantification of selegiline.

A novel human monoamine oxidase B (hMAO B) based biosensor for inhibitory measurements was developed. It allows both the characterisation of the type of enzyme inhibition and the sensitive and simple determination of inhibitors like selegiline hydrochloride. The sensor consists of a screen printed carbon working electrode modified with 20% manganese dioxide (MnO2) and the enzyme hMAO B, which was immobilised on the electrode via a dialysis membrane (regenerated cellulose, molecular weight cut-off 14000). Inhibition of hMAO B is evaluated by adding different concentrations of the inhibitor selegiline hydrochloride to the enzyme and applying a defined amount of the hMAO B substrate phenylethylamine (PEA). The enzymatically formed H2O2 is amperometrically detected at 0.4V vs. Ag/AgCl in a flow injection analysis (FIA) system. With 100µM PEA the sensor showed a linear correlation between peak height and inhibitor concentration in a range of 0.51-3.25µg/mL selegiline hydrochloride. LOD and LOQ were determined to be 0.15 and 0.51µg/mL, respectively. The sensor showed a repeatability of 3.7% and an intermediate precision of 8.1%. The inhibition-based biosensor was successfully employed to quantify selegiline hydrochloride in pharmaceutical samples. Kinetic studies via Lineweaver-Burk plot and enzyme quantity vs. current plot revealed that the inhibition is irreversible.

Targeting of IL-2 and GM-CSF immunocytokines to a tumor vaccine leads to increased anti-tumor activity.

Fusion proteins combining antibodies with cytokines such as IL-2 and GM-CSF appear to be promising reagents for tumor therapy. In this study, we combined such immunocytokines with the tumor vaccine ATV-NDV consisting of irradiated tumor cells infected with Newcastle disease virus (NDV). The two fusion proteins bsF-GMCSF and tsHN-IL2-GM-CSF, binding, respectively, to the viral fusion protein (F) or to hemagglutinin-neuraminidase (HN) expressed on the surface of the vaccine cells and containing GM-CSF or GM-CSF and IL-2-activities were produced by recombinant antibody technology. The purified molecules showed the expected binding specificity and biological activity inherent to the respective cytokine. Using a newly established in vitro tumor neutralisation assay (TNA), we showed improved antitumoral effect through tumor growth inhibition when human peripheral blood mononuclear cells from healthy donors were stimulated with immunocytokine modified versus non-modified tumor vaccine cells. These effects induced by the fusion proteins, in the presence of a suboptimal T cell activation signal 1 provided by bsHN-CD3, occured only when these were bound to the tumor vaccine. Furthermore, it was shown that CD14+ monocytes could be activated by the GM-CSF cytokine fused within the recombinant proteins and that they contributed essentially to the antitumor effect in the TNA. The data presented here suggest an easy way for a broad clinical development and application of tumor-targeted IL-2- and GM-CSF-based immunocytokines based on the associated increase of anti-tumor activity mediated by T cells and monocytes.

A gene signature distinguishing CD133hi from CD133- colorectal cancer cells: essential role for EGR1 and downstream factors.

AIMS: In colorectal cancer (CRC), CD133 expression is an independent prognostic marker associated with adverse clinical outcome. The CD133 epitope AC133 allowed isolating stem cells from normal and cancerous tissues, although its use in colon was questioned. We aimed to identify differences between AC133 and AC133 cells. METHODS: We analysed the gene expression profiles of EpCAM/CEA/AC133 and EpCAM/CEA/AC133 cells from primary CRC and liver metastasis tissues (n = 5). Immunohistochemistry confirmed these results in a validation set. RESULTS: We identified 68 genes differentially expressed between both populations, including genes of notorious importance in CRC pathogenesis, and several candidates not previously shown to play a major role in CRC. Notably, EGR1 belonged to the most highly expressed genes in AC133 cells. In the validation set, the presence of EGR1 and CD133 correlated (r = 0.625). Since EGR1 regulates Wnt through up-regulation of TCF4, which induces stem cell marker LGR5, the potential association between LGR5, EGR1 and CD133 was investigated. The presence of LGR5 correlated with the presence of EGR1 and CD133. Strong signals for LGR5 were detected throughout tumour invasion fronts. CONCLUSIONS: The study suggests a connection between CD133 and EGR1 and emphasises the importance of the EGR1/TCF4/CD133/LGR5 network in CRC.

Transcriptome analysis and cytokine profiling of naive T cells stimulated by a tumor vaccine via CD3 and CD25.

T-cell receptor engagement by peptide/MHC complexes constitutes the main signal for the activation of naive T cells, but for a productive generation and maintenance of effector cells, full activation requires additional signals driven by costimulatory molecules present on activated antigen-presenting cells. Herein we describe T cell costimulation via CD25, the interleukin (IL)-2 receptor, during priming of naive T cells with a tumor vaccine. To this end, we produced, purified and characterized the fusion protein bsHN-IL2 which contains the IL-2 cytokine and an antibody scFv fragment directed towards the Hemagglutinin-Neuraminidase (HN) protein of Newcastle Disease Virus (NDV). Tumor vaccine cells were modified by infection with this virus which allows the attachment of the immunocytokine bsHN-IL2. In the presence of CD3-mediated signal 1, the vaccine/bsHN-IL2 provided via CD25 a strong bystander antitumor effect in vitro leading to tumor growth inhibition, even stronger than the vaccine/bsHN-CD28 which provides costimulation via CD28. Transcriptome analysis of naive T cells which were stimulated with the vaccine/bsHN-IL2 showed, similarly to the vaccine/bsHN-CD28, upregulation of 71 genes belonging to different signalling pathways, including PLC-γ1, Grb-2, Vav-1 and PDE-4A. Analysis of the supernatants of activated T cells with ligand-bound tumor vaccine showed that the vaccine/bsHN-IL2, in contrast to the vaccine/bsHN-CD28, did not lead to the production of additional IL-2. We report here the first transcriptome analysis of IL-2 receptor mediated costimulatory signals. The findings provide new insights into mechanisms of function of IL-2 during T cell priming.

Optimization studies for the coupling of bispecific antibodies to viral anchor molecules of a tumor vaccine.

Tumor vaccines have to provide several signals for T cell activation. Among them, signal 1 (through TCR/CD3) and signal 2 (through CD28) are the most important. We herein describe a procedure to introduce anti-CD3 and anti-CD28 signals into any tumor cell which is susceptible to infection by Newcastle disease virus (NDV). We developed the ATV-NDV tumor vaccine which consists of patient-derived tumor cells (ATV) modified through infection by NDV. We tested for further improvement of vaccine efficiency the addition of two bispecific single-chain antibodies. They bind with one arm to the viral hemagglutinin-neuraminidase (HN) or fusion (F) protein of NDV expressed at the surface of the vaccine cells while the second arm is directed either against CD3 or CD28 of T cells. The aim of this study was to optimize the coupling of these new reagents to the tumor vaccine. When anti-CD3 and anti-CD28 molecules bind to the same anchoring viral molecule (e.g. HN), competition for binding could occur under certain conditions. This was not the case when the bispecific reagents bound to separate viral molecules (HN or F, respectively). When using transfectants expressing HN and F either separately or on the same cell, we show that T cell activation works best when anti-CD3 and anti-CD28 are attached to the same stimulator cell. The clinical application of such a combined therapy with ATV-NDV vaccine cells and bi-specific antibodies allows to modify the strength of signal 1 and 2 in a quantitative and predictable way according to the immune status of the T cells and the requirements of the patients' immune system.

Meta-analysis shows that detection of circulating tumor cells indicates poor prognosis in patients with colorectal cancer.

BACKGROUND & AIMS: The prognostic significance of circulating (CTCs) and disseminated tumor cells in patients with colorectal cancer (CRC) is controversial. We performed a meta-analysis of available studies to assess whether the detection of tumor cells in the blood and bone marrow (BM) of patients diagnosed with primary CRC can be used as a prognostic factor. METHODS: We searched the Medline, Biosis, Science Citation Index, and Embase databases and reference lists of relevant articles (including review articles) for studies that assessed the prognostic relevance of tumor cell detection in the peripheral blood (PB), mesenteric/portal blood (MPB), or BM of patients with CRC. Meta-analyses were performed using a random effects model, with hazard ratio (HR) and 95% confidence intervals (95% CIs) as effect measures. RESULTS: A total of 36 studies, including 3094 patients, were eligible for final analyses. Pooled analyses that combined all sampling sites (PB, MPB, and BM) associated the detection of tumor cells with poor recurrence-free survival (RFS) (HR = 3.24 [95% CI: 2.06-5.10], n = 26, I(2) = 77%) and overall survival (OS) (2.28 [1.55-3.38], n = 21, I(2) = 66%). Stratification by sampling site showed that detection of tumor cells in the PB compartment was a statistically significant prognostic factor (RFS: 3.06 [1.74-5.38], n = 19, I(2) = 78%; OS: 2.70 [1.74-4.20], n = 16, I(2) = 59%) but not in the MPB (RFS: 4.12 [1.01-16.83], n = 8, I(2) = 75%; OS: 4.80 [0.81-28.32], n = 5, I(2) = 82%) or in the BM (RFS: 2.17 [0.94-5.03], n = 4, I(2) = 78%; OS: 1.50 [0.52-4.32], n = 3, I(2) = 84%). CONCLUSION: Detection of CTCs in the PB indicates poor prognosis in patients with primary CRC.

Antigen-specific Tregs control T cell responses against a limited repertoire of tumor antigens in patients with colorectal carcinoma.

Spontaneous antitumor T cell responses in cancer patients are strongly controlled by Tregs, and increased numbers of tumor-infiltrating Tregs correlate with reduced survival. However, the tumor antigens recognized by Tregs in cancer patients and the impact of these cells on tumor-specific T cell responses have not been systematically characterized. Here we used a broad panel of long synthetic peptides of defined tumor antigens and normal tissue antigens to exploit a newly developed method to identify and compare ex vivo the antigen specificities of Tregs with those of effector/memory T cells in peripheral blood of colorectal cancer patients and healthy subjects. Tregs in tumor patients were highly specific for a distinct set of only a few tumor antigens, suggesting that Tregs exert T cell suppression in an antigen-selective manner. Tumor-specific effector T cells were detectable in the majority of colorectal cancer patients but not in healthy individuals. We detected differences in the repertoires of antigens recognized by Tregs and effector/memory T cells in the majority of colorectal cancer patients. In addition, only effector/memory T cell responses against antigens recognized by Tregs strongly increased after Treg depletion. The selection of antigens according to preexisting T cell responses may improve the efficacy of future immunotherapies for cancer and autoimmune disease.

An effective tumor vaccine optimized for costimulation via bispecific and trispecific fusion proteins.

T cell costimulation has great therapeutic potential if it can be optimized and controlled. To achieve this, we engineered T cell-activating fusion proteins and immunocytokines that specifically attach to viral antigens of a virus-infected tumor vaccine. We employed the avian Newcastle Disease Virus because this agent is highly efficient for human tumor cell infection, and leads to introduction of viral hemagglutinin-neuraminidase (HN) molecules at the tumor cell surface. Here, we demonstrated the strong potentiation of the T cell stimulatory activity of such a vaccine upon attachment of bispecific or trispecific fusion proteins which bind with one arm to viral HN molecules of the vaccine, and with the other arm either to CD3 (signal 1), to CD28 (costimulatory signal 2a), or to interleukin-2 receptor (costimulatory signal 2b) on T cells. A vaccine with a combination of all three signals triggered the strongest activation of naïve human T cells, thereby inducing the most durable bystander antitumor activity in vitro. Adoptive transfer of such polyclonally activated cells into immunodeficient mice bearing human breast carcinoma caused tumor regression. Furthermore, tumor-reactive memory T cells from draining lymph nodes of carcinoma patients could be efficiently reactivated in a short-term ELISpot assay using an autologous tumor vaccine with optimized signals 1 and 2, but not with a similarly modified vaccine from an unrelated tumor cell line. Our data describe new bioactive molecules which in combination with an established virus-modified tumor vaccine greatly augments the antitumor activity of T cells from healthy donors and cancer patients.