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Forschungsbereich C

Al18F labeling at room temperature of 2-aminomethylpiperidinebased chelators for PET imaging

ChemMedChem, 2019, publiziert am 16.12.2019
Positron emission tomography (PET) is a non-invasive molecular imaging technology constantly expanding, with a high demand for specific antibody-derived imaging probes. The use of tracers based on temperature-sensitive molecules (i.e. Fab, svFab, nanobodies) is increasing and leads us to design a class of chelators based on the structure of 2-aminomethylpiperidine (AMP) with acetic and/or hydroxybenzyl pendant arms (2-AMPTA, NHB-2-AMPDA, and 2-AMPDA-HB), which have been investigated as such for {Al18F}2+-core chelation efficiency. All the compounds were characterized by HPLC-MS analysis and NMR spectroscopy. The AlF-18 labeling reactions were performed in different conditions (pH/temperature), and the radiolabeled chelates were purified and characterized by radio-TLC and radio-HPLC. The stability of labeled chelates was investigated up to 240min in human serum (HS), EDTA 5mM, PBS and 0.9% NaCl solutions. In vivo stability of [Al18F(2- AMPDA-HB)]-  was assessed in healthy nude mice (n=6). RCYs between 55% and 81% were obtained at pH 5 and rt. High stability in HS was measured for [Al18F(2-AMPDA-HB)]- , with 90% of F-18 complexed after 120min. High stability in vivo, a fast hepatobiliary and renal excretion, with irrelevant accumulation of free F-18 in the bones were measured. Thus, this new Al18F-chelator may have a great impact on immunoPET radiopharmacy, by facilitating tdevelopment of new fluorine-18 labeled heat-sensitive biomolecules.

 

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Heat-inducible mesenchymal stem cell (MSC)-mediated sodium iodide symporter (NIS) gene therapy

2019, publiziert am 18.06.2019
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Stimulation of tumour growth by thyroid hormone depends on integrin αvβ3 expression

Department of Internal Medicine IV, LMU Munich, Munich, Germany, 2019, publiziert am 12.12.2019
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Snail drives the cell cycle and determines immune cell profile in pancreatic ductal adenocarcinoma

2019, publiziert am 30.01.2019
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Mesenchymal stem cell-mediated sodium iodide symporter (NIS) reporter gene delivery in an orthotopic glioblastoma mouse model

1Medizinische Klinik und Poliklinik IV and 2Neurochirurgische Klinik und Poliklinik, University Hospital of Munich, LMU Munich, Germany, 2018, publiziert am 18.12.2018
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Heat-inducible mesenchymal stem cell-mediated sodium iodide symporter (NIS) gene transfer

2018, publiziert am 18.12.2018
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Adaptive rewiring of oncogenic signaling in response to MTOR blockade in pancreatic cancer

2018, publiziert am 12.12.2018
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T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target

2018, publiziert am 15.10.2018
Cancer immunotherapy has proven high efficacy in treating diverse cancer entities by immune checkpoint modulation and adoptive T-cell transfer. However, patterns of treatment response differ substantially from conventional therapies, and reliable surrogate markers are missing for early detection of responders versus non-responders. Current imaging techniques using 18F-fluorodeoxyglucose-positron-emmission-tomograpy (18F-FDG-PET) cannot discriminate, at early treatment times, between tumor progression and inflammation. Therefore, direct imaging of T cells at the tumor site represents a highly attractive tool to evaluate effective tumor rejection or evasion. Moreover, such markers may be suitable for theranostic imaging.

Methods: We mainly investigated the potential of two novel pan T-cell markers, CD2 and CD7, for T-cell tracking by immuno-PET imaging. Respective antibody- and F(ab´)2 fragment-based tracers were produced and characterized, focusing on functional in vitro and in vivo T-cell analyses to exclude any impact of T-cell targeting on cell survival and antitumor efficacy.

Results: T cells incubated with anti-CD2 and anti-CD7 F(ab´)2 showed no major modulation of functionality in vitro, and PET imaging provided a distinct and strong signal at the tumor site using the respective zirconium-89-labeled radiotracers. However, while T-cell tracking by anti-CD7 F(ab´)2 had no long-term impact on T-cell functionality in vivo, anti-CD2 F(ab´)2 caused severe T-cell depletion and failure of tumor rejection.

Conclusion: This study stresses the importance of extended functional T-cell assays for T-cell tracer development in cancer immunotherapy imaging and proposes CD7 as a highly suitable target for T-cell immuno-PET imaging.

Keywords: T-cell imaging, cancer immunotherapy, T-cell function, immuno-PET

 

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Molecular similarities and differences from human pulmonary fibrosis and corresponding mouse model: MALDI imaging mass spectrometry in comparative medicine

Laboratory Investigation, 2018, doi:10.1038/labinvest.2017.110 publiziert am 19.02.2018
Animal models can reproduce some model-specific aspects of human diseases, but some animal models translate poorly or fail to translate to the corresponding human disease. Here, we develop a strategy to systematically compare human and mouse tissues, and conduct a proof-of-concept experiment to identify molecular similarities and differences using patients with idiopathic pulmonary fibrosis and a bleomycin-induced fibrosis mouse model. Our novel approach employs highthroughput tissue microarrays (TMAs) of humans and mice, high-resolution matrix-assisted laser desorption/ionization- Fourier transform-ion cyclotron resonance-mass spectrometry imaging (MALDI-FT-ICR-MSI) to spatially resolve mass spectra at the level of specific metabolites, and hierarchical clustering and pathway enrichment analysis to identify functionally similar/different molecular patterns and pathways in pathological lesions of humans and mice. We identified a large number of common molecules (n = 1366) and fewer exclusive molecules in humans (n = 83) and mice (n = 54). Among the common molecules, the ‘ascorbate and aldarate metabolism’ pathway had the highest similarity in human and mouse lesions. This proof-of-concept study demonstrates that our novel strategy employing a reliable and easy-toperform experimental design accurately identifies pathways and factors that can be directly compared between animal models and human diseases.

 

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MTOR inhibitor-based combination therapies for pancreatic cancer

BRITISH JOURNAL OF CANCER, 2018, 1–12, doi: 10.1038/bjc.2017.421 publiziert am 02.01.2018
Background: Although the mechanistic target of rapamycin (MTOR) kinase, included in the mTORC1 and mTORC2 signalling hubs, has been demonstrated to be active in a significant fraction of patients with pancreatic ductal adenocarcinoma (PDAC), the value of the kinase as a therapeutic target needs further clarification.

Methods: We used Mtor floxed mice to analyse the function of the kinase in context of the pancreas at the genetic level. Using a dual-recombinase system, which is based on the flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies, we generated a novel cellular model, allowing the genetic analysis of MTOR functions in tumour maintenance. Cross-species validation and pharmacological intervention studies were used to recapitulate genetic data in human models, including primary human 3D PDAC cultures.

Results: Genetic deletion of the Mtor gene in the pancreas results in exocrine and endocrine insufficiency. In established murine PDAC cells, MTOR is linked to metabolic pathways and maintains the glucose uptake and growth. Importantly, blocking MTOR genetically as well as pharmacologically results in adaptive rewiring of oncogenic signalling with activation of canonical extracellular signal-regulated kinase and phosphoinositide 3-kinase-AKT pathways. We provide evidence that interfering with such adaptive signalling in murine and human PDAC models is important in a subgroup. Conclusions: Our data suggest developing dual MTORC1/TORC2 inhibitor-based therapies for subtype-specific intervention.

 

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Oncogenic signalling of cancer drivers - Context matters

Nature Reviews Cancer, 2016, publiziert am 14.11.2016
How can we treat cancer more effectively? Traditionally, tumours from the same anatomical site are treated as one tumour entity. This concept has been challenged by recent breakthroughs in cancer genomics and translational research enabling molecular tumour profiling. The identification and validation of cancer drivers, which are shared between different tumour types, spurred the new paradigm to target driver pathways across anatomical sites by off-label drug use, or within so called “basket or umbrella trials”, assuming that findings in one tumour entity can be extrapolated to all others. Recent clinical and preclinical studies suggest however, that this assumption is not generally valid due to tissue- and cell type-specific differences in tumourigenesis and the organization of oncogenic signalling pathways. In this Opinion article, we focus on the molecular, cellular, systemic and environmental determinants of organ-specific tumourigenesis and mechanisms of context-specific oncogenic signalling outputs. Investigation, recognition and in-depth biological understanding of these differences will be vital for the design of next-generation clinical trials and the implementation of rational molecularly guided cancer therapies in the future.

 

 



Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry

NATURE COMMUNICATIONS, 2016, DOI: 10.1038/ncomms13404 publiziert am 14.11.2016
Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient’s tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.

 

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Class I histone deacetylases regulate p53/NF-κB crosstalk in cancer cells

Elsevier Inc., 2016, publiziert am 09.11.2016
The transcription factors NF-κB and p53 as well as their crosstalk determine the fate of tumor cells upon therapeutic interventions. Replicative stress and cytokines promote signaling cascades that lead to the co-regulation of p53 and NF-κB. Consequently, nuclear p53/NF-κB signaling complexes activate NF-κB-dependent survival genes. The 18 histone deacetylases (HDACs) are epigenetic modulators that fall into four classes (I-IV). Inhibitors of histone deacetylases (HDACi) become increasingly appreciated as anti-cancer agents. Based on their effects on p53 and NF-κB, we addressed whether clinically relevant HDACi affect the NF-κB/p53 crosstalk. The chemotherapeuticshydroxyurea, etoposide, and fludarabine halt cell cycle progression, induce DNA damage, and lead to DNA fragmentation. These agents co-induce p53 and NF-κB-dependent gene expression in cell lines from breast and colon cancer and in primary chronic lymphatic leukemia (CLL) cells. Using specific HDACi, we find that the class I subgroup of HDACs, but not the class IIb deacetylase HDAC6, are required for the hydroxyurea-induced crosstalk between p53 and NF-κB. HDACi decrease the basal and stress-induced expression of p53 and block NF-κB-regulated gene expression. We further show that class I HDACi induce senescence in pancreatic cancer cells with mutant p53.

 

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Evaluierung und prognostische Bedeutung multiparametrischer Magnetresonanz-Bildgebungsmethoden im PDAC

1Institut für allgemeine Pathologie und pathologische Anatomie, TU München, 2Institut für diagnostische und interventionelle Radiologie, Klinikum rechts der Isar, München, 3II. Medizinische Klinik, Klinikum rechts der Isar, München, 4Nuklearmedizinis, 2016, publiziert am 04.11.2016
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Co-clinical assessment of tumor cellularity in pancreatic cancer

2016, (pdf) publiziert am 03.11.2016
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HDAC1 and HDAC2 integrate the expression of p53 mutants in pancreatic cancer

Oncogene advance online publication, 2016, doi:10.1038/onc.2016.344 publiziert am 10.10.2016
Mutation of p53 is a frequent genetic lesion in pancreatic cancer being an unmet clinical challenge. Mutants of p53 have lost the tumour-suppressive functions of wild type p53. In addition, p53 mutants exert tumour-promoting functions, qualifying them as important therapeutic targets. Here, we show that the class I histone deacetylases HDAC1 and HDAC2 contribute to maintain the expression of p53 mutants in human and genetically defined murine pancreatic cancer cells. Our data reveal that the inhibition of these HDACs with small molecule HDAC inhibitors (HDACi), as well as the specific genetic elimination of HDAC1 and HDAC2, reduce the expression of mutant p53 mRNA and protein levels. We further show that HDAC1, HDAC2 and MYC directly bind to the TP53 gene and that MYC recruitment drops upon HDAC inhibitor treatment. Therefore, our results illustrate a previously unrecognized class I HDAC-dependent control of the TP53 gene and provide evidence for a contribution of MYC. A combined approach targeting HDAC1/HDAC2 and MYC may present a novel and molecularly defined strategy to target mutant p53 in pancreatic cancer.

 

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Anovel pseudotyped vesicular stomatitis virus vector for improved safety and efficacy for therapy of hepatocellular carcinoma

2016, publiziert am 14.09.2016
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Immuno-PET Imaging of Engineered Human T Cells in Tumors

American Association for Cancer Research, 2016, doi: 10.1158/0008-5472.CAN-15-2784 publiziert am 15.07.2016
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Concepts to target MYC in pancreatic cancer

Medizinische Klinik, Technische Universität München, München, 81675, Germany 2Institute of Pharmacy, Faculty of Chemistry and Pharmacy, University of Regensburg, 93040, Germany 3Department of Toxicology, University of Mainz Medical Center, Mainz, 551, 2016, publiziert am 12.09.2016

Current data suggest that MYC is an important signaling hub and driver in pancreatic ductal adenocarcinoma (PDAC), a tumor entity with a strikingly poor prognosis. No targeted therapies with a meaningful clinical impact were successfully developed against PDAC so far. This points to the need to establish novel concepts targeting the relevant drivers of PDAC, like KRAS or MYC. Here, we discuss recent developments of direct or indirect MYCinhibitors and their potential mode of action in PDAC.

 

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Development of imaging strategies for investigation of TCR with defined antitumor reactivity in vivo

Medizinische Klinik III, Klinikum rechts der Isar, Technische Universität München, Munich, Germany 2 Institute of Biological and Medical Imaging, Helmholtz Zentrum München, Germany, 2016, publiziert am
T-cell based immunotherapies are novel and promising therapeutic approaches for a variety of malignant diseases. However, diverse approaches including those using T-cell receptor (TCR)- and chimeric antigen receptor (CAR)-transgenic T cells show highly different characteristics in vitro and in vivo. Preclinical in vivo models providing high predictive value with respect to tumor reactivity and toxicity or treatment failure due to tumor evasion are currently missing. For surveillance of therapeutic efficacy of adoptive T cell transfer, nuclear imaging has been used as a non-invasive and sensitive cell tracking technology, although limits in spatial resolution are given. We aimed to develop optoacoustic imaging as an alternative non-invasive and novel method to track TCR-transgenic T-cell responses in vivo. Multispectral optoacoustical imaging (MSOT) operates in the near-infrared (NIR) spectral region and allows deep penetration in tissue with high resolution. Cell dyes and reporter genes were tested as suitable tracers for detecting T cells with MSOT. T cells labeled with DiR, a stable cell membrane dye, presented bright fluorescence and strong absorption in the NIR spectrum. As an alternative labeling method, T cell were stably transduced with variations of the reporter gene near-infrared fluorescent protein (iRFP), in which the variation iRFP720 showed a higher brightness and a detectable signal by MSOT due to its higher emission in the near-infrared spectrum. T cells labeled with DiR, T cells expressing iRFP720 and T cells harboring both tracers were compared with respect to the limit of detection by MSOT in agarose phantoms and in vivo. For the in vivo analysis, T cells mixed with matrigel were subcutaneously injected in the back of a mouse. T cells labeled with DiR presented the most sensitive detection by MSOT both in phantoms and in vivo. However, in case of DiR-labeled T cells simultaneously expressing iRFP720, the detection of the DiR signal by MSOT was highly impaired and the sensitivity of the method decreased around 10 times. For T cells expressing iRFP alone, up to 2,5x106 cells could be detected in vivo by MSOT. A xenogenic mouse model of myeloid sarcoma was used and human central memory T cells (TCM) transgenic for the leukemia-specific TCR2.5D6 and subsequently labeled with DiR were adoptively transferred. MSOT imaging was performed at different time points post TCM cell transfer in order to investigate TCM-distribution in vivo over time. Tumor rejection correlated with the infiltration of DiR-labeled TCM cells in the ML2 B7 tumor, which was observed by epi-fluorescence imaging and confirmed with fluorescence microscopy and immunohistochemistry (IHC). MSOT analysis of the tumor region alone allowed detection of specific DiR-labeled TCM cell signal in the ML2 B7 tumor only, which correlates with the oxygenated areas of this tumor. Further analyses are necessary to confirm the infiltration and concentration of TCM cells preferentially in tumor areas with high oxygenation.

 

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Noninvasive In Vivo Imaging and Biologic Characterization of Thyroid Tumors by ImmunoPET Targeting of Galectin-3

American Association for Cancer Research, 2016, doi: 10.1158/0008-5472.CAN-15-3046 publiziert am 20.06.2016
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New drugs for medullary thyroid cancer: new promises?

Endocr Relat Cancer, 2016, doi: 10.1530/ERC-16-0104 publiziert am 16.05.2016
Medullary thyroid cancer (MTC) is a rare tumor arising from the calcitonin-producing parafollicular C cells of the thyroid gland, occurring either sporadically or alternatively in a hereditary form based on germline RET mutations in approximately one-third of cases. Historically, patients with advanced, metastasized MTC have had a poor prognosis, partly due to limited response to conventional chemotherapy and radiation therapy. In the past decade, however, considerable progress has been made in identifying key genetic alterations and dysregulated signaling pathways paving the way for the evaluation of a series of multitargeted kinase inhibitors that have started to meaningfully impact clinical practice. Two drugs, vandetanib and cabozantinib, are now approved in the US and EU for use in advanced, progressive MTC, with additional targeted agents also showing promise or awaiting results from clinical trials. However, the potential for toxicities with significant reduction in quality of life and lack of curative outcomes has to be carefully weighed against potential for benefit. Despite significant PFS prolongation observed in randomized clinical trials, most patients even with metastatic disease enjoy indolent courses with slow progression observed over years, wherein watchful waiting is still the preferred strategy. As advanced, progressive MTC is a rare and complex disease, a multidisciplinary approach centered in specialized centers providing interdisciplinary expertise in the individualization of available therapeutic options is preferred. In this review, we summarize current concepts of the molecular pathogenesis of advanced MTC and discuss results from clinical trials of targeted agents and also cytotoxic chemotherapy in the context of clinical implications and future perspectives.

 

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Modeling Therapy Response and Spatial Tissue Distribution of Erlotinib in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is likely the most aggressive and therapy-resistant of all cancers. The aim of this study was to investigate the emerging technology of matrixassisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) as a powerful tool to study drug delivery andspatial tissue distribution in PDAC. We utilized an established genetically engineered mouse model of spontaneous PDAC to examine the distribution of the small-molecule inhibitor erlotinib in healthy pancreas and PDAC. MALDI IMS was utilized on sections of single-dose or long-term–treated mice to measure drug tissue distribution. Histologic and statistical analyses were performed to correlate morphology, drug distribution, and survival. We found that erlotinib levels were significantly lower in PDAC compared with healthy tissue (P ¼ 0.0078). Survival of long-term–treated mice did not correlate with overall levels of erlotinib or with overall histologic tumor grade but did correlate both with the percentage of atypical glands in the cancer (P ¼ 0.021, rs ¼ 0.59) and the level of erlotinib in those atypical glands (P ¼ 0.019, rs ¼ 0.60). The results of this pilot study present MALDI IMS as a reliable technology to study drug delivery and spatial distribution of compounds in a preclinical setting and support drug imaging– based translational approaches. Mol Cancer Ther; 15(5); 1–8. 2016 AACR.

 

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Determination of the detection limit of T cell receptor-transgenic effector T cells for quantification of the effective dose by PET/CT imaging

1. Department of Nuclear Medicine, Klinikum rechts der Isar, Technische Universität München, Munich, Germany 2. Department of Medicine lll, Klinikum rechts der Isar , Technische Universität München, Munich, Germany, 2016, publiziert am 12.09.2016
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KrasG12D induces EGFR-MYC cross signaling in murine primary pancreatic ductal epithelial cells

Oncogene © 2015 Macmillan Publishers Limited, 2015, doi:10.1038/onc.2015.437 publiziert am 23.11.2015

Epidermal growth factor receptor (EGFR) signaling has a critical role in oncogenic Kras-driven pancreatic carcinogenesis. However, the downstream targets of this signaling network are largely unknown. We developed a novel model system utilizing murine primary pancreatic ductal epithelial cells (PDECs), genetically engineered to allow time-specific expression of oncogenic KrasG12D from the endogenous promoter. We show that primary PDECs are susceptible to KrasG12D-driven transformation and form pancreatic ductal adenocarcinomas in vivo after Cdkn2a inactivation. In addition, we demonstrate that activation of KrasG12D induces an EGFR signaling loop to drive proliferation. Interestingly, pharmacological inhibition of EGFR fails to decrease KrasG12D-activated ERK or PI3K signaling. Instead our data provide novel evidence that EGFR signaling is needed to activate the oncogenic and pro-proliferative transcription factor c-MYC. EGFR and c-MYC have been shown to be essential for pancreatic carcinogenesis. Importantly, our data link both pathways and thereby explain the crucial role of EGFR for KrasG12D-driven carcinogenesis in the pancreas.

 

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Combined inhibition of BET family proteins and histone deacetylases as a potential epigenetics-based therapy for pancreatic ductal adenocarcinoma

AACR-Konferenz, 2015, publiziert am 15.10.2015
 
Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers and shows resistance to any therapeutic strategy used. Here we tested small-molecule inhibitors targeting chromatin regulators as possible therapeutic agents in PDAC. We show that JQ1, an inhibitor of the bromodomain and extraterminal (BET) family of proteins, suppresses PDAC development in mice by inhibiting both MYC activity and inflammatory signals. The histone deacetylase (HDAC) inhibitor SAHA synergizes with JQ1 to augment cell death and more potently suppress advanced PDAC. Finally, using a CRISPR-Cas9–based method for gene editing directly in the mouse adult pancreas, we show that de-repression of p57 (also known as KIP2 or CDKN1C) upon combined BET and HDAC inhibition is required for the induction of combination therapy–induced cell death in PDAC. SAHA is approved for human use, and molecules similar to JQ1 are being tested in clinical trials. Thus, these studies identify a promising epigenetic- based therapeutic strategy that may be rapidly implemented in fatal human tumors.

 

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PI3K signaling in pancreatic cancer progression and maintenance

47th Annual Meeting of the European Pancreatic Club, 2015, publiziert am 03.08.2015
Genetically engineered mouse models have dramatically improved our understanding of tumor evolution and therapeutic resistance. Recently, we showed that cell-autonomous PI3K and Pdk1 are key effectors of oncogenic Kras in the pancreas, mediating formation of pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma (PDAC). Pdk1 deletion blocked pancreatic carcinogenesis completely in a Cre/loxP-based model indicating its importance in tumor initiation. However, sequential genetic manipulation of gene expression is almost impossible using traditional Cre/loxP-based models.

 

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Model Matters: Differences in Orthotopic Rat Hepatocellular Carcinoma Physiology Determine Therapy Response to Sorafenib

American Association for Cancer Research, 2015, doi: 10.1158/1078-0432.CCR-14-2018 publiziert am 20.05.2015
Purpose: Preclinical model systems should faithfully reflect the complexity of the human pathology. In hepatocellular carcinoma (HCC), the tumor vasculature is of particular interest in diagnosis and therapy. By comparing two commonly applied preclinical model systems, diethylnitrosamine induced (DEN) and orthotopically implanted (McA) rat HCC, we aimed to measure tumor biology noninvasively and identify differences between the models. Experimental Design: DEN and McA tumor development was monitored by MRI and PET. A slice-based correlation of imaging and histopathology was performed. Array CGH analyses were applied to determine genetic heterogeneity. Therapy response to sorafenib was tested in DEN and McA tumors. Results: Histologically and biochemically confirmed liver damage resulted in increased 18F-fluorodeoxyglucose (FDG) PET uptake and perfusion in DEN animals only. DEN tumors exhibited G1–3 grading compared with uniform G3 grading of McA tumors. Array comparative genomic hybridization revealed a highly variable chromosomal aberration pattern in DEN tumors. Heterogeneity of DEN tumors was reflected in more variable imaging parameter values. DEN tumors exhibited lower mean growth rates and FDG uptake and higher diffusion and perfusion values compared with McA tumors. To test the significance of these differences, the multikinase inhibitor sorafenib was administered, resulting in reduced volume growth kinetics and perfusion in the DEN group only. Conclusions: This work depicts the feasibility and importance of in depth preclinical tumor model characterization and suggests the DEN model as a promising model system of multifocal nodular HCC in future therapy studies. Clin Cancer Res; 21(19);1–11. _2015 AACR.See related commentary by Weber et al., p. 4254

 

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STAT3 inhibition reduces toxicity of oncolytic VSV and provides a potentially synergistic combination therapy for hepatocellular carcinoma

Cancer Gene Therapy advance online publication, 2015, doi:J 0.J 038/cgt.2015.23 publiziert am 01.05.2015
Hepatocellular carcinoma(HCC) is a refractory malignancy with a high mortality and increasing worldwide incidence rates, including the United States and central Europe. In this study, we demonstrate that a specific inhibitor of signal transducer and activator of transcription 3 (STAT3), NSC748S9, efficiently reduces HCC cell proliferation and can be successfully combined with oncolyic virotherapy using vehicular stomatitis virus (VSV). The potential benefits of this combination treatment are strengthened by the ability of NSC74859 to protect primary hepatocytes and nervous system cells against virus-induced cytotoxicity, with an elevation of the VSV maximum tolerated dose in mice. Hereby we propose a strategy for improving the current regimen for HCC treatment and seek to further explore the molecular mechanisms underlying selective oncolytic specificity of VSV.

 

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In vivo molecular imaging of chemokine receptor CXCR4 expression in patients with advanced multiple myeloma

EMBO Mol Med, 2015, doi: 10.15252/emmm.201404698 publiziert am 06.03.2015
CXCR4 is a G-protein-coupled receptor that mediates recruitment of blood cells toward its ligand SDF-1. In cancer, high CXCR4 expression is frequently associated with tumor dissemination and poor prognosis. We evaluated the novel CXCR4 probe [68Ga]Pentixafor for in vivo mapping of CXCR4 expression density in mice xenografted with human CXCR4-positive MM cell lines and patients with advanced MM by means of positron emission tomography (PET). [68Ga]Pentixafor PET provided images with excellent specificity and contrast. In 10 of 14 patients with advanced MM [68Ga]Pentixafor PET/CT scans revealed MM manifestations, whereas only nine of 14 standard [18F]fluorodeoxyglucose PET/CT scans were rated visually positive. Assessment of blood counts and standard CD34+ flow cytometry did not reveal significant blood count changes associated with tracer application. Based on these highly encouraging data on clinical PET imaging of CXCR4 expression in a cohort of MM patients, we conclude that [68Ga]Pentixafor PET opens a broad field for clinical investigations on CXCR4 expression and for CXCR4-directed therapeutic approaches in MM and other diseases.

 

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Disclosing the CXCR4 Expression in Lymphoproliferative Diseases by Targeted Molecular Imaging

Theranostics 2015; 5(6):618-630, 2015, doi:10.7150/thno.11251 publiziert am 06.03.2015
Chemokine ligand-receptor interactions play a pivotal role in cell attraction and cellular trafficking, both in normal tissue homeostasis and in disease. In cancer, chemokine receptor-4 (CXCR4) expression is an adverse prognostic factor. Early clinical studies suggest that targeting CXCR4 with suitable high-affinity antagonists might be a novel means for therapy. In addition to the preclinical evaluation of [68Ga]Pentixafor in mice bearing human lymphoma xenografts as an exemplary CXCR4-expressing tumor entity, we report on the first clinical applications of [68Ga]Pentixafor-Positron Emission Tomography as a powerful method for CXCR4 imaging in cancer patients. [68Ga]Pentixafor binds with high affinity and selectivity to human CXCR4 and exhibits a favorable dosimetry. [68Ga]Pentixafor-PET provides images with excellent specificity and contrast. This non-invasive imaging technology for quantitative assessment of CXCR4 expression allows to further elucidate the role of CXCR4/CXCL12 ligand interaction in the pathogenesis and treatment of cancer, cardiovascular diseases and autoimmune and inflammatory disorders.

 

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Biodistribution and Radiation Dosimetry for the Chemokine Receptor CXCR4-Targeting Probe 68Ga-Pentixafor

J Nucl Med, 2015, doi: 10.2967/jnumed.114.151647 publiziert am 06.03.2015
68Ga-pentixafor is a promising PET tracer for imaging the expression of the human chemokine receptor 4 (CXCR4) in vivo. The whole-body distribution and radiation dosimetry of 68Ga-pentixafor were evaluated. Methods: Five multiple-myeloma patients were injected intravenously with 90–158 MBq of 68Ga-pentixafor (mean ± SD, 134 ± 25 MBq), and a series of 3 rapid multiple-bed-position whole-body scans were acquired immediately afterward. Subsequently, 4 static whole-body scans followed at 30 min, 1 h, 2 h, and 4 h after administration of the radiopharmaceutical. Venous blood samples were obtained. Time-integrated activity coefficients were determined from multiexponential regression of organ region-of-interest data normalized to the administered activity, for example, the time-dependent percentages of the injected activity per organ. Mean organ-absorbed doses and effective doses were calculated using OLINDA/EXM. Results: The effective dose based on 150 MBq of 68Ga-pentixafor was 2.3 mSv. The highest organ-absorbed doses (for 150 MBq injected) were found in the urinary bladder wall (12.2 mGy), spleen (8.1 mGy), kidneys (5.3 mGy), and heart wall (4.0 mGy). Other organ mean absorbed doses were as follows: 2.7 mGy, liver; 2.1 mGy, red marrow; 1.7 mGy, testes; and 1.9 mGy, ovaries. Conclusion: 68Ga-pentixafor exhibits a favorable dosimetry, delivering absorbed doses to organs that are lower than those delivered by 18F-FDG– or 68Ga-labeled somatostatin receptor ligands.

 

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PET Imaging of Oncolytic VSV Expressing the Mutant HSV-1 Thymidine Kinase Transgene in a Preclinical HCC Rat Model

The American Society of Gene & Cell Therapy, 2015, doi:10.1038/mt.2015.12 publiziert am 17.02.2015
Hepatocellular carcinoma (HCC) is the most predominant form of liver cancer and the third leading cause of cancerrelated death worldwide. Due to the relative ineffectiveness of conventional HCC therapies, oncolytic viruses have emerged as novel alternative treatment agents. Our previous studies have demonstrated significant prolongation of survival in advanced HCC in rats after oncolytic vesicular stomatitis virus (VSV) treatment. In this study, we aimed to establish a reporter system to reliably and sensitively image VSV in a clinically relevant model of HCC for clinical translation. To this end, an orthotopic, unifocal HCC model in immune-competent Buffalo rats was employed to test a recombinant VSV vector encoding for an enhanced version of the herpes simplex virus 1 (HSV-1) thymidine kinase (sr39tk) reporter, which would allow the indirect detection of VSV via positron emission tomography (PET). The resulting data revealed specific tracer uptake in VSV-HSV1-sr39tk–treated tumors. Further characterization of the VSV-HSV1-sr39tk vector demonstrated its optimal detection time-point after application and its detection limit via PET. In conclusion, oncolytic VSV expressing the HSV1-sr39tk reporter gene allows for highly sensitive in vivo imaging via PET. Therefore, this imaging system may be directly translatable and beneficial in further clinical applications. Received 8 August 2014; accepted 12 January 2015; advance onlinepublication 17 February 2015. doi:10.1038/mt.2015.

 

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Direct Parametric Image Reconstruction in Reduced Parameter Space for Rapid Multi-Tracer PET Imaging

IEEE Trans Med Imaging, 2015, doi: 10.1109/TMI.2015.2403300 publiziert am 06.03.2015
The separation of multiple PET tracers within an overlapping scan based on intrinsic differences of tracer pharmacokinetics is challenging, due to limited signal-to-noise ratio (SNR) of PET measurements and high complexity of fitting models. In this study, we developed a direct parametric image reconstruction (DPIR) method for estimating kinetic parameters and recovering single tracer information from rapid multi-tracer PET measurements. This is achieved by integrating a multi-tracer model in a reduced parameter space (RPS) into dynamic image reconstruction. This new RPS model is reformulated from an existing multi-tracer model and contains fewer parameters for kinetic fitting. Ordered-subsets expectation-maximization (OSEM) was employed to approximate log-likelihood function with respect to kinetic parameters. To incorporate the multi-tracer model, an iterative weighted nonlinear least square (WNLS) method was employed. The proposed multi-tracer DPIR (MTDPIR) algorithm was evaluated on dual-tracer PET simulations ([18F]FDG and [11C]MET) as well as on preclinical PET measurements ([18F]FLT and [18F]FDG). The performance of the proposed algorithm was compared to the indirect parameter estimation method with the original dual-tracer model. The respective contributions of the RPS technique and the DPIR method to the performance of the new algorithm were analyzed in detail. For the preclinical evaluation, the tracer separation results were compared with single [18F]FDG scans of the same subjects measured 2 days before the dual-tracer scan. The results of the simulation and preclinical studies demonstrate that the proposed MT-DPIR method can improve the separation of multiple tracers for PET image quantification and kinetic parameter estimations.

 

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Disruption of the PRKCD-FBXO25-HAX-1 axis attenuates the apoptotic response and drives lymphomagenesis

Nature Medicine, 2014, doi: 10.1038/nm.3740 publiziert am 06.03.2015
We searched for genetic alterations in human B cell lymphoma that affect the ubiquitin-proteasome system. This approach identified FBXO25 within a minimal common region of frequent deletion in mantle cell lymphoma (MCL). FBXO25 encodes an orphan F-box protein that determines the substrate specificity of the SCF (SKP1-CUL1-F-box)(FBXO25) ubiquitin ligase complex. An unbiased screen uncovered the prosurvival protein HCLS1-associated protein X-1 (HAX-1) as the bona fide substrate of FBXO25 that is targeted after apoptotic stresses. Protein kinase Cδ (PRKCD) initiates this process by phosphorylating FBXO25 and HAX-1, thereby spatially directing nuclear FBXO25 to mitochondrial HAX-1. Our analyses in primary human MCL identify monoallelic loss of FBXO25 and stabilizing HAX1 phosphodegron mutations. Accordingly, FBXO25 re-expression in FBXO25-deleted MCL cells promotes cell death, whereas expression of the HAX-1 phosphodegron mutant inhibits apoptosis. In addition, knockdown of FBXO25 significantly accelerated lymphoma development in Eμ-Myc mice and in a human MCL xenotransplant model. Together we identify a PRKCD-dependent proapoptotic mechanism controlling HAX-1 stability, and we propose that FBXO25 functions as a haploinsufficient tumor suppressor and that HAX1 is a proto-oncogene in MCL.

 

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GP130 activation induces myeloma and collaborates with MYC

J Clin Invest, 2014, doi: 10.1172/JCI69094 publiziert am 06.03.2015
Multiple myeloma (MM) is a plasma cell neoplasm that results from clonal expansion of an Ig-secreting terminally differentiated B cell. Advanced MM is characterized by tissue damage that involves bone, kidney, and other organs and is typically associated with recurrent genetic abnormalities. IL-6 signaling via the IL-6 signal transducer GP130 has been implicated as an important driver of MM pathogenesis. Here, we demonstrated that ectopic expression of constitutively active GP130 (L-GP130) in a murine retroviral transduction-transplantation model induces rapid MM development of high penetrance. L-GP130-expressing mice recapitulated all of the characteristics of human disease, including monoclonal gammopathy, BM infiltration with lytic bone lesions, and protein deposition in the kidney. Moreover, the disease was easily transplantable and allowed different therapeutic options to be evaluated in vitro and in vivo. Using this model, we determined that GP130 signaling collaborated with MYC to induce MM and was responsible and sufficient for directing the plasma cell phenotype. Accordingly, we identified Myc aberrations in the L-GP130 MM model. Evaluation of human MM samples revealed recurrent activation of STAT3, a downstream target of GP130 signaling. Together, our results indicate that deregulated GP130 activity contributes to MM pathogenesis and that pathways downstream of GP130 activity have potential as therapeutic targets in MM.

 

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A next-generation dual-recombinase system for time- and host-specific targeting of pancreatic cancer

Nature America, 2014, doi:10.1038/nm.3646 publiziert am 17.02.2015
Genetically engineered mouse models (GEMMs) have dramatically improved our understanding of tumor evolution and therapeutic resistance. However, sequential genetic manipulation of gene expression and targeting of the host is almost impossible using conventional Cre-loxP–based models. We have developed an inducible dual-recombinase system by combining flippase-FRT (Flp-FRT) and Cre-loxP recombination technologies to improve GEMMs of pancreatic cancer. This enables investigation of multistep carcinogenesis, genetic manipulation of tumor subpopulations (such as cancer stem cells), selective targeting of the tumor microenvironment and genetic validation of therapeutic targets in autochthonous tumors on a genome-wide scale. As a proof of concept, we performed tumor cell–autonomous and nonautonomous targeting, recapitulated hallmarks of human multistep carcinogenesis, validated genetic therapy by 3-phosphoinositide-dependent protein kinase inactivation as well as cancer cell depletion and show that mast cells in the tumor microenvironment, which had been thought to be key oncogenic players, are dispensable for tumor formation.

 

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A novel approach of MALDI drug imaging, immunohistochemistry, and digital image analysis for drug distribution studies in tissues

Anal. Chem., Just Accepted Manuscript, 2014, DOI: 10.1021/ac502177y publiziert am 28.09.2014
Drug efficacy strongly depends on the presence of the drug substance at the target site. As vascularization is an important factor for the distribution of drugs in tissues, we analyzed drug distribution as a function of blood vessel localization in tumor tissue. In order to explore distribution of the anti-cancer drugs afatinib, erlotinib, and sorafenib, a combined approach of matrix-assisted laser desorption/ionization (MALDI) drug imaging and immunohistochemical vessel staining was applied and examined by digital image analysis. Two xenograft models were investigated: (1) mice carrying squamous cell carcinoma (FaDu) xenografts (ntumor=13) were treated with afatinib or erlotinib, and (2) sarcoma (A673) xenograft bearing mice (ntumor=8) received sorafenib treatment. MALDI drug imaging revealed a heterogeneous distribution of all anti-cancer drugs. The tumor regions containing high drug levels were associated with a higher degree of vascularization than the regions without drug signals (p<0.05). When correlating the impact of blood vessel size to drug abundance in the sarcoma model, a higher amount of small vessels was detected in the tumor regions with high drug levels compared to the tumor regions with low drug levels (p<0.05). With the analysis of co-registered MALDI imaging and CD31 immunohistochemical data by digital image analysis, we demonstrate for the first time the potential of correlating MALDI drug imaging and immunohistochemistry. Here we describe a specific and precise approach for correlating histological features and pharmacokinetic properties of drugs at microscopic level, that will provide information for the improvement of drug design, administration formula or treatment schemes.

 

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Myc-induced SUMOylation is a therapeutic vulnerability for B-cell lymphoma

Blood: 124 (13), 2014, http://dx.doi.org/10.1182/blood-2014-06-584524 publiziert am 25.09.2014
Myc oncogenic transcription factors (c-Myc, N-Myc, and L-Myc) coordinate the control of cell growth, division, and metabolism. In cancer, Myc overexpression is often associated with aggressive disease, which is in part due to the destruction of select targets by the ubiquitin-proteasome system (eg, SCFSkp2-directed destruction of the Cdk inhibitor p27Kip1). We reasoned that Myc would also regulate SUMOylation, a related means of posttranslational modification of proteins, and that this circuit would play essential roles in Myc-dependent tumorigenesis. Here, we report marked increases in the expression of genes that encode regulators and components of the SUMOylation machinery in mouse and human Myc-driven lymphomas, resulting in hyper-SUMOylation in these tumors. Further, inhibition of SUMOylation by genetic means disables Myc-induced proliferation, triggering G2/M cell-cycle arrest, polyploidy, and apoptosis. Using genetically defined cell models and conditional expression systems, this response was shown to be Myc specific. Finally, in vivo loss-of-function and pharmacologic studies demonstrated that inhibition of SUMOylation provokes rapid regression of Myc-driven lymphoma. Thus, targeting SUMOylation represents an attractive therapeutic option for lymphomas with MYC involvement.

 

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MYC and EGR1 synergize to trigger tumor cell death by controlling NOXA and BIM transcription upon treatment with the proteasome inhibitor bortezomib

Nucleic Acids Research, 2014, Vol. 42, No. 16, 10433–10447, 2014, DOI 10.1093/nar/gku763 publiziert am 21.08.2014
The c-MYC (MYC afterward) oncogene is well known for driving numerous oncogenic programs. However, MYC can also induce apoptosis and this function of MYC warrants further clarification. We report here that a clinically relevant proteasome inhibitor significantly increases MYC protein levels and that endogenous MYC is necessary for the induction of apoptosis. This kind of MYC-induced cell death is mediated by enhanced expression of the pro-apoptotic BCL2 family members NOXA and BIM. Quantitative promoter-scanning chromatin immunoprecipitations (qChIP) further revealed binding of MYC to the promoters of NOXA and BIM upon proteasome inhibition, correlating with increased transcription. Both promoters are further characterized by the presence of tri-methylated lysine 4 of histone H3, marking active chromatin. We provide evidence that in our apoptosis models cell death occurs independently of p53 or ARF. Furthermore, we demonstrate that recruitment of MYC to the NOXA as well as to the BIM gene promoters depends on MYC’s interaction with the zinc finger transcription factor EGR1 and an EGR1-binding site in both promoters. Our study uncovers a novel molecular mechanism by showing that the functional cooperation of MYC with EGR1 is required for bortezomib-induced cell death. This observation may be important for novel therapeutic strategies engaging the inherent pro-death function of MYC.

 

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BET and HDAC inhibitors induce similar genes and biological effects and synergize to kill in Myc-induced murine lymphoma

PNAS, 2014, doi: 10.1073/pnas.1406722111 publiziert am 16.06.2014

The bromodomain and extraterminal (BET) domain family of proteins binds to acetylated lysines on histones and regulates gene transcription. Recently, BET inhibitors (BETi) have been developed that show promise as potent anticancer drugs against various solid and hematological malignancies. Here we show that the structurally novel and orally bioavailable BET inhibitor RVX2135 inhibits proliferation and induces apoptosis of lymphoma cells arising in Myc-transgenic mice in vitro and in vivo. We find that BET inhibition exhibits broad transcriptional effects in Myc-transgenic lymphoma cells affecting many transcription factor networks. By examining the genes induced by BETi, which have largely been ignored to date, we discovered that these were similar to those induced by histone deacetylase inhibitors (HDACi). HDACi also induced cell-cycle arrest and cell death of Myc-induced murine lymphoma cells and synergized with BETi. Our data suggest that BETi sensitize Myc-overexpressing lymphoma cells partly by inducing HDAC-silenced genes, and suggest synergistic and therapeutic combinations by targeting the genetic link between BETi and HDACi.

 

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Advanced radioiodine-refractory differentiated thyroid cancer: the sodium iodide symporter and other emerging therapeutic targets.

Lancet Diabetes Endocrinol, 2014, DOI: 10.1016/S2213-8587(14)70051-8 publiziert am 02.06.2014
Approximately 30% of patients with advanced, metastatic differentiated thyroid cancer have radioiodine-refractory disease, based on decreased expression of the sodium iodide symporter SLC5A5 (NIS), diminished membrane targeting of NIS, or both. Patients with radioiodine-refractory disease, therefore, are not amenable to (131)I therapy, which is the initial systemic treatment of choice for non-refractory metastatic thyroid cancer. Patients with radioiodine-refractory cancer have historically had poor outcomes, partly because these cancers often respond poorly to cytotoxic chemotherapy. In the past decade, however, considerable progress has been made in delineating the molecular pathogenesis of radioiodine-refractory thyroid cancer. As a result of the identification of key genetic and epigenetic alterations and dysregulated signalling pathways, multiple biologically targeted drugs, in particular tyrosine-kinase inhibitors, have been evaluated in clinical trials with promising results and have begun to meaningfully impact clinical practice. In this Review, we summarise the current knowledge of the molecular pathogenesis of advanced differentiated thyroid cancer and discuss findings from clinical trials of targeted drugs in patients with radioiodine-refractory disease. Additionally, we focus on the molecular basis of loss of NIS expression, function, or both in refractory disease, and discuss preclinical and clinical data on restoration of radioiodine uptake.

 

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Positron emission tomographic monitoring of dual phosphatidylinositol-3-kinase and mTOR inhibition in anaplastic large cell lymphoma.

NCBI, 2014, 7:789-98, doi: 10.2147/OTT.S59314 publiziert am 23.05.2014

BACKGROUND:

Dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition offers an attractive therapeutic strategy in anaplastic large cell lymphoma depending on oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) signaling. We tested the efficacy of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in two anaplastic large cell lymphoma cell lines in vitro and in vivo and performed an early response evaluation with positron emission tomography (PET) imaging using the standard tracer, 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) and the thymidine analog, 3'-deoxy-3'-[(18)F] fluorothymidine (FLT).

METHODS:

The biological effects of BGT226 were determined in vitro in the NPM-ALK positive cell lines SU-DHL-1 and Karpas299 by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, propidium iodide staining, and biochemical analysis of PI3K and mTOR downstream signaling. FDG-PET and FLT-PET were performed in immunodeficient mice bearing either SU-DHL-1 or Karpas299 xenografts at baseline and 7 days after initiation of treatment with BGT226. Lymphomas were removed for immunohistochemical analysis of proliferation and apoptosis to correlate PET findings with in vivo treatment effects.

RESULTS:

SU-DHL-1 cells showed sensitivity to BGT226 in vitro, with cell cycle arrest in G0/G1 phase and an IC50 in the low nanomolar range, in contrast with Karpas299 cells, which were mainly resistant to BGT226. In vivo, both FDG-PET and FLT-PET discriminated sensitive from resistant lymphoma, as indicated by a significant reduction of tumor-to-background ratios on day 7 in treated SU-DHL-1 lymphoma-bearing animals compared with the control group, but not in animals with Karpas299 xenografts. Imaging results correlated with a marked decrease in the proliferation marker Ki67, and a slight increase in the apoptotic marker, cleaved caspase 3, as revealed by immunostaining of explanted lymphoma tissue.

CONCLUSION:

Dual PI3K/mTOR inhibition using BGT226 is effective in ALK-positive anaplastic large cell lymphoma and can be monitored with both FDG-PET and FLT-PET early on in the course of therapy.

 

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Week one FLT-PET response predicts complete remission to R-CHOP and survival in DLBCL

Impact Journals - Oncotarget, 2014, Online ISSN: 1949-2553 publiziert am 19.05.2014

Despite improved survival in the Rituximab (R) era, a considerable number of patients with diffuse large B-cell lymphoma (DLBCL) ultimately die from the disease. Functional imaging using [18F]fluorodeoxyglucose-PET is suggested for assessment of residual viable tumor very early during treatment but is compromised by non-specific tracer retention in inflammatory lesions. The PET tracer [18F]fluorodeoxythymidine (FLT) as surrogate marker of tumor proliferation may overcome this limitation. We present results of a prospective clinical study testing FLT-PET as superior and early predictor of response to chemotherapy and outcome in DLBCL. 54 patients underwent FLT-PET prior to and one week after the start of R-CHOP chemotherapy. Repetitive FLT-PET imaging was readily implemented into the diagnostic work-up. Our data demonstrate that the reduction of FLT standard uptake valuemean (SUVmean) and SUVmax one week after chemotherapy was significantly higher in patients achieving complete response (CR, n=48; non-CR, n=6; p<0.006). Martingale-residual and Cox proportional hazard analyses showed a significant monotonous decrease of mortality risk with increasing change in SUV. Consistent with these results, early FLT-PET response showed relevant discriminative ability in predicting CR. In conclusion, very early FLT-PET in the course of R-CHOP chemotherapy is feasible and enables identification of patients at risk for treatment failure.

 

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Therapeutic targeting of naturally presented myeloperoxidase-derived HLA peptide ligands on myeloid leukemia cells by TCR-transgenic T cells

Leukemia, 2014, 1-12, doi:10.1038/leu.2014.131 publiziert am 13.05.2014
T cells have been proven to be therapeutically effective in patients with relapsed leukemias, although target antigens on leukemic cells as well as T-cell receptors (TCRs), potentially recognizing those antigens, are mostly unknown. We have applied an immunopeptidomic approach and isolated human leukocyte antigen (HLA) ligands from primary leukemia cells. We identified a number of ligands derived from different genes that are restrictedly expressed in the hematopoietic system. We exemplarily selected myeloperoxidase (MPO) as a potential target and isolated a high-avidity TCR with specificity for a HLA-B*07:02-(HLA-B7)-restricted epitope of MPO in the single HLA-mismatched setting. T cells transgenic for this TCR demonstrated high peptide and antigen specificity as well as leukemia reactivity in vitro and in vivo. In contrast, no significant on- and off-target toxicity could be observed. In conclusion, we here demonstrate, exemplarily for MPO, that leukemia-derived HLA ligands can be selected for specific effector tool development to redirect T cells to be used for graft manipulation or adoptive T-cell therapies in diverse transplant settings. This approach can be extended to other HLA ligands and HLA molecules in order to provide better treatment options for this life-threatening disease.

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Sorting out Pandora´s Box: discerning the dynamic roles of liver microenvironment in oncolytic virus therapy for hepatocellular carcinoma

frontiers in Oncology, 2014, 4, doi: 10.3389 / fonc.2014.00085 publiziert am 22.04.2014
Oncolytic viral therapies have recently found their way into clinical application for hepatocellular carcinoma (HCC), a disease with limited treatment options and poor prognosis. Adding to the many intrinsic challenges of in vivo oncolytic viral therapy, is the complex microenvironment of the liver, which imposes unique limitations to the successful delivery and propagation of the virus. The normal liver milieu is characterized by an intricate network of hepatocytes and non-parenchymal cells including Kupffer cells, stellate cells, and sinusoidal endothelial cells, which can secrete anti-viral cytokines, provide a platform for non-specific uptake, and form a barrier to efficient viral spread. In addition, natural killer cells are greatly enriched in the liver, contributing to the innate defense against viruses.The situation is further complicated when HCC arises in the setting of underlying hepatitis virus infection and/or hepatic cirrhosis, which occurs in more than 90% of clinical cases. These conditions pose further inhibitory effects on oncolytic virus (OV) therapy due to the presence of chronic inflammation, constitutive cytokine expression, altered hepatic blood flow, and extracellular matrix deposition. In addition, OVs can modulate the hepatic microenvironment, resulting in a complex interplay between virus and host. The immune system undoubtedly plays a substantial role in the outcome of OV therapy, both as an inhibitor of viral replication, and as a potent mechanism of virus-mediated tumor cell killing.This review will discuss the particular challenges of oncolytic viral therapy for HCC, as well as some potential strategies for modulating the immune system and synergizing with the hepatic microenvironment to improve therapeutic outcome.

 

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Limited-projection-angle hybrid fluorescence molecular tomography of multiple molecules

Journal of Biomedical Optics, April 2014, 19 (4), 046016-1 publiziert am 01.04.2014
An advantage of fluorescence methods over other imaging modalities is the ability to concurrently resolve multiple moieties using fluorochromes emitting at different spectral regions. Simultaneous imaging of spectrally separated agents is helpful in interrogating multiple functions or establishing internal controls for accurate measurements. Herein, we investigated multimoiety imaging in the context of a limited-projection-angle hybrid fluorescence molecular tomography (FMT), and x-ray computed tomography implementation and the further registration with positron emission tomography (PET) data. Multichannel FMT systems may image fluorescent probes of varying distribution patterns. Therefore, it is possible that different channels may require different use of priors and regularization parameters. We examined the performance of automatically estimating regularization factors implementing priors, using data-driven regularization specific for limited-projectionangle schemes. We were particularly interested in identifying the implementation variations between hybrid FMT channels due to probe distribution variation. For this reason, initial validation of the data-driven algorithm on a phantom was followed by imaging different agent distributions in animals, assuming superficial and deep seated activity. We further demonstrate the benefits of combining hybrid FMT with PET to gain multiple readings on the molecular composition of disease.

 

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Molecular imaging for early prediction of response to Sorafenib treatment in sarcoma

AJNMMI, 2013, 70-79, PMCID: PMC3867731 publiziert am 15.12.2013

The role of [18F]fluorodeoxyglucose ([18F]FDG) PET in staging of sarcoma is well established. The aim of this preclinical study was to compare [18F]fluorothymidine ([18F]FLT) PET to [18F]FDG PET regarding early metabolic changes of sarcoma in the course of targeted cancer therapy. SCID mice bearing sarcoma A673 xenotransplants were used for investigation of tumor response after treatment with the multikinase inhibitor Sorafenib. [18F]FLT and/or [18F]FDG-PET were performed prior to and early after initiation of treatment. Tumoral uptake (% Injected Dose per gram (%ID/g) of [18F]FLT-PET was compared to [18F]FDG-PET. Results were correlated with histopathology and in vitro data including cellular uptake, cell cycle-related protein expression, cell cycle distribution and apoptosis. In vitro experiments showed that A673 cells were sensitive to Sorafenib. In vivo, tumor growth was inhibited in comparison to a 4-fold increase of the tumor volume in control mice. Using [18F]FDG as tracer, a moderate reduction in tracer uptake (n=15, mean relative %ID/g 74%, range 35%-121%, p=0.03) was observed. The decrease in %ID/g using [18F]FLT-PET was significantly higher (p=0.003). The mean relative %ID/g in [18F]FLT uptake on day + 5 was significantly reduced to 54% compared to baseline (n=15, range 24%-125%, SD=29%). The PET analysis 24 hr after therapy showed a significant reduction of the mean [18F]FLT-%ID/g (p=0.04). The reduction of %ID/g on day + 1 in [18F]FDG-PET was not statistically significant (p=0.99). In conclusion, both [18F]FDG- and [18F]FLT-PET were able to predict response to Sorafenib treatment. In contrast to [18F]FDG-PET, [18F]FLT-PET was more predictive for very early response to treatment.

 

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Antifibrotic Properties of Transarterial Oncolytic VSV Therapy for Hepatocellular Carcinoma in Rats with Thioacetamide-Induced Liver Fibrosis

Molecular Therapy, 2013, mt.2013.181, doi:10.1038 publiziert am 24.09.2013
Recombinant  vesicular  stomatitis  virus  (VSV)  shows promise for the treatment of hepatocellular carcinoma  (HCC), but its safety and efficacy when administered in a setting of hepatic fibrosis, which occurs in the majority of clinical cases, is unknown. We hypothesized that VSV could provide a novel benefit to the underlying fibrosis, due to its ability to replicate and cause cell death specifically in activated hepatic stellate cells. In addition to the ability of VSV to produce a significant oncolytic response in HCC-bearing rats in the background of thioacetamideinduced hepatic fibrosis without signs of hepatotoxicity, we observed a significant downgrading of fibrosis stage, a decrease in collagen content in the liver, and modulation  of  gene  expression  in  favor  of  fibrotic  regression. Together, this work  suggests  that  VSV  is  not  only  safe and effective for the treatment of HCC with underlying fibrosis, but it could potentially be developed for clinical application as a novel antifibrotic agent.

Synthetic lethal metabolic targeting of cellular senescence in cancer therapy

Nature, 2013, doi: 10.1038, nature12437 publiziert am 14.08.2013
Activated oncogenes and anticancer chemotherapy induce cellular senescence, a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation (H3K9me3). Although therapy-induced senescence (TIS) improves long-term outcomes, potentially harmful properties of senescent tumour cells make their quantitative elimination a therapeutic priority. Here we use the Em-myc transgenic mouse lymphoma model in which TIS depends on the H3K9 histone methyltransferase Suv39h1 to show the mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescenceinducing chemotherapy, TIS-competent lymphomas but not TIS-incompetent Suv39h1 lymphomas showincreased glucose utilization and much higher ATP production.We demonstrate that this is linked tomassive proteotoxic stress,whichis a consequence of the senescenceassociated secretory phenotype (SASP) described previously. SASPproducing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energyconsuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, were more sensitive to blocking glucose utilization or autophagy, which led to their selective elimination through caspase-12- and caspase-3-mediated endoplasmicreticulum-related apoptosis.Consequently, pharmacological targeting of thesemetabolic demands on TISinductionin vivoprompted tumour regression and improved treatment outcomes further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting.

Selective Requirement of PI3K/PDK1 Signaling for Kras Oncogene-Driven Pancreatic Cell Plasticity and Cancer

Cancer Cell, 2013, 23, 406-420 publiziert am 18.03.2013
Oncogenic Kras activates a plethora of signaling pathways, but our understanding of critical Ras effectors is still very limited. We show that cell-autonomous phosphoinositide 3-kinase (PI3K) and 3-phosphoinositide-dependent protein kinase 1 (PDK1), but not Craf, are key effectors of oncogenic Kras in the pancreas, mediating cell plasticity, acinar-to-ductal metaplasia (ADM), and pancreatic ductal adenocarcinoma (PDAC) formation. This contrasts with Kras-driven non-small cell lung cancer, where signaling via Craf, but not PDFK1, is an essential tumor-initiating event. These in vivo genetic studies together with pharmacologic treatment studies in models of human ADM and PDAC demostrate tissue-specific differences of oncogenic Kras signaling and define PI3K/PDK1as a suitable target for therapeutic intervention specifically in PDAC.

Efemp1 and p27 Kip1 modulate responsiveness of pancreatic cancer cells towards a dual PI3K/mTOR inhibitor in preclinical models

Impact Journals, 2013, Vol. 4, No 2, 277-288 publiziert am 26.02.2013
Pancreatic ductal adenocarcinoma (PDAC) remains a dismal disease with a poor prognosis and targeted therapies have failed in the clinic so far. Several evidences point to the phosphatidylinositol 3-kinase (PI3K)-mTOR pathway as a promising signaling node for targeted therapeutic intervention. Markers, which predict responsiveness of PDAC cells towards PI3K inhibitors are unknown. However, such markers are needed and critical to better stratify patients in clinical trials.

We used a large murine KrasG12D - and PI3K (p110α H1047R )-driven PDAC cell line platform to unbiased define modulators of responsiveness towards the dual PI3K-mTOR inhibitor Bez235. In contrast to other tumor models, we show that KrasG12D - and PI3K (p110α H1047R )-driven PDAC cell lines are equally sensitive towards Bez235. In an unbiased approach we found that the extracellular matrix protein Efemp1 controls sensitivity of murine PDAC cells towards Bez235. We show that Efemp1 expression is connected to the cyclin-dependent kinase inhibitor p27Kip1 . In a murine KrasG12D -driven PDAC model, p27Kip1 haploinsufficiency accelerates cancer development in vivo. Furthermore, p27Kip1 controls Bez235 sensitivity in a gene dose-dependent fashion in murine PDAC cells and lowering of p27Kip1 decreases Bez235 responsiveness in murine PDAC models.

Together, we define the Efemp1-p27Kip1 axis as a potential marker module of PDAC cell sensitivity towards dual PI3K-mTOR inhibitors, which might help to better stratify patients in clinical trials.



[18F]FLT is superior to [18F]FDG for predicting early response to antiporliferative treatment in high-grade lymphoma in a dose-dependent manner

Springer Link, 2012, 40:34–43, Eur J Nucl Med Mol Imaging (2013) publiziert am 05.10.2012
Purpose Positron emission tomography (PET) with the thymidine analogue [18F]fluorothymidine ([18F]FLT) has been shown to detect early response to chemotherapy in highgrade lymphoma. In this preclinical in vitro and in vivo study we compared [18F]FLT to the glucose analogue [18F] fluorodeoxyglucose ([18F]FDG) regarding dose-dependent visualization and prediction of early therapy response.

FLT-PET Is Superior to FDG-PET for Very Early Response Prediction in NPM-ALK-Positive Lymphoma Treated with Targeted Therapy

Cancer Res, 2012, publiziert am 08.08.2012
 

The prognosis of relapsed or refractory aggressive lymphoma is poor. The huge variety of currently evolving targeted treatment approaches would benefit from tools for early prediction of response or resistance. We used various ALK-positive anaplastic large cell lymphoma (ALCL) cell lines to evaluate two inhibitors, the HSP90 inhibitor NVP-AUY922, and the mTOR inhibitor everolimus, both of which have shown to interfere with ALK-dependent oncogenic signal transduction. Their therapeutic effect was determined in vitro by MTT assay, [18F]fluorodeoxyglucose- (FDG) and [18F]fluorothymidine- (FLT) uptake, and by biochemical analysis of ALK-induced signalling. Micro FDG- and FLT-PET imaging studies in immunodeficient mice bearing ALCL xenotransplants were performed with the cell lines SUDHL-1 and Karpas299 to assess early treatment response to NVP-AUY922 or everolimus in vivo. SUDHL-1 cells showed sensitivity to both inhibitors in vitro. Importantly, we detected a significant reduction of FLT-uptake in SUDHL-1 bearing animals using both inhibitors compared to baseline as early as 5 days after initiation of targeted therapy. Immunostaining showed a decrease in Ki-67 and an increase in cleaved caspase-3 staining. In contrast, FDG-uptake did not significantly decrease at early time points. Karpas299 xenotransplants, which are resistant to NVP-AUY922 and sensitive to everolimus treatment, showed an increase of mean FLT-uptake on day 2 after administration of NVP-AUY299, but a significant reduction in FLT-uptake upon everolimus treatment. In conclusion, we show that FLT- but not FDG-PET is able to predict response to treatment with specific inhibitors very early in the course of treatment and thus enables early prediction of treatment efficacy.



Disclosure of erlotinib as a multikinase inhibitor in pancreatic ductal adenocarcinoma

Neoplasia., 2011, 13(11), 1026-34 publiziert am 01.11.2011
 
A placebo-controlled phase 3 trial demonstrated that the epidermal growth factor receptor (EGFR) inhibitor erlotinib in combination with gemcitabine was especially efficient in a pancreatic ductal adenocarcinoma (PDAC) subgroup of patients developing skin toxicity. However, EGFR expression was not predictive for response, and markers to characterize an erlotinib-responding PDAC group are currently missing. In this work, we observed high erlotinib IC50 values in a panel of human and murine PDAC cell lines. Using EGFR small interfering RNA, we detected that the erlotinib response was marginally influenced by EGFR. To find novel EGFR targets, we used an unbiased chemical proteomics approach for target identification and quality-controlled target affinity determination combined with quantitative mass spectrometry based on stable isotope labeling by amino acids in cell culture. In contrast to gefitinib, we observed a broad target profile of erlotinib in PDAC cells by quantitative proteomics. Six protein kinases bind to erlotinib with similar or higher affinity (K(d) = 0.09-0.358 µM) than the EGFR (K(d) 0.434 µM). We provide evidence that one of the novel erlotinib targets, ARG, contributes in part to the erlotinib response in a PDAC cell line. Our data show that erlotinib is a multikinase inhibitor, which can act independent of EGFR in PDAC. These findings may help to monitor future erlotinib trials in the clinic.

Assessing antiangiogenic therapy response by DCE-MRI: development of a physiology driven multi-compartment model using population pharmacometrics.

PLoS One., 2011, 6(10), e26366 publiziert am 18.10.2011
 
Dynamic contrast enhanced (DCE-) MRI is commonly applied for the monitoring of antiangiogenic therapy in oncology. Established pharmacokinetic (PK) analysis methods of DCE-MRI data do not sufficiently reflect the complex anatomical and physiological constituents of the analyzed tissue. Hence, accepted endpoints such as Ktrans reflect an unknown multitude of local and global physiological effects often rendering an understanding of specific local drug effects impossible. In this work a novel multi-compartment PK model is presented, which for the first time allows the separation of local and systemic physiological effects. DCE-MRI data sets from multiple, simultaneously acquired tissues, i.e. spinal muscle, liver and tumor tissue, of hepatocellular carcinoma (HCC) bearing rats were applied for model development. The full Markov chain Monte Carlo (MCMC) Bayesian analysis method was applied for model parameter estimation and model selection was based on histological and anatomical considerations and numerical criteria. A population PK model (MTL3 model) consisting of 3 measured and 6 latent (unobserved) compartments was selected based on Bayesian chain plots, conditional weighted residuals, objective function values, standard errors of model parameters and the deviance information criterion. Covariate model building, which was based on the histology of tumor tissue, demonstrated that the MTL3 model was able to identify and separate tumor specific, i.e. local, and systemic, i.e. global, effects in the DCE-MRI data. The findings confirm the feasibility to develop physiology driven multi-compartment PK models from DCE-MRI data. The presented MTL3 model allowed the separation of a local, tumor specific therapy effect and thus has the potential for identification and specification of effectors of vascular and tissue physiology in antiangiogenic therapy monitoring.

Free-breathing quantitative dynamic contrast-enhanced magnetic resonance imaging in a rat liver tumor model using dynamic radial T(1) mapping

Invest Radiol., 2011, 46(10), 624-31 publiziert am 01.10.2011
 
OBJECTIVES:

The high sensitivity to motion artifacts is a major limiting factor for applying the dynamic 3D T1-weighted gradient-echo (3D T1w GRE) technique for dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) experiments in small rodents. Dynamic quantification of the relaxation rate R1 (1/T1) presents an alternative approach to reduce these motion artifacts. In this work, an optimized 2D single-shot Look-Locker based T1 mapping technique, named GOLD, applying radial sampling in the golden-angle view order and contrast-enhancing k-space filter was evaluated for its use in free-breathing quantitative DCE-MRI of rat liver on a clinical 1.5 T MRI system.

 

MATERIALS AND METHODS:

In vitro measurements and initial in vivo experiments in healthy rats were performed to evaluate the accuracy and resilience of the GOLD technique to motion artifacts. Unifocal hepatocellular carcinoma (HCC) was established in 20 male Buffalo rats. Twelve days after tumor cell implantation, animals were screened for intrahepatic tumor nodules by high-resolution T2-weighted MRI. Quantitative DCE-MRI experiments applying bolus injected gadopentetate dimeglumine were performed in 11 HCC-bearing rats using the GOLD technique. For comparison, a standard 3D T1w GRE sequence was applied in 6 additional rats.

 

RESULTS:

Phantom experiments showed good agreement for T1 values measured by the GOLD method and an inversion recovery spectroscopy measurement. The in vivo experiments in healthy rats confirmed the robustness of the GOLD method in T1 value determination and its resilience to motion artifacts. Gadopentetate dimeglumine concentration (CGd) time curves determined from free-breathing GOLD-based DCE-MRI experiments of HCC-bearing rats allowed reliable and robust pharmacokinetic modeling (K, ve, lag time Td, and slow washout rate rwo) of tumor, liver, and spinal muscle. In comparison to the dynamic 3D T1w GRE, the GOLD method showed less variation and jitter in the CGd time curves and significantly increased accuracy (in terms of the goodness of fit) in the pharmacokinetic modeling. Significant differences were detected for K and ve with the 3D T1w GRE method apparently underestimating those parameters.

 

CONCLUSIONS:

The GOLD technique allowed dynamic sampling of 2D axial T1 maps of the rat abdomen with 6-second temporal resolution enabling simultaneous and robust pharmacokinetic modeling of HCC, normal liver, and spinal muscle.



In vivo diagnosis of murine pancreatic intraepithelial neoplasia and early-stage pancreatic cancer by molecular imaging

Proc Natl Acad Sci U S A, 2011, 108(24), 9945-50 publiziert am 14.06.2011
 

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor patient outcome often resulting from late diagnosis in advanced stages. To date methods to diagnose early-stage PDAC are limited and in vivo detection of pancreatic intraepithelial neoplasia (PanIN), a pre-invasive precursor of PDAC, is impossible. Using a cathepsin-activatable near-infrared probe in combination with flexible confocal lasermicroscopy (CFL) in a genetically defined mouse model of PDAC we were able to detect and grade murine PanIN lesions in real-time in vivo. Our diagnostic approach is highly sensitive and specific and proved superior to clinically established fluorescein-enhanced imaging. Translation of this endoscopic technique into the clinic should tremendously improve detection of pancreatic neoplasia thus reforming management of patients at risk for PDAC.

 



Predictive value of initial 18F-FLT uptake in patients with aggressive non-Hodgkin lymphoma receiving R-CHOP treatment

J Nucl Med., 2011, 52(5), 690-6 publiziert am 01.05.2011
 
R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone)-like chemotherapy is the standard therapy in aggressive B-cell lymphoma. (18)F-FDG PET has high prognostic implications at treatment completion but is limited as an early predictor. Here, we present the results of a prospective study correlating the initial uptake of the in vivo proliferation marker 3'-deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) with the clinical outcome of patients with aggressive non-Hodgkin lymphoma treated with R-CHOP.

 

METHODS:

Sixty-six eligible patients were evaluated prospectively with (18)F-FLT PET before R-CHOP. PET was performed 45 min after injection of 300-370 MBq of (18)F-FLT. Mean and maximum standardized uptake values (SUVs) were calculated on a lesion-by-lesion basis. Response was assessed at the end of therapy. International Prognostic Index (IPI) scores and clinical parameters (Ann Arbor stage, lactate dehydrogenase, performance status, extranodal disease) were determined in all patients. Response was assessed according to revised response criteria after the end of therapy. After treatment, patients were followed in intervals from 4 wk to 6 mo (mean follow-up, 23.1 mo [range, 1-63 mo]), and progression-free and overall survival were determined.

 

RESULTS:

All lymphoma lesions identified by a reference method ((18)F-FDG PET/CT or multislice CT of the trunk) showed increased focal tracer uptake (mean (18)F-FLT SUV, 7.3 ± 2.5). Response assessment revealed progressive disease in 4, partial response in 3, and complete response (CR) in the remaining 55 patients. The IPI score was predictive for achieving CR (P = 0.034). Importantly, initial mean SUV was also significantly higher in patients who showed progressive disease and partial response than in patients who achieved CR (P = 0.049). In addition, we found a significant correlation between IPI score and initial (18)F-FLT uptake.

 

CONCLUSION:

Taken together, high (18)F-FLT uptake is a negative predictor of response to R-CHOP treatment in aggressive B-cell non-Hodgkin lymphoma and correlates with the IPI score. Thus, (18)F-FLT PET may represent a useful tool for implementing risk-adapted treatment in these patients.



Optical imaging of integrin αVß3 detects pancreatic cancer in endogenous mouse models

publiziert am 01.05.2011
 
Pancreatic ductal adenocarcinoma remains one of the most lethal malignacies in the Western World with a 5-year survival rate Iower then 4%. Late diagnosis and poor response to all available chemotherapies are main causes for the devastating clinical course. Development of methods for early detection and evaluation of new therapies is of great importance. Here, we used optical imaging for detection of αVß3 integrin in pancreatic cancer in endogenous mouse models. To model stages of PDAC development in humans, following genetically engineered animal models (GEMM) were used: mice with pancreas specific activation of oncogenic Kras (p48+/Cre Kras +/LSL-G12D , CK) that develop preneoplastic lesions preceding the cancer and mice with concomitant deletion of p53 (p48+/Cre Kras+/LSL-Gi2Dp53Lox/Lox^c/cp) that develop PDAC already at 6-8 weeks of age. Pancreatic aVß3 integrin expression was analysed by immunohistochemistry and Western blot. For optical imaging, animals were injected with Integrisense680, Near Infrared fluorochrome labelled agent that specifically binds to aVß3. After 24h, animals were subjected to intraoperative epi-illumination optical imaging procedure. Sacrificed animals were whole-mount frozen for analysis of Integrisense intratissue distribution via muitispectral cryoslicing optical imaging. For comparison, T2 weighted (T2w) images of the abdomen were generated with 1,5T clinical MRI Scanner and a dedicated surface coil. ß3 protein expression was dramatically increased in CKP pancreatic tumors compared to CK pancreata. ß3 positive cells were found among malignant cells and in the reactive stroma. In CK animals, ß3 expression was specifically limited to ductal and preneoplastic lesions and surrounding stroma with no expression in normal tissue. Intraoperative optical imaging of Integrisense correlated well with the high ß3 expression in the tumor and demonstrated very strong and specific fluorescence Signals at the tumor surface with excellent demarcation of tumor borders and good contrast to surrounding tissues. CK pancreas was well visualized and spotty fluorescence pattern of Integrisense was observed. Cryoslicing imaging of the animals confirmed that Integrisense680 penetrated well into all parts of the tumor and accumulated very specifically in cancer tissue in CKPanimals. In CK pancreata, spots of fluorescence were also observed throughout the tissue confirming intraoperative imaging. Spots of fluorescence correlated well with the foci of ductal and preneoplastic lesions embedded in healthy tissue äs confirmed by histopathological analysis. In contrast, preneoplastic lesions were difficult to differentiate on T2w-MRI images due to low spatial resolution. Tumors appeared relative homogenously hyperintense on T2w images and tumor borders were in certain cases difficult to identify, when adjacent structures, i.e. gut, were of similar Signal intensity.

Monitoring of pancreatic ductal adenocarcinoma in mouse models via multiparametric magnetic resonance imaging

publiziert am 01.05.2011
 
Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease due to the late diagnosis and the lack of effective chemo/radiotherapy. The development of strategies for early tumor detection and evaluation of new treatments in models with high predictive value is of outmost importance. To characterize the spontaneous course of tumor progression, we monitored tumor growth kinetics and physiology in genetically engineered mouse models (GEMMs) of PDAC by multiparametric magnetic resonance imaging (MRI).

For generation of conditional endogenous PDAC models, Ptfla+/Cre mice were crossed to Kras+/LSL-G12D (K)Tgfα(T), K;p53(P)+/fl, K;Pfl/fl, K;P+/R172H;T and K;P+/fl;T GEMMs and subjected to serial T2- weighted (T2w) MRI from 4-6 weeks of age onwards. Upon detection of solid tumor, diffusion- weighted imaging and dynamic contrast enhanced-MRI protocols were implemented. Survival analysis and histopathological characterization of PDAC were performed and correlated with MRI data. Tumor growth kinetics as determined by semi-automated segmentation of TIW data showed good correlation with the animal genotype. In K;P+/fl animals, tumors were first detected at 12-15 weeks of age while significant tumor burden was observed around weck 20. Tumor appearance was highly heterogeneous, with cystic and solid tumor parts clearly distinguishable. Additional TGFα overexpression led to earlier onset, faster tumor growth and significantly shorter lifespan.



Endogenous mouse models of pancreatic adenocarcinoma as a preclinical trial platform characterized by multiparametric magnetic resonance imaging

publiziert am 01.05.2011
 
Development of strategies for early tumor detection and evaluation of new therapies in preclinical models is one of the main goals of current cancer research. Pancreatic Ductal Adenocarcinoma (PDAC) is a lethal disease, with no effective treatment plan. PDAC progression can be very well mimicked by established genetically engineered mouse models (GEMMs), representing an excellent platform for preclinical studies. Therefore tumor physiology, including growth kinetics, tumor perfusion and tumor composition, were characterized in several GEMM. In addition tumor response to the Standard therapeutic agent Gemcitabine was monitored by multiparametric magnetic resonance imaging (MRI). Ptf1a+/Cre mice were crossed to Kras+/LSL-G12D(K)Tgfα(T), K;p53(P)+/fl, K;Pfl/fl, K;P+/R172H;T and K;P+/fl;T GEMMs to generate conditional endogenous PDAC models. Animals were subjected to serial T2-weighted (T2w) MRI from 4-6 weeks of age onwards using 1,5 T clinical Scanner. Upon detection of solid tumor, longitudinal diffusion-weighted imaging (DWI) and dynamic contrast enhanced-MRI (DCE-MRI with Gd-DTPA) protocols were applied. For the evaluation of therapy response monitoring, K;Pfl/fl mice were injected twice a week with 120 mg/kg body weight with Gemcitabine or 0,9% NaCI solution. MRI data were correlated with survival analysis, vascular staining and histopathology of tumor specimens. Volume analysis was performed using semi-automatic segmentation of solid and cystic compartments. K;Pfl/fl, and K;P+/fl;T models showed 100% tumor incidence, detected by MRI and verified by histology. Moreover, K;P+/fl;T animals often presented with metastatic disease to lung and liver, easily identified on T2w scans, while no metastases were observed in K;Pfl/fl mice. The final tumor volume ranged between 800 and 4500

Validation of preclinical multiparametric imaging for prediction of necrosis in hepatocellular carcinoma after embolization.

J Hepatol., 2011, 55(5), 1034-40 publiziert am 24.02.2011
 
BACKGROUND & AIMS:

The hepatocellular carcinoma (HCC) exhibits varying degrees of vascularization with more poorly differentiated carcinoma commonly exhibiting high amounts of vascularization. Transcatheter arterial embolization (TAE) of HCC tumor nodules results in varying amounts of tumor necrosis. Reliable quantification of necrosis after TAE, would aid in treatment planning and testing of novel combinatorial treatment regimen. The aim of this work was to validate different imaging parameters as individual or combined predictors of tumor necrosis after TAE in an orthotopic rat HCC tumor model.

 

METHODS:

Unifocal rat HCC was imaged by T(2)-weighted MRI, quantitative dynamic contrast enhanced (DCE) MRI, diffusion weighted MRI (DWI) and [(18)F]-FDG PET imaging before (day-1) and after (days 1 and 3) TAE. Univariate and multivariate regression analyses were carried out to analyze the ability of each imaging parameter to predict the percent residual vital tumor (vtu) and vital tissue (vti) as determined by quantitative histopathology.

 

RESULTS:

TAE induced a wide range of tumor necrosis. Tumor volume was the only parameter showing a correlation with vti (r(2) = 0.63) before TAE. After TAE, moderate correlations were found for FDG tracer uptake (r(2) = 0.56) and plasma tissue transfer constant (r(2) = 0.55). Correlations were higher for the extravascular extracellular volume fraction (v(e), r(2) = 0.68) and highest for the apparent diffusion coefficient (ADC, r(2) = 0.86). Multivariate analyses confirmed highest correlation of ADC and v(e) with vtu and vti.

 

CONCLUSIONS:

DWI and DCE-MRI with the respective parameters ADC (day 3) and v(e) (day 1) were identified as the most promising imaging techniques for the prediction of necrosis. This study validates a preclinical platform allowing for the improved tumor stratification after TAE and thus the testing of novel combinatorial therapy approaches in HCC.



Reference Region-Based Pharmacokinetic Modeling in Quantitative Dynamic Contract-Enhanced MRI Allows Robust Treatment Monitoring in a Rat Liver Tumor Model Despite Cardiovascular Changes

Magn Reson Med., 2010, publiziert am 24.09.2010
 

In this work, two pharmacokinetic modeling techniques, population arterial input function model, and reference region model, were applied to dynamic contract-enhanced MRI data, to test the influence of a change in heart rate on modeling parameters. A rat population arterial input function was generated by dynamic contrast-enhanced computed tomography measurements using the MR contrast agent gadolinium diethylenetriamine penta-acetic acid. Then, dynamic contract-enhanced MRI was used for treatment monitoring in two groups of hepatocellular carcinoma bearing rats. Whereas group and pre, post checked 1 had the same heart rate as animals analyzed for the population arterial input function (263 6 20 bpm), group 2 had a higher heart rate (369 6 11 bpm) due to a different anesthesia protocol. The pharmacokinetic modeling parameters volume transfer constant Ktrans and relative extravascular extracellular space ve were calculated with both models and statistically compared. For group 1, good correlation and agreement was found between the models showing no difference in Ktrans and ve (DKtrans:4 6 19% and Dve:4 6 12%, P 5 0.2). In contrast, for group 2, abias in parameter values for the population arterial input func- tion model was detected (DKtrans: 245 6 7% and Dve: 231 6 7%, P ! 0.001). The presented work underlines the value of the reference region model in longitudinal treatment monitoring and provides a straightforward approach for the generation of a rat population arterial input function.

 

Magn Reson Med 000:000–000, 2010. V C 2010 Wiley-Liss, Inc.



First demonstration of 3-D lymphatic mapping in breast cancer using freehand SPECT

European Journal of Nuclear Medicine and Molecular Imaging, 2010, 37(8), 1452-61 publiziert am 01.08.2010
 
Freehand SPECT is a 3D tomographic imaging modality based on data acquisition with a hand-held detector that is moved freely, in contrast to conventional, fixed gamma camera systems. In this pilot study, the feasibility of freehand SPECT for 3D lymphatic mapping in breast cancer was evaluated. Methods: A total of 85 patients (pts) (age, 29–88 years) with an initial diagnosis of invasive breast cancer and no clinical evidence of nodal involvement prospectively underwent sentinel lymph node (SLN) biopsy. Preoperative lymphatic mapping (35–87 MBq 99mTc-Nanocoll) included tomographic imaging with a SPECT/CT device (Siemens Symbia T6) serving as reference. Initially, the freehand SPECT approach was assessed in a pilot study consisting of 50 pts. The quality of each freehand SPECT acquisition was assessed and ranked as good, intermediate, or poor. In another series comprising a further 35 pts (validation study), a guidance system for the acquisition was implemented based on the results of the pilot study, ensuring acquisitions with good quality. For 3D tomographic image reconstruction, ad hoc models and iterative reconstruction algorithms were used in all 85 pts. To allow for adequate comparison, SPECT/CT data and freehand SPECT data were registered within the same coordinate system. Results: In the pilot study, freehand SPECT enabled mapping of 24/83 SLNs in 20/44 pts (3 drop-outs, 3 pts without SLN neither in SPECT/CT nor in freehand SPECT). Using SPECT/CT as reference, the accuracy of freehand SPECT was 77.8% (7/9 nodes) in scans with good quality, while for intermediate and poor quality scans, the accuracy was reduced to 34.3% and 12.8%, respectively. In the validation study, quality feedback improved the results significantly and freehand SPECT enabled the mapping of at least one SLN in 87.5% of the pts (28/32 – 3 drop-outs). Compared to the reference method, freehand SPECT showed a sensitivity of 83.3% (35/42 nodes). False negative findings were related to insufficient scanning time, insufficient coverage of the axillary region, close proximity of the SLN to the injection site, and low tracer uptake in the SLNs. Conclusions: In this preliminary study, we could demonstrate that 3D localization of SLNs is feasible using freehand SPECT technology. Prerequisites for acquisition of a good scan quality, most likely allowing precise SLN mapping, have been defined. This approach has high potential to allow image-guided biopsy and further standardization of SLN dissection, thus bringing 3D nuclear imaging into the operating room.

Cell Cycle Progression or Translation Control Is Not Essential for Vesicular Stomatitis Virus Oncolysis of Hepatocellular Carcinoma

PLoS ONE | www.plosone.org, 2010, 5(6), e10988 publiziert am 02.06.2010
 

The intrinsic oncolytic specificity of vesicular stomatitis virus (VSV) is currently being exploited to develop alternative therapeutic strategies for hepatocellular carcinoma (HCC). Identifying key regulators in diverse transduction pathways that define VSV oncolysis in cancer cells represents a fundamental prerequisite to engineering more effective oncolytic viral vectors and adjusting combination therapies. After having identified defects in the signalling cascade of type I interferon induction, responsible for attenuated antiviral responses in human HCC cell lines, we have now investigated the role of cell proliferation and translation initiation. Cell cycle progression and translation initiation factors eIF4E and eIF2Bε have been recently identified as key regulators of VSV permissiveness in T-lymphocytes and immortalized mouse embryonic fibroblasts, respectively. Here, we show that in HCC, decrease of cell proliferation by cell cycle inhibitors or siRNA-mediated reduction of G(1) cyclin-dependent kinase activities (CDK4) or cyclin D1 protein expression, do not significantly alter viral growth. Additionally, we demonstrate that translation initiation factors eIF4E and eIF2Bε are negligible in sustaining VSV replication in HCC. Taken together, these results indicate that cellular proliferation and the initiation phase of cellular protein synthesis are not essential for successful VSV oncolysis of HCC. Moreover, our observations indicate the importance of cell-type specificity for VSV oncolysis, an important aspect to be considered in virotherapy applications in the future.



Aurora kinases A and B are up-regulated by Myc and are essential for maintenance of the malignant state

Blood, 2010, publiziert am 02.06.2010
 

Myc oncoproteins promote continuous cell growth, in part by controlling the transcription of key cell cycle regulators. Here, we report that c-Myc regulates the expression of Aurora A and B kinases (Aurka and Aurkb), and that Aurka and Aurkb transcripts and protein levels are highly elevated in Myc-driven B cell lymphomas in both mice and man. The induction of Aurka by Myc is transcriptional and is directly mediated via E-boxes, while Aurkb is regulated indirectly. Blocking Aurka/b kinase activity with a selective Aurora kinase inhibitor triggers transient mitotic arrest, polyploidization and apoptosis of Myc-induced lymphomas. These phenotypes are selectively bypassed by a kinase inhibitor-resistant Aurkb mutant, demonstrating that Aurkb is the primary therapeutic target in the context of Myc. Importantly apoptosis provoked by Aurk inhibition was p53 independent, suggesting that Aurka/Aurkb inhibitors will show efficacy in treating primary or relapsed malignancies having Myc involvement and/or loss of p53 function.



Antifungal Therapy with Itraconazole Impairs the Anti-Lymphoma Effects of Rituximab by Inhibiting Recruitment of CD20 to Cell Surface Lipid Rafts

Cancer Res, 2010, 70(11), OF1-5 ©2010 AACR. publiziert am 11.05.2010
 
Immunotherapy with rituximab alone or in conjunction with chemotherapy has significantly improved the treatment outcome of B-cell lymphoma patients. Nevertheless, a subpopulation of patients does not respond to rituximab. The reason for treatment failure as well as the exact mechanism of action is still uncertain. The function of rituximab has long been associated with the partitioning of CD20 molecules to membrane micro-domains. Here, we show that concomitant antifungal treatment with itraconazole impairs the rituximab anti-lymphoma effect both in vitro and in vivo. At the molecular level, recruitment of CD20 to lipid rafts is inhibited in the presence of itraconazole. Furthermore, calcium influx, which is crucial for rituximab-mediated cell death, was nearly completely abolished by itraconazole treatment. In contrast, the antifungal drug caspofungin did not inhibit CD20 recruitment to lipid rafts, nor did it affect calcium influx or the cytotoxic effect of rituximab. The finding that itraconazole also abolished the cytotoxic effects of other therapeutic antibodies directed against lipid raft–associated molecules (i.e., CD20 and CD52) but not those against the non–raft-associated molecule CD33 further supported our proposed mechanism of action. Our results argue that concomitant medications must be adjusted carefully to achieve optimal antitumor effects with monoclonal antibodies.

 

Published OnlineFirst on May 11, 2010 as 10.1158/0008-5472.CAN-10-0259



Targeted Radioiodine Therapy of Neuroblastoma Tumors following Systemic Nonviral Delivery of the Sodium Iodide Symporter Gene

Clin Cancer Res, 2009, 15(19), 6079–8 publiziert am 01.10.2009
 
We recently reported the significant therapeutic efficacy of radioiodine therapy in various tumor mouse models following transcriptionally targeted sodium iodide symporter (NIS) gene transfer. These studies showed the high potential of NIS as a novel diagnostic and therapeutic gene for the treatment of extrathyroidal tumors. As a next crucial step towards clinical application of NIS-mediated radionuclide therapy we aim at systemic delivery of the NIS gene to target extrathyroidal tumors even in the metastatic stage.

Vollversion

PI3K Signaling Maintains c-myc Expression to Regulate Transcription of E2F1 in Pan-creatic Cancer Cells

MOLECULAR CARCINOGENESIS, 2009, 48, 1149–1158 publiziert am 14.07.2009
 
Phosphatidylinositol 3-kinase (PI3K) signaling controls survival and proliferation of cancer cells and is activated in around 60% of pancreatic ductal adenocarcinomas (PDACs). Al-though not entirely clarified, PI3K signaling is linked to cell cycle progression of PDAC cells. In this study we demonstrate that PI3K signaling controls transcription of the E2F1 gene and show that E2F1 is essential for S-phase progression of PDAC cells. On the molecular level, PI3K signalling controls c-myc protein abundance in a glycogen synthase kinase-3 (GSK3)-dependent fashion. c-myc binds to the E-box of the E2F1 gene in PDAC cells and this binding is under control of the PI3K-signaling pathway. Together, we demonstrate that PI3K–GSK3-dependent control of c-myc protein expression is connected to the transcription of the E2F1 gene in PDAC cells, leading to S-phase progression of the cell cycle. © 2009 Wiley-Liss, Inc.

Gene editing enables T cell engineering to redirect antigen specificity for potent tumor rejection

2019, publiziert am 12.03.2019
Adoptive transfer of T cell receptor (TCR) transgenic T cells holds great promise for treating various cancers. So far, mainly semi-randomly integrating vectors have been used to genetically modify T cells. These carry the risk of insertional mutagenesis and the sole addition of an exogenous TCR potentially results in the mispairing of TCR chains with endogenous ones. Existing approaches using non-viral vectors, such as transposons, already reduce the risk of insertional mutagenesis, but have not accomplished site-specific integration. Here, we used CRISPR-Cas9 ribonucleoproteins and Adeno-associated virus 6 for gene-targeting to deliver an engineered TCR gene specifically to the T cell receptor alpha constant (TRAC) locus, thus placing it under endogenous transcriptional control. Our data demonstrate that this approach replaces the endogenous TCR, functionally redirects the edited T cells’ specificity in vitro, and facilitates potent tumor rejection in an in vivo xenograft model.

 

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