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Forschungsbereich B

Membrane-associated heat shock proteins in oncology: from basic research to new theranostic targets

Cells, 2020, publiziert am 27.05.2020
Heat shock proteins (HSPs) constitute a large family of conserved proteins acting as molecular chaperones that play a key role in intracellular protein homeostasis, regulation of apoptosis, and protection from various stress factors (including hypoxia, thermal stress, oxidative stress). Apart from their intracellular localization members of different HSP families such as small HSPs, HSP40, HSP60, HSP70 and HSP90 have been found to be localized on the plasma membrane of malignantly transformed cells. In the current article, the role of membrane-associated molecular chaperones in normal and tumor cells is comprehensively reviewed with implication of these proteins as plausible targets for cancer therapy and diagnostics.

 

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Imaging glioma biology: spatial comparison of amino acid PET, amide proton transfer, and perfusion-weighted MRI in newly diagnosed gliomas

European Journal of Nuclear Medicine and Molecular Imaging, 2020, publiziert am 20.01.2020

 

Purpose Imaging glioma biology holds great promise to unravel the complex nature of these tumors. Besides well-established imaging techniques such O-(2-[18F]fluoroethyl)-L-tyrosine (FET)-PET and dynamic susceptibility contrast (DSC) perfusion imaging, amide proton transfer–weighted (APTw) imaging has emerged as a promising novel MR technique. In this study, we aimed to better understand the relation between these imaging biomarkers and how well they capture cellularity and vascularity in newly diagnosed gliomas.

Methods Preoperative MRI and FET-PET data of 46 patients (31 glioblastoma and 15 lower-grade glioma) were segmented into contrast-enhancing and FLAIR-hyperintense areas. Using established cutoffs, we calculated hot-spot volumes (HSV) and their spatial overlap. We further investigated APTw and CBV values in FET-HSV. In a subset of 10 glioblastoma patients, we

compared cellularity and vascularization in 34 stereotactically targeted biopsies with imaging. Results In glioblastomas, the largest HSV was found for APTw, followed by PET and CBV (p < 0.05). In lower-grade gliomas, APTw–HSV was clearly lower than in glioblastomas. The spatial overlap of HSV was highest between APTw and FET in both tumor entities and regions. APTw correlated significantly with cellularity, similar to FET, while the association with vascularitywas more pronounced in CBV and FET.

Conclusions We found a relevant spatial overlap in glioblastomas between hotspots of APTw and FET both in contrast-enhancing and FLAIR-hyperintense tumor. As suggested by earlier studies, APTw was lower in lower-grade gliomas compared with glioblastomas. APTw meaningfully contributes to biological imaging of gliomas.

 

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7Hsp70 serum levels in pet dogs—a potential diagnostic biomarker for spontaneous round cell tumors

Springer Netherlands, 2019, doi.org/10.1007/s12192-019-01024-9 publiziert am 03.08.2019
The concentration of circulating heat shock protein 70 (Hsp70) was measured in liquid biopsies of canine tumor patients as a potential biomarker. Compared with rodent tumor models, spontaneously occurring tumors in pet dogs reflect the clinical situation of human patients better, as dogs cohabitate with their owners in the same environment, reach a much older age than rodents, can provide blood samples much more frequently, and receive up-to-date medical care and, similar to humans, their tumors show a high genetic heterogeneity. Due to the species-specific sequence homology of human and canine Hsp70, two human enzyme-linked immunosorbent assay (ELISA) systems (R&D and lipHsp70) were used to measure canine Hsp70 concentrations in serum and plasma. In general, higher Hsp70 concentrations were found in serum compared with plasma samples of dogs, and the lipHsp70 ELISA detected higher peak concentrations of Hsp70 in a broader range than the R&D ELISA. Compared with a tumor-free control group, serum Hsp70 concentrations were higher in tumor-bearing dogs, irrespective of breed, age, body weight, and gender. A sub-classification of the different tumors according to their cytological characteristics revealed significantly elevated Hsp70 serum concentrations in dogs with round cell tumors (p < 0.01), a heterogeneous group of malignancies with hematopoietic origin such as mast cells, plasma cells, lymphocytes, histiocytes, and melanomas. Future studies with larger patient cohorts and well-defined tumor sizes are necessary to elucidate the role of serum Hsp70 as a biomarker for tumor detection and monitoring of outcome in pet animals.

 

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Increased heat shock protein 70 (Hsp70) serum levels and low NK cell counts after radiotherapy – potential markers for predicting breast cancer recurrence?

Radiation Oncology, 2019, publiziert am 10.05.2019
Background: Breast cancer is the most common invasive tumor in women worldwide and the second cause of cancer-related deaths. After breast conserving surgery the tumor bed gets irradiated. Radiation-induced tumor cell death has been found to be associated with the release of damage-associated molecular patterns (DAMPs) including free Hsp70 that can stimulate inflammatory immune responses. Therefore, Hsp70 serum levels as well as the composition of lymphocyte subpopulations have been measured in breast cancer patients during therapy and in the follow-up period as potential predictors for clinical outcome. Methods: The serum of 40 breast cancer patients, who received a breast-conserving surgery and adjuvant radiotherapy (RT) was examined for soluble, free Hsp70 using the R&D Human HSP70 DuoSet and lipHsp70 ELISA. Lymphocyte subpopulations and total lymphocyte counts were analysed by multiparameter flow cytometry in the peripheral blood. Blood samples were collected before (t1), after 30 Gy (t2) and 60 Gy (t3), 6 weeks (t4), 6 months (t5) and 1 year (t6) after RT. Clinical responses were assessed regularly up to 5 years after RT. Results: Patients who developed a contralateral recurrence or metastases within the first 2 years after RT had significantly higher serum Hsp70 values at the end of RT (t3; p = 0.03) up to 6 weeks after RT (t4; p = 0.007) compared to patients who either remained disease-free or developed a secondary endometrial carcinoma. Clinicopathological parameters such as age, tumor size, grading and TNM-stage of the resected tumors, adjuvant chemotherapy and irradiation dose did not affect serum Hsp70 levels. Elevated free Hsp70 levels might be indicative for a chronic inflammatory response which could support tumor recurrence. Lymphocyte subpopulation analysis revealed lower NK cell counts after RT in recurrence/metastases patients as compared to disease-free patients. In contrast, no significant changes were observed in the proportion of T and B cells. Conclusion: Longitudinal elevated serum levels of free Hsp70 up to 6 weeks after RT and dropping NK cell counts might be predictive for an unfavourable prognosis in patients with breast cancer.

Keywords: Breast carcinoma prediction, DAMPs, Free Hsp70, Immunophenotyping, Radiotherapy

 

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Radiochemotherapy combined with NK cell transfer followed by second-line PD-1 inhibition in a patient with NSCLC stage IIIb inducing long-term tumor control: a case study

Springer Berlin Heidelberg, 2019, doi.org/10.1007/s00066-019-01434-9 publiziert am 19.02.2019
Background Membrane heat shock protein 70 (mHsp70) is indicative of high-risk tumors and serves as a tumor-specific target for natural killer (NK) cells stimulated with Hsp70 peptide (TKD) and Interleukin(IL)-2. Radiochemotherapy (RCT), mHsp70-targeting NK cells, and programmed death(PD)-1 inhibition were combined to improve the efficacy of tumor-specific immune cells in a non-small cell lung carcinoma (NSCLC) patient. Patient Following simultaneous RCT (64.8 Gy), a patient with inoperable NSCLC (cT4, cN3, cM0, stage IIIb) was treated with 4 cycles of autologous ex vivo TKD/IL-2-activated NK cells and the PD-1 antibody nivolumab as a second-line therapy. Blood samples were taken for immunophenotyping during the course of therapy. Results Adoptive transfer of ex vivo TKD/IL-2-activated NK cells after RCT combined with PD-1 blockade is well tolerated and results in superior overall survival (OS). No viable tumor cells but a massive immune cell infiltration in fibrotic tissue was detected after therapy. Neither tumor progression nor distant metastases were detectable by CT scanning 33 months after diagnosis. Therapy response was associated with significantly increased CD3– /NKG2D+/CD94+ NK cell counts, elevated CD8+ to CD4+ T cell and CD3–/CD56bright to CD3– /CD56dim NK cell ratios, and significantly reduced regulatory T cells (Tregs) in the peripheral blood. Conclusion A combined therapy consisting of RCT, mHsp70-targeting NK cells, and PD-1 antibody inhibition is well tolerated, induces anti-tumor immunity, and results in long-term tumor control in one patient with advanced NSCLC. Further, randomized studies are necessary to confirm the efficacy of this combination therapy.

Keywords Membrane Hsp70 · Radiotherapy · Lung cancer · Immune checkpoint inhibition · Adoptive NK cell transfer

 

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Hsp70 interactions with membrane lipids regulate cellular functions in health and disease

Elsevier Ltd., 2019, publiziert am 30.01.2019
Beyond guarding the cellular proteome the major stress inducible heat shock protein Hsp70 has been shown to interact with lipids. Non-cytosolic Hsp70 stabilizes membranes during stress challenges and, in pathophysiological states, facilitates endocytosis, counteracts apoptotic mechanisms, sustains survival pathways or represents a signal that can be recognized by the immune system. Disease-coupled lipid-associated functions of Hsp70 may be targeted via distinct subcellular localizations of Hsp70 itself or its specific interacting lipids. With a special focus on interacting lipids, here we discuss localization-dependent roles of the membranebound Hsp70 in the context of its therapeutic potential, particularly in cancer and neurodegenerative diseases.

 

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Membrane_Hsp70 A_Novel_Target_for_the_Isolation_of_Circulating_Tumor_Cells_After_Epithelial-to-Mesenchymal_Transition

Frontiers in Oncology, 2018, doi: 10.3389/fonc.2018.00497 publiziert am 01.11.2018
The presence of circulating tumor cells (CTCs) in the peripheral blood is a pre-requisite for progression, invasion, and metastatic spread of cancer. Consequently, the enumeration and molecular characterization of CTCs from the peripheral blood of patients with solid tumors before, during and after treatment serves as a valuable tool for categorizing disease, evaluating prognosis and for predicting and monitoring therapeutic responsiveness. Many of the techniques for isolating CTCs are based on the expression of epithelial cell surface adhesion molecule (EpCAM, CD326) on tumor cells. However, the transition of adherent epithelial cells to migratory mesenchymal cells (epithelial-to-mesenchymal transition, EMT)—an essential element of the metastatic process—is frequently associated with a loss of expression of epithelial cell markers, including EpCAM. A highly relevant proportion of mesenchymal CTCs cannot therefore be isolated using techniques that are based on the “capture” of cells expressing EpCAM. Herein, we provide evidence that a monoclonal antibody (mAb) directed against a membrane-bound form of Hsp70 (mHsp70)—cmHsp70.1—can be used for the isolation of viable CTCs from peripheral blood of tumor patients of different entities in a more quantitative manner. In contrast to EpCAM, the expression of mHsp70 remains stably upregulated on migratory, mesenchymal CTCs, metastases and cells that have been triggered to undergo EMT. Therefore, we propose that approaches for isolating CTCs based on the capture of cells that express mHsp70 using the cmHsp70.1 mAb are superior to those based on EpCAM expression.

 

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Preclinical_Evaluation_of_the_Hsp70_Peptide_Tracer_TPP-PEG24-DFO_89Zr__for_Tumor-Specific_PET_CT_Imaging

American Association for Cancer Research., 2018, doi: 10.1158/0008-5472.CAN-18-0707 publiziert am 01.11.2018
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Synthesis and preclinical characterization of the PSMA-targeted hybrid tracer PSMA-I&F for nuclear and fluorescence imaging of prostate cancer

2018, publiziert am 11.09.2018
The PSMA-targeted radiotracers 68Ga/177Lu-PSMA-I&T and 99mTc-PSMA-I&S are currently successfully employed for clinical PET imaging, radionuclide therapy and radioguided surgery of metastatic prostate cancer. To additionally exploit the high sensitivity and spatial resolution of fluorescence imaging for improved surgical guidance, a first PSMA-I&T-based hybrid tracer, PSMA-I&F (DOTAGA-k(Sulfo-Cy5)-y-nal-k-Sub-KuE), has been developed and evaluated.

The in vitro PSMA-targeting efficiency of PSMA-I&F, the reference PSMA-I&T and their corresponding natGa-/68Ga- and natLu/177Lu-counterparts was determined in LNCaP cells via competitive binding assays (IC50), dual-tracer radioligand- and fluorescence internalization studies. Biodistribution and small-animal PET imaging studies were performed in CB17-SCID and LNCaP xenograft bearing SHO mice, respectively, and complemented by intraoperative far-red fluorescence imaging using a clinical laparoscope. Additionally, fully automated serial cryosectioning and fluorescence imaging of one tumor-bearing animal as well as PSMA immunohistochemistry (IHC) and fluorescence microscopy of organ cryosections (tumor, kidney, spleen) were also performed.

Compared to the parent PSMA-I&T analogs, the PSMA-affinities of PSMA-I&F and its natGa-/natLu-complexes remained high and unaffected by dye conjugation (7.9<IC50<10.5 nM for all ligands). The same was observed for the internalization of 68Ga- and 177Lu-PSMA-I&F. In vivo, blood clearance of 68Ga- and 177Lu-PSMA-I&F was only slightly delayed by high plasma protein binding (94-95%), and very low accumulation in non-target organs was observed already at 1h p.i.. Dynamic PET imaging confirmed PSMA-specific (as demonstrated by coinjection of 2-PMPA) uptake into the LNCaP xenograft (4.5±1.8%iD/g) and the kidneys (106±23%iD/g). Tumor/background ratios of 2.1, 5.2, 9.6 and 9.6 for blood, liver, intestines and muscle, respectively, at 1h p.i. led to excellent imaging contrast in 68Ga-PSMA-I&F PET and in intraoperative fluorescence imaging. Furthermore, fluorescence imaging of tissue cryosections allowed high-resolution visualization of intraorgan PSMA-I&F distribution in vivo and its correlation with PSMA expression as determined by IHC.

Thus, with its high PSMA-targeting efficiency and favorable pharmacokinetic profile, 68Ga/177Lu-PSMA-I&F serves as an excellent proof-of-concept compound for the general feasibility of PSMA-I&T-based hybrid imaging. The PSMA-I&T scaffold represents a versatile PSMA-targeted lead structure, allowing relatively straightforward adaptation to the different structural requirements of dedicated nuclear or hybrid imaging agents.

 

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Modeling and predicting tumor response in radioligand therapy

Journal of Nuclear Medicine, 2018, doi:10.2967/jnumed.118.210377 publiziert am 26.09.2018
The aim of this work was to develop a theranostic method that allows predicting PSMA-positive tumor volume after radioligand therapy (RLT) based on a pretherapeutic PET/CT measurement and physiologically based pharmacokinetic/dynamic (PBPK/PD) modeling at the example of RLT using 177Lulabeled PSMA for imaging and therapy (PSMA I&T). Methods: A recently developed PBPK model for 177Lu PSMA I&T RLT was extended to account for tumor (exponential) growth and reduction due to irradiation (linear quadratic model). Data of 13 patients with metastatic castration-resistant prostate cancer (mCRPC) were retrospectively analyzed. Pharmacokinetic/dynamic parameters were simultaneously fitted in a Bayesian framework to PET/CT activity concentrations, planar scintigraphy data and tumor volumes prior and post (6 weeks) therapy. The method was validated using the leave-one-out Jackknife method. The tumor volume post therapy was predicted based on pre-therapy PET/CT imaging and PBPK/PD modeling. Results: The relative deviation of the predicted and measured tumor volume for PSMA-positive tumor cells (6 weeks post therapy) was 1±40% excluding one patient (PSA negative) from the population. The radiosensitivity for the PSA positive patients was determined to be 0.0172±0.0084 Gy-1. Conclusion: The proposed method is the first attempt to solely use PET/CT and modeling methods to predict the PSMA-positive tumor volume after radioligand therapy. Internal validation shows that this is feasible with an acceptable accuracy. Improvement of the method and external validation of the model is ongoing.

Keywords PBPK/PD model; radioligand therapy; 177Lu-PSMA; tumor response;

 

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The effect of total tumor volume on the biologically effective dose of tumor and kidneys for 177Lu-labelled PSMA peptides

Journal of Nuclear Medicine, 2018, doi:10.2967/jnumed.117.203505 publiziert am 06.03.2018
Aim: To simulate the effect of PSMA positive total tumor volume (TTV) on the tumor and  organs at risk (OARs) biologically effective doses (BEDs) in patients with metastatic  castration resistant prostate cancer (mCRPC) in 177Lu-PSMA radioligand therapy.

Methods: A physiologically-based pharmacokinetic model was fitted to data of 13  patients treated with 177Lu-PSMA I&T. The tumor, kidneys and salivary glands BEDs  were simulated for TTVs of 0.1-10 l. The activity and peptide amounts leading to an optimal tumor-to-kidneys BED ratio were also investigated. Results: Increasing the TTV from 0.3 to 3 l, the simulated BEDs for tumor, kidneys,  parotid and submandibular glands decreased from (22±15) Gy1.49 to (11.0±6.0) Gy1.49, (6.5±2.3) Gy2.5 to (3.7±1.4) Gy2.5, (10.9±2.7) Gy4.5 to (6.4±1.9) Gy4.5, and (11.0±2.7) Gy4.5 to (6.3±1.9) Gy4.5, respectively. The BED for the red marrow increased from 13 (0.17±0.05) Gy15 to (0.32±0.11) Gy15. For patients with TTV > 0.3 l, the optimal amounts  of peptide and activity were (273±136) nmol labeled with (10.4±4.4) GBq. 

Conclusion: This simulation study suggests that in patients with large PSMA positive  tumor volumes higher activity and peptide amounts could be safely administered to  maximize tumor BEDs without exceeding the tolerable BED for the OARs.

Key words: Prostate specific membrane antigen (PSMA); biologically effective dose (BED); total tumor volume (TTV); Physiologically-based pharmacokinetic (PBPK)  modeling.

 

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Hyperkalemia in patients treated with endoradiotherapy combined with amino acid infusion is associated with severe metabolic acidosis

Pfob et al. EJNMMI Research, 2018, doi.org/10.1186/s13550-018-0370-z publiziert am 21.09.2018
Background:

Amino acid co-infusion for renal protection in endoradiotherapy (ERT) applied as prostate-specific membrane antigen (PSMA)-targeted radioligand therapy (RLT) or peptide receptor radionuclide therapy (PRRT) has been shown to cause severe hyperkalemia. The pathophysiology behind the rapid development of hyperkalemia is not well understood. We hypothesized that the hyperkalemia should be associated with metabolic acidosis.

Results: Twenty-two patients underwent ERT. Prior to the first cycle, excretory kidney function was assessed by mercapto-acetyltriglycine (MAG-3) renal scintigraphy, serum biochemistry, and calculated glomerular filtration rate (eGFR). All patients received co-infusion of the cationic amino acids L-arginine and L-lysine for nephroprotection. Clinical symptoms, electrolytes, and acid-base status were evaluated at baseline and after 4 h. No patient developed any clinically relevant side effects. At baseline, acid base status and electrolytes were normal in all patients. Excretory kidney function was normal or only mildly impaired in all except two patients with stage 3 renal insufficiency. All patients developed hyperkalemia. Base excess and HCO3 − were significantly lower after 4 h. In parallel, mean pH dropped from 7.36 to 7.29. There was a weak association between calculated (r = − 0.21) as well as MAG-3-derived GFR (r = − 0.32) and the rise in potassium after 4 h.

Conclusion: Amino acid co-infusion during ERT leads to severe metabolic acidosis which induces hyperkalemia by potassium hydrogen exchange. This novel finding implies that commercially available bicarbonate solutions might be an easy therapeutic option to correct metabolic acidosis rapidly.

Keywords: Hyperkalemia, Radiotherapy, PRRT, RLT, Amino acid, Prostate cancer, Metabolic acidosis

 

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68Ga-PSMA-HBED-CC uptake in cervical, coeliac and sacral ganglia as an important pitfall in prostate cancer PET imaging.

Journal of Nuclear Medicine, 2018, DOI: 10.2967/jnumed.117.204677 publiziert am 25.01.2018
The study aims to investigate the presence of physiological prostate-specific membrane antigen (PSMA)-ligand uptake on positron-emission-tomography (PET) in cervical, coeliac and sacral ganglia of the sympathetic trunk as a pitfall for lymph node metastases in prostate cancer imaging. Methods: 407 patients who underwent "Glu-NH-CO-NH-Lys" radio-labeled with [68Ga]gallium N,N-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N-diacetic acid (68Ga-PSMA-HBED-CC) PET (combined with a diagnostic computed tomography (CT)) were retrospectively analyzed. The number of PSMA PET-positive cervical, coeliac and sacral ganglia was determined and the configuration and maximum standardized uptake value (SUVmax) of each ganglion was measured. In addition, the configuration and SUVmax of adjacent lymph node metastases in the respective region (cervical, coeliac or sacral) were determined. Results: PSMA-ligand uptake above background was detected in 401 (98.5%) patients in any peripheral ganglia, in 369 (92%) patients in cervical ganglia, in 363 (89%) patients in coeliac ganglia, and in 183 (46%) patients in sacral ganglia. The PSMA-ligand uptake was highest in coeliac (mean SUVmax 2.9±0.8 vs. cervical (mean SUVmax 2.4±0.6) and sacral (mean SUVmax 1.7±0.5), both p<0.0001) ganglia. Intra-individually there was a statistically significant, but weak to moderate correlation between the PSMA-ligand uptake in cervical vs. coeliac ganglia (R=0.34, p<0.0001), cervical vs. sacral (R=0.52, p<0.0001) and coeliac vs. sacral (R=0.16, p<0.05). The PSMA-ligand uptake was significantly more intense in adjacent lymph node metastases compared to the respective ganglia (cervical: 18.0±16.2 vs. 2.4±0.6, p<0.0001; coeliac: 13.5±12.3 vs. 2.9±0.8, p<0.0001; sacral: 13.4±11.6 vs. 1.7±0.5, p<0.0001). Furthermore, ganglia predominantly exhibit a band-shaped (71.2%), followed by a teardrop (26.8%) and only rarely a nodular configuration (2.0%). Conversely, lymph node metastases are only rarely band-shaped (1.1%), but more often show teardrop (40.3%) or nodular appearance (58.6%) (p<0.00001). Conclusion: PSMA-ligand uptake in ganglia along the sympathetic trunk as assessed by 68Ga-PSMA-HBED-CC PET represents an important pitfall in prostate cancer PET imaging. The PSMA-ligand uptake is higher in coeliac ganglia compared to cervical or sacral ganglia and the level of PSMA-ligand uptake seems to be patient-related. For the differentiation between lymph node metastases and sympathetic ganglia both intensity of PSMA-ligand-uptake as well as exact localization and configuration of the respective lesion should be examined carefully.

 

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Prostate Cancer Molecular Imaging Standardized Evaluation (PROMISE): Proposed miTNM Classification for the Interpretation of PSMA-Ligand PET/CT

THE JOURNAL OF NUCLEAR MEDICINE, 2018, DOI: 10.2967/jnumed.117.198119 publiziert am 10.01.2018

Prostate-specific membrane antigen (PSMA)–ligand PET imaging provides unprecedented accuracy for whole-body staging of prostate cancer. As PSMA-ligand PET/CT is increasingly adopted in clinical trials and routine practice worldwide, a unified language for image reporting is urgently needed. We propose a molecular imaging TNM system (miTNM, version 1.0) as a standardized reporting framework for PSMA-ligand PET/CT or PET/MRI. miTNM is designed to organize findings in comprehensible categories to promote the exchange of information among physicians and institutions. Additionally, flowcharts integrating findings of PSMA-ligand PET and morphologic imaging have been designed to guide image interpretation. Specific applications, such as assessment of prognosis or impact on management, should be evaluated in future trials. miTNM is a living framework that evolves with clinical experience and scientific data.

 

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Targeting experimental orthotopic glioblastoma with chitosan-based superparamagnetic iron oxide nanoparticles (CS-DX-SPIONs)

Technische Universität München, Klinikum rechts der Isar, Germany, Munich, 2018, publiziert am 08.01.2018
Glioblastoma is the most devastating primary brain tumor of the central nervous system in adults. Magnetic nanocarriers may help not only for a targeted delivery of chemotherapeutic agents into the tumor but also provide contrast enhancing properties for diagnostics using magnetic resonance imaging (MRI). Synthesized hybrid chitosan-dextran superparamagnetic nanoparticles (CS-DX-SPIONs) showed a uniform diameter of 55 nm under transmission electron microscopy and superparamagentic characteristics as determined by T1 and T2 proton relaxation times. Application of the chitosan increased the charge from +8.9 to +19.3 mV of the dextran-based SPIONs. The nonlinear magnetic response at second harmonic of CS-DX-SPIONs following the slow change of stationary magnetic fields with very low hysteresis evidenced superparamagnetic state of particles at ambient temperatures. Confocal microscopy and flow cytometry studies showed an enhanced internalization of the chitosan-based nanoparticles in U87, C6 glioma and HeLa cervix carcinoma cells as compared to dextran-coated particles. After internalization cytotoxicity increased in a dose-dependent manner. Acceptable toxicity profiles of the synthesized nanoparticles were observed up to a concentration of 10 μg/ml. Intraveneously administered CS-DX-SPIONs in orthotopic C6 gliomas in rats accumulated in the tumor as shown by high-resolution MR imaging (11.0 T). Retention of nanoparticles resulted in a significant contrast enhancement of the tumor image that was accompanied with a dramatic drop in T2 values (P<0.001). Subsequent histological studies proved an accumulation of the nanoparticles inside glioblastoma cells. Hybrid chitosan-dextran magnetic particles demonstrated high MR contrast enhancing properties for the delineation of the brain tumor. Due to a significant retention of the particles in the tumor an application of the CS-DX-SPIONs could not only improve the tumor imaging but also could allow a targeted delivery of chemotherapeutic agents.

 

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Efficacy, Predictive Factors, and Prediction Nomograms for 68Ga-labeled Prostate-specific Membrane Antigen–ligand Positron-emission Tomography/Computed Tomography in Early Biochemical Recurrent Prostate Cancer After Radical Prostatectomy

European Association of Urology. Published by Elsevier B.V., 2018, doi.org/10.1016/j.eururo.2018.01.006 publiziert am 03.01.2018
Recently, 68Ga-labeled prostate-specific membrane antigen (PSMA)–ligand positron-emission tomography (PET) imaging has been shown to improve detection rates in recurrent prostate cancer (PC). However, published studies include only small patient numbers at low prostate-specific antigen (PSA) values. For this study, 272 consecutive patients with biochemical recurrence after radical prostatectomy and PSA value between 0.2 and 1 ng/ml were included. The 68Ga-PSMA-ligand PET/computed tomography (CT) was evaluated, and detection rates were determined and correlated to various clinical variables using univariate and multivariable analyses. Subgroups of patients with very low (0.2–0.5 ng/ml) and low (>0.5–1.0 ng/ml) PSA values were analyzed. In total, lesions indicative of PC recurrence were detected in 55% (74/134) and 74% (102/138) with very low and low PSA values, respectively. Main sites of recurrence were pelvic or retroperitoneal lymph nodes metastases, followed by local recurrence and bone metastases with higher probability in the low versus very low PSA subgroup. Detection rates significantly increased with higher PSA values, primary pT 3a, primary pN+ disease, grade group 4, previous radiation therapy, and concurrent androgen deprivation therapy (ADT) in univariate analysis. In a multivariable logistic regression model, concurrent ADT and PSA values were identified as most relevant predictors of positive 68Ga-PSMA-ligand PET/CT. Further, prediction nomograms were established, which may help in estimating pretest PSMA-ligand PET positivity in clinical practice. Patient summary: In our study, 68Ga-labeled prostate-specific membrane antigen (PSMA)–ligand positron- emission tomography (PET)/computed tomography (CT) detected recurrent disease after radical prostatec- tomy in 55% (74/134) and 74% (102/138) of patients with very low (0.2–0.5 ng/ml) and low (>0.5–1.0 ng/ml) prostate-specific antigen values, respectively. On the basis of these data, it seems reasonable to perform 68Ga- PSMA-ligand PET/CT also in patients with early biochemical recurrence, as it can tailor further therapy decisions (eg, local vs systemic treatment). The established prediction nomograms can further assist urologists in discussions on the use of 68Ga-PSMA-ligand PET/CT with their patients in specific clinical settings.

 

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Head-to-head comparison of 68Ga-PSMA-11 and 18F-Fluciclovine PET/CT in a case series of 10 patients with prostate cancer recurrence

Journal of Nuclear Medicine, 2017, doi:10.2967/jnumed.117.203257 publiziert am 21.12.2017
 

Purpose: This is a head-to-head comparison between Gallium-68 Prostate Specific Membrane Antigen (68Ga-PSMA-11) and 18F-Fluciclovine Positron Emission Tomography/Computed Tomography (PET/CT) in a series of 10 patients with prostate cancer (PCa) recurrence. Methods: 288 patients with PCa recurrence were enrolled in a prospective study of 68Ga-PSMA-11 PET/CT imaging for recurrent disease localization (NCT02940262). We retrospectively identified 10 patients who underwent clinical indicated 18F-Fluciclovine PET/CT prior enrollment. Results: The median time between´ both scans was 2.3 months (range 0.2-4.2). The median PSA value was 1.0 ng/ml (mean 4.7 ng/ml; range 0.13-18.1) and 1.1 ng/ml (mean 6.2 ng/ml; range 0.24-31.3) at the time of 18F-Fluciclovine and 68Ga-PSMA-11 PET/CT, respectively. 5/10 patients (50%) were negative on 18F-Fluciclovine PET/CT but showed positive results on 68Ga-PSMA-11 PET/CT. 2/10 patients (20%) had both positive 18F-Fluciclovine and 68Ga-PSMA-11 PET/CT but 68Ga-PSMA-11 PET/CT showed additional lymph nodes (LN) metastasis. 3/10 patients (30%) had both negative 18F-Fluciclovine and 68Ga-PSMA-11 PET/CT. Conclusion: This case series suggests improved detection rates of 68Ga-PSMA-11 when compared to 18F-Fluciclovine PET/CT in patients with recurrent PCa. Prospective trials designed to directly compare 68Ga-PSMA-11 and 18F-Fluciclovine PET/CT should be initiated.

 

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Actual impact of 68Ga-PSMA-11 PET/CT on the management of prostate cancer patients with biochemical recurrence.

Journal of Nuclear Medicine, 2017, doi: 10.2967/jnumed.117.202945 publiziert am 14.12.2017
Purpose: In this prospective survey of referring physicians, we investigated whether and how Gallium-68 Prostate Specific Membrane Antigen Positron Emission Tomography/Computed Tomography (PSMA-11 PET/CT) affects the actually implemented management of prostate cancer patients wih biochemical recurrence (BCR). Methods: We conducted a prospective survey of physicians (NCT02940262) who referred 161 patients with prostate cancer BCR (median Prostate Specific Antigen (PSA) value 1.7 ng/ml (range 0.05-202)). Referring physicians completed one questionnaire prior to the scan to indicate the treatment plan without PSMA-11 PET/CT information (Q1; n = 101); one immediately after the scan to denote intended management changes (Q2; n = 101); and one 3 to 6 months later to document the final implemented management (Q3; n = 56). Implemented management was also obtained via electronic chart review and/or patient contact (n = 45). Results: Complete documented management strategy (Q1+Q2+implemented management) was available in 101/161 patients (63%). Seventy-six of these (75%) had a positive PSMA-11 PET/CT study. The actually implemented management differed from the pre-scan intended treatment plan (Q1) in 54/101 patients (53%). The post-scan intended management (Q2) differed from the pre-scan intended management (Q1) in 62/101 patients (61%); however, these intended changes were not implemented in 29/62 patients (47%). Pelvic nodal and extra-pelvic metastatic disease on PSMA-11 PET/CT (PSMA T0N1M0 and PSMA T0N1M1 patterns) were significantly associated with implemented management changes (P = 0,001, 0.05). Conclusion: PSMA-11 PET/CT results in actually implemented management changes in more than 50% of prostate cancer patients with BCR (54/101; 53%). However, intended management changes early after PSMA-11 PET/CT frequently differ from actually implemented management changes.

 

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Animal models of MEN1

Bioscientifica Ltd., 2017, DOI: 10.1530/ERC-17-0249 publiziert am 20.11.2017
Animal models of cancer have been instrumental in advancing our understanding of the biology of tumor initiation and progression, in studying gene function and in performing preclinical studies aimed at testing novel therapies. Several animal models of the MEN1 syndrome have been generated in different organisms by introducing loss-of-function mutations in the orthologues of the human MEN1 gene. In this review, we will discuss MEN1 and MEN1-like models in Drosophila, mice and rats. These model systems with their specific advantages and limitations have contributed to elucidate the function of Menin in tumorigenesis, which turned out to be remarkably conserved from flies to mammals, as well as the biology of the disease. Mouse models of MEN1 closely resemble the human disease in terms of tumor spectrum and associated hormonal changes, although individual tumor frequencies are variable. Rats affected by the MENX (MEN1-like) syndrome share some features with MEN1 patients albeit they bear a germline mutation in Cdkn1b (p27) and not in Men1. Both Men1-knockout mice and MENX rats have been exploited for therapy-response studies testing novel drugs for efficacy against neuroendocrine tumors (NETs) and have provided promising leads for novel therapies. In addition to presenting well-established models of MEN1, we also discuss potential models which, if implemented, might broaden even further our knowledge of neuroendocrine tumorigenesis. In the future, patient-derived xenografts in zebrafish or mice might allow us to expand the tool-box currently available for preclinical studies of MEN1-associated tumors.

 

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Characterization of neuroendocrine tumors in heterozygous mutant MENX rats: a novel model of invasive medullary thyroid carcinoma

Society for Endocrinology, 2017, publiziert am 15.11.2017
Rats affected by the MENX syndrome spontaneously develop multiple neuroendocrine tumors (NETs) including adrenal, pituitary and thyroid gland neoplasms. MENX was initially reported to be inherited as a recessive trait and affected rats were found to be homozygous for the predisposing Cdkn1b mutation encoding p27. We here report that heterozygous MENX mutant rats (p27+/mut) develop the same spectrum of NETs seen in the homozygous (p27mut/mut) animals but with slower progression. Consequently, p27+/mut rats have a significantly shorter lifespan compared with their wild-type (p27+/+) littermates. In the tumors of p27+/mut rats, the wild-type Cdkn1b allele is neither lost nor silenced, implying that p27 is haploinsufficient for tumor suppression in this model. Transcriptome profiling of rat adrenal (pheochromocytoma) and pituitary tumors having different p27 dosages revealed a tissue-specific, dose-dependent effect of p27 on gene expression. In p27+/mut rats, thyroid neoplasms progress to invasive and metastatic medullary thyroid carcinomas (MTCs) accompanied by increased calcitonin levels, as in humans. Comparison of expression signatures of late-stage versus early-stage MTCs from p27+/mut rats identified genes potentially involved in tumor aggressiveness. The expression of a subset of these genes was evaluated in human MTCs, and found associated with aggressive RET-M918T-positive tumors. Altogether, p27 haploinsufficiency in MENX rats uncovered a novel, representative model of invasive and metastatic MTC exploitable for translational studies of this often aggressive and incurable cancer.

 

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68Ga-PSMA-11 PET/CT mapping of prostate cancer biochemical recurrence following radical prostatectomy in 270 patients with PSA<1.0ng/ml: Impact on Salvage Radiotherapy Planning

Journal of Nuclear Medicine, 2017, doi:10.2967/jnumed.117.201749 publiziert am 09.11.2017

Background: Target volume delineations for prostate cancer (PCa) salvage radiotherapy (SRT) after radical prostatectomy are usually drawn in the absence of visibly recurrent disease. Gallium-68 Prostate Specific Membrane Antigen Positron Emission Tomography/Computed Tomography (68Ga-PSMA-11 PET/CT) detects recurrent PCa with sensitivity superior to standard of care imaging at serum prostate specific antigen (PSA) values low enough to impact target volume delineations for routine SRT. Objective: To i) map the recurrence pattern of PCa early biochemical recurrence (BCR) after radical prostatectomy with 68Ga-PSMA-11 PET/CT in patients with serum PSA levels <1 ng/ml, ii) determine how often consensus clinical target volumes (CTV) based on the Radiation Therapy Oncology Group (RTOG) guidelines cover 68Ga-PSMA-11 PET/CT-defined disease, and iii) assess the potential impact of 68Ga-PSMA-11 PET/CT on SRT. Patients and Methods: This is a post-hoc analysis of an intention-to-treat population of 270´patients who underwent 68Ga-PSMA-11 PET/CT at 4 institutions for BCR after prostatectomy without prior radiotherapy at PSA<1 ng/mL. RTOG consensus CTV that included both the prostate bed and pelvic lymph nodes were contoured on the CT dataset of the PET/CT by a radiation oncologist blinded to the PET component. 68Ga-PSMA-11 PET/CT images were analyzed by a nuclear medicine physician. PSMA-positive lesions not covered by planning volumes based on the consensus CTV were considered to have a major potential impact on treatment planning. Results: The median PSA at the time of 68Ga-PSMA-11 PET/CT was 0.48 ng/ml (range 0.03-1). One-hundred-thirty-two/270 patients (49%) had a positive 68Ga-PSMA-11 PET/CT. Fifty-two/270 (19%) had at least one PSMA-positive lesion not covered by the consensus CTV. Thirty-three/270 (12%) had extra-pelvic PSMA-positive lesions and 19/270 (7%) had PSMA-positive lesions within the pelvis but not covered by consensus CTV. The two most common 68Ga-PSMA-11 PETpositive lesion locations outside the consensus CTV were bone (23/52, 44%) and perirectal lymph nodes (16/52, 31%). Conclusion: Post-hoc analysis of 68Ga-PSMA-11 PET/CT implies a major impact on SRT planning in 52/270 patients (19%) with PCa early BCR (PSA<1.0 ng/ml). This justifies a randomized imaging trial of SRT with or without 68Ga-PSMA-11 PET/CT investigating its potential benefit on clinical outcome.

 

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The role of p27 in pheochromocytoma development

1Helmholtzzentrum München, Institute of Diabetes and Cancer, Munich, Germany 2University Nice, Institute of molecular and cellular Pharmacology, Valbonne, France 3Helmholtzzentrum München, Institute of Computational Biology, Munich, Germany, 2016, publiziert am 04.11.2016
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Targeting PI3K/mTOR signaling exerts potent antitumor activity in pheochromocytoma in vivo

Endocr Relat Cancer, 2016, publiziert am 03.11.2016
Pheochromocytomas (PCCs) are mostly benign tumors, amenable to complete surgical resection. However, 10-17% of cases can become malignant, and once metastasized, there is no curative treatment for this disease. Given the need to identify effective therapeutic approaches for PCC, we evaluated the antitumor potential of the dual PI3K/mTOR inhibitor BEZ235 against these tumors. We employed an in vivo model of endogenous PCCs (MENX mutant rats), which closely recapitulate the human tumors. Mutant rats with PCCs were treated with 2 doses of BEZ235 (20 and 30 mg/kg), or with placebo, for 2 weeks. Treatment with BEZ235 induced cytostatic and cytotoxic effects on rat PCCs, which could be appreciated by both staining the tumors ex vivo with appropriate markers, and non-invasively by functional imaging (diffusion weighted-magnetic resonance imaging) in vivo. Transcriptomic analyses of tumors from rats treated with BEZ235 or placebo identified potential mediators of therapy response. Slc6a2, encoding the norepinephrine transporter (NET), was downregulated in a dose-dependent manner by BEZ235 in rat PCCs. Moreover, BEZ235 reduced Slc6a2/NET expression also in PCC cell lines (MPC). Studies of a BEZ235-resistant derivative of the MPC cell line confirmed that the reduction of NET expression associates with the response to the drug. Reduction of NET expression following BEZ235 treatment in vivo could be monitored by Positron Emission Tomography (PET) using a tracer targeting NET. Altogether, we here demonstrate the efficacy of BEZ235 against PCC in vivo, and show that functional imaging can be employed to monitor the response of PCC to PI3K/mTOR inhibition therapy.

Anticalins directed against vascular endothelial growth factor receptor 3 (VEGFR-3) with picomolar affinities show potential for medical therapy and in vivo imaging

Biological Chemistry, 2016, DOI: 10.1515/hsz-2016-0195 publiziert am 16.08.2016
Members of the vascular endothelial growth factor receptor (VEGFR) family play a central role in angiogenesis as well as lymphangiogenesis and are crucial for tumor growth and metastasis. In particular, VEGFR-3 expression is induced in endothelial cells during tumor angiogenesis. We report the design of Anticalins that specifically recognize the ligandbinding domains 1 and 2 of VEGFR-3. To this end, a library of the lipocalin 2 scaffold with 20 randomized positions distributed across its binding site was subjected to phage display selection and ELISA screening using the VEGF-C binding fragment (D1-2) or the entire extracellular region (D1-7) of VEGFR-3 as target proteins. Promising Anticalin candidates were produced in E. coli and biochemically characterized. Three variants with different receptor binding modes were identified, and two of them were optimized with regard to target affinity as well as folding efficiency. The resulting Anticalins show dissociation constants down to the single-digit picomolar range. Specific recognition of VEGFR-3 on cells was demonstrated by immunofluorescence microscopy. Competitive binding versus VEGF-C was demonstrated for two of the Anticalins with Ki values in the low nanomolar range. Based on these data, VEGFR-3 specific Anticalins provide promising reagents for the diagnosis and/or therapeutic intervention of tumor-associated vessel growth.

 

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Zero-valent Fe confined mesoporous silica nanocarriers (Fe(0) @ MCM-41) for targeting experimental orthotopic glioma in rats

www.nature.com/scientificreports/, 2016, DOI: 10.1038/srep29247 publiziert am 08.07.2016
Mesoporous silica nanoparticles (MSNs) impregnated with zero-valent Fe (Fe(0) @ MCM-41) represent an attractive nanocarrier system for drug delivery into tumor cells. The major goal of this work was to assess whether MSNs can penetrate the blood-brain barrier in a glioblastoma rat model. Synthesized MSNs nanomaterials were characterized by energy dispersive X-ray spectroscopy, measurements of X-ray diffraction, scanning electron microscopy and Mössbauer spectroscopy. For the detection of the MSNs by MR and for biodistribution studies MSNs were labeled with zero-valent Fe. Subsequent magnetometry and nonlinear-longitudinal-response-M2 (NLR-M2) measurements confirmed the MR negative contrast enhancement properties of the nanoparticles. After incubation of different tumor (C6 glioma, U87 glioma, K562 erythroleukemia, HeLa cervix carcinoma) and normal cells such as fibroblasts and peripheral blood mononuclear cells (PBMCs) MSNs rapidly get internalized into the cytosol. Intracellular residing MSNs result in an enhanced cytotoxicity as Fe(0) @ MCM-41 promote the reactive oxygen species production. MRI and histological studies indicated an accumulation of intravenously injected Fe(0) @ MCM-41 MSNs in orthotopic C6 glioma model. Biodistribution studies with measurements of second harmonic of magnetization demonstrated an increased and dose-dependent retention of MSNs in tumor tissues. Taken together, this study demonstrates that MSNs can enter the blood-brain barrier and accumulate in tumorous tissues.

 

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70-kDa heat shock protein coated magnetic nanocarriers as a nanovaccine for induction of anti-tumor immune response in experimental glioma

Elsevier B.V., 2015, doi.org/10.1016/j.jconrel.2015.10.051 publiziert am 29.10.2015
Nanovaccines based on superparamagnetic iron oxide nanoparticles (SPIONs) provide a novel approach to induce the humoral and cell-based immune system to fight cancer. Herein, we increased the immunostimulatory capacity of SPIONs by coating themwith recombinant heat shock protein 70 (Hsp70) which is known to chaperone antigenic peptides. After binding, Hsp70–SPIONs deliver immunogenic peptides from tumor lysates to dendritiс cells (DCs) and thus stimulate a tumor-specific, CD8+ cytotoxic T cell response. We could show that binding activity of Hsp70–SPIONs to the substrate-binding domain (SBD) is highly dependent on the ATPase activity of its nucleotide-binding domain NBD), as shown by 31P NMR spectroscopy. Immunization of C6 glioma-bearing rats with DCs pulsedwith Hsp70–SPIONs and tumor lysates resulted in a delayed tumor progression (as measured byMRI) and an increased overall survival. In parallel an increased IFNγ secretion were detected in the serum of these animals and immunohistological analysis of subsequent cryosections of the glioma revealed an enhanced infiltration of memory CD45RO+ and cytotoxic CD8+ T cells. Taken together the study demonstrates that magnetic nanocarriers such as SPIONs coated with Hsp70 can be applied as a platform for boosting anti-cancer immune responses.

 

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Anticalins directed against the fibronectin extra domain B as diagnostic tracers for glioblastomas

International Journal of Cancer, 2015, DOI: 10.1002/ijc.29874 publiziert am 30.09.2015
The standard of care for diagnosis and therapy monitoring of gliomas is magnetic resonance imaging (MRI), which however, provides only an indirect and incomplete representation of the tumor mass, offers limited information for patient stratification according to WHO-grades and may insufficiently indicate tumor relapse after antiangiogenic therapy. Anticalins are alternative binding proteins obtained via combinatorial protein design from the human lipocalin scaffold that offer novel diagnostic reagents for histology and imaging applications. Here, the Anticalins N7A, N7E and N9B, which possess exquisite specificity and affinity for oncofetal fibronectin carrying the extra domain B (ED-B), a well-known proangiogenic extracellular matrix protein, were applied for immunohistochemical studies. When investigating ED-B expression in biopsies from 41 patients with confirmed gliomas of WHO grades I to IV, or in non-neoplastic brain samples, we found that Anticalins specifically detect ED-B in primary glioblastoma multiforme (GBM; WHO IV) but not in tumors of lower histopathological grade or in tumor-free brain. In primary GBM samples, ED-B specific Anticalins locate to fibronectin-rich perivascular areas that are associated with angiogenesis. Anticalins specifically detect ED-B both in fixed tumor specimen and on vital cells, as evidenced by cytofluorometry. Beyond that, we labeled an Anticalin with the c-emitter 123I and demonstrated specific binding to GBM-tissue samples using in vitro autoradiography. Overall, our data indicate that ED-B specific Anticalins are useful tools for the diagnosis of primary GBM and related angiogenic sites, presenting them as promising tracers for molecular tumor imaging.

 

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Targeting PI3K/mTOR Signaling Displays Potent Antitumor Efficacy against Nonfunctioning Pituitary Adenomas

American Association for Cancer Research, 2015, DOI: 10.1158/1078-0432.CCR-15-0288 publiziert am 02.04.2015
Purpose: Novel therapeutic approaches are needed to improve the postoperative management of residual nonfunctioning pituitary adenomas (NFPA), given their high relapse rate. Here, we evaluated the antitumor efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in the only available model of spontaneous NFPAs (MENX rats). Experimental Design: Organotypic cultures of rat primary NFPAs were incubated with NVP-BEZ235 and assessed for cell viability, proliferation, apoptosis, and PI3K/mTOR inhibition. NVP-BEZ235, or placebo, was administered to MENX rats and tumor response was monitored noninvasively by diffusion weighted-magnetic resonance imaging (DW-MRI). Following treatment, tumor tissues were investigated for cell proliferation, apoptosis, and PI3K/mTOR inhibition. Genes mediating the cytotoxic activity of NVP-BEZ235 were identified by gene-expression profiling. Among them, Defb1, encoding beta-defensin 1, was further studied for its role in pituitary cells and in human pancreatic neuroendocrine tumor (NET) cells. Results: NVP-BEZ235 showed antiproliferative and pro-cell death activities against NFPAs both in vitro and in vivo, and the response to the drug correlated with inhibition of the PI3K pathway. DW-MRI identified early functional changes (decreased cellularity) in the adenomas before their size was affected and emerged as a useful modality to assess therapy response. The cytotoxic effect of PI3K/mTOR blockade inNFPA was mediated by several genes, including Defb1. NVP-BEZ235 treatment induced Defb1 expression in NFPAs in vitro and in vivo, and in pancreatic NET cells. High Defb1 levels sensitized NET cells to PI3K/mTOR inhibition. Conclusions: Our findings provide rationale for clinical investigation of PI3K/mTOR inhibition in NFPAs and identify novel effectors of PI3K-mediated neuroendocrine cell survival. Clin CancerRes; 1–12. _2015 AACR.

 

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SSTR3 is a putative target for the medical treatment of gonadotroph adenomas of the pituitary

Endocrine-Related Cancer, 2014, DOI: 10.1530/ERC-14-0472 publiziert am 27.01.2015
Gonadotroph pituitary adenomas (GPAs) often present as invasive macroadenomas not amenable to complete surgical resection. Radiotherapy is the only post-operative option for patients with large invasive or recurrent lesions. No medical treatment is available for these patients. The somatostatin analogs (SSAs) octreotide and lanreotide that preferentially target somatostatin receptor type 2 (SSTR2) have little effect on GPAs. It is widely accepted that the expression of specific SSTR subtypes determines the response to SSAs. Given that previous studies on mRNA and protein expression of SSTRs in GPAs have generated conflicting results, we investigated the expression of SSTR2, SSTR3, and SSTR5 (the main targets of available SSAs) in a clinically and pathologically well-characterized cohort of 108 patients with GPAs. A total of 118 samples were examined by immunohistochemistry using validated and specific MABs. Matched primary and recurrent tissues were available for ten patients. The results obtained were validated in an independent cohort of 27 GPAs. We observed that SSTR3 was significantly more abundant than SSTR2 (P<0.0001) in GPAs, while full-length SSTR5 was only expressed in few tumors. Expression of SSTR3 was similar in primary and recurrent adenomas, was high in potentially aggressive lesions, and did not change significantly in adenomas that recurred after irradiation. In conclusion, low levels of expression of SSTR2 may account for the limited response of GPAs to octreotide and lanreotide. Given the potent anti-proliferative, pro-apoptotic, and anti-angiogenic activities of SSTR3, targeting this receptor with a multireceptor ligand SSA such as pasireotide may be indicated for potentially aggressive GPAs.

 

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α-Radioimmunotherapy with 213-Bi-anti-CD38 immunoconjugates is effective in a mouse model of human multiple myeloma

Oncotarget, Advance Publications 2014, 2014, publiziert am 09.02.2015
In spite of development of molecular therapeutics, multiple myeloma (MM) is fatal in most cases. CD38 is a promising target for selective treatment of MM. We tested radioimmunoconjugates consisting of the α-emitter 213-Bi coupled to an anti-CD38 MAb in preclinical treatment of MM. Efficacy of 213-Bi-anti-CD38-MAb was assayed towards different MM cell lines with regard to induction of DNA double-strand breaks, induction of apoptosis and initiation of cell cycle arrest. Moreover, mice bearing luciferase-expressing MM xenografts were treated with 213-Bi-anti-CD38-MAb. Therapeutic efficacy was monitored by bioluminescence imaging, overall survival and histology. 213-Bi-anti-CD38-MAb treatment induced DNA damage which did not result in activation of the G2 DNA-damage-response checkpoint, but instead in mitotic arrest and subsequent mitotic catastrophe. The anti-tumor effect of 213-Bi-anti-CD38-MAb correlated with the expression level of CD38 in each MM cell line. In myeloma xenografts, treatment with 213-Bi-anti-CD38-MAb suppressed tumor growth via induction of apoptosis in tumor tissue and significantly prolonged survival compared to controls. The major organ systems did not show any signs of 213-Bi-induced toxicity. Preclinical treatment of MM with 213-Bi-anti-CD38-MAb turned out as an effective therapeutic option.

 

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ALDH1A1 expression increases in recurrent Glioblastoma patients and predicts a chemoresistant phenotype

Technische Universität München & Klinikum Rechts der Isar der Technischen Universität München, 2015, publiziert am 30.11.2014
Glioblastomas (GBM) are the most common brain tumors in humansandare effectively incurable. The defining hallmarks of the aggressiveness of GBMarelocal invasion and neo-angiogenesis. Limited efficacy of patient treatmentstrategiesis due to rapid invasion of surrounding tissue and the fast appearance of chemo-andradioresistant tumor cells often termed cancer stem cells (CSC). Our laboratoryhasrecently shown that Aldehyde Dehydrogenase 1 A1 (ALDH1A1) expressing GBM cells show a CS Cphenotype and a nincreased resistance to Temozolomid (TMZ) compared to ALDH1A1 negative cells.

 

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Selective In Vivo Imaging of Syngeneic, Spontaneous, and Xenograft Tumors Using a Novel Tumor Cell–Specific Hsp70 Peptide-Based Probe

Cancer Research, 2014, doi:10.1158/0008-5472.CAN-14-0413 publiziert am 09.10.2014
Although in vivo targeting of tumors using florescently labeled probes has greatly gained in importance over the last few years, most of the clinically applied reagents lack tumor cell specificity. Our novel tumor cell – penetrating peptide-based probe (TPP) recognizes an epitope of Hsp70 that is exclusively present on the cell surface of a broad variety of human and mouse tumors and metastases, but not on normal tissues. Because of the rapid turnover rate of membrane Hsp70, fluorescently labeled TPP is continuously internalized into syngeneic, spontaneous, chemically/genetically induced and xenograft tumors following intravenous administration, thereby enabling site-specific labeling of primary tumors and metastases. In contrast with the commercially available nonpeptide small molecule a v b 3 -integrin antagonist IntegriSense, TPP exhibits a significantly higher tumor-to-background contrast and stronger tumor-specific signal intensity in all tested tumor models. Moreover, in contrast with IntegriSense, TPP reliably differentiates between tumor cells and cells of the tumor microenvironment, such as tumor-associated macrophages and fibroblasts, which were found to be membrane-Hsp70 negative. Therefore, TPP provides a useful tool for multimodal imaging of tumors and metastases that might help to improve our understanding of tumorigenesis and allow the establishment of improved diagnostic procedures and more accurate therapeutic monitoring. TPP might also be a promising platform for tumor-specific drug delivery and other Hsp70-based targeted therapies.

 

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Technical considerations on the validity of blood oxygenation level-dependent-based MR assessment of vascular deoxygenation

Wiley Online Library, 2014, DOI 10.1002/nbm.3131 publiziert am 09.05.2014
A blood oxygenation level-dependent (BOLD)-based apparent relative oxygen extraction fraction (rOEF) as a semi-quantitative marker of vascular deoxygenation has recently been introduced in clinical studies of patients with glioma and stroke, yielding promising results. These rOEF measurements are based on independent quantification of the transverse relaxation times T 2 and T 2 * and relative cerebral blood volume (rCBV). Simulations demonstrate that small errors in any of the underlying measures may result in a large deviation of the calculated rOEF. Therefore, we investigated the validity of such measurements. For this, we evaluated the quantitative measurements of T 2 and T 2 * at 3 T in a gel phantom, in healthy subjects and in healthy tissue of patients with brain tumors. We calculated rOEF maps covering large portions of the brain from T 2 , T 2 * and rCBV [routinely measured in patients using dynamic susceptibility contrast (DSC)], and obtained rOEF values of 0.63 ± 0.16 and 0.90 ± 0.21 in healthy-appearing gray matter (GM) and white matter (WM), respectively; values of about 0.4 are usually reported. Quantitative T 2 mapping using the fast, clinically feasible, multi-echo gradient spin echo (GRASE) approach yields significantly higher values than much slower multiple single spin echo (SE) experiments. Although T 2 * mapping is reliable in magnetically homogeneous tissues, uncorrectable macroscopic background gradients and other effects (e.g. iron deposition) shorten T 2 *. Cerebral blood volume (CBV) measurement using DSC and normalization to WM yields robust estimates of rCBV in healthy-appearing brain tissue; absolute quantification of the venous fraction of CBV, however, is difficult to achieve. Our study demonstrates that quantitative measurements of rOEF are currently biased by inherent difficulties in T 2 and CBV quanti fi cation, but also by inadequacies of the underlying model. We argue, however, that standardized, reproducible measurements of apparent T 2 , T 2 * and rCBV may still allow the estimation of a meaningful apparent rOEF, which requires further validation in clinical studies.

 

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Hypoxia upregulates aldehyde dehydrogenase isoform 1 (ALDH1) expression and induces functional stem cell characteristics in human glioblastoma cells

Brain Tumor Pathology, 2013, DOI 10.1007/s10014-013-0170-0 publiziert am 06.11.2013
Aldehyde dehydrogenase 1 (ALDH1) has been used to isolate tumorigenic stem-like cells in a large number of tumors, including glioblastoma multiforme (GBM). We recently showed that human glioblastoma cells with high ALDH1 (ALDH1high) activity contain stem-cell-like characteristics. In the study reported here, we isolated established and primary human glioblastoma cells based on their ALDH1 expression. When tested for asymmetric division, only cells with ALDH1high expression were able to restore heterogeneous populations after a few days, whereas cells with ALDH1low levels could not. Most interestingly, the capacity of cells with ALDH1low levels to divide asymmetrically into cells with either ALDH1high or ALDH1low expression could be restored after exposure to hypoxic culture conditions. Consequently, we found neurosphere formation reinstated in posthypoxic, formerly ALDH1low, cells. The direct involvement of ALDH1 could be confirmed by ALDH1 small hairpin ribonucleic acid (shRNA) knockdown, suggesting ALDH1 as an intracellular marker for the identification and isolation of stem-like glioblastoma cells. In summary, we show that ALDH1 expression correlates well with asymmetric division capacity and tumor sphere formation. Furthermore, we demonstrated that hypoxic culture conditions induce and/or upregulate ALDH1 expression in established and primary GBM cell lines.

 

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MR-based hypoxia measures in human glioma

Journal of Neuro-Oncology (J Neurooncol (2013) 115:197–207), 2013, DOI 10.1007/s11060-013-1210-7 publiziert am 07.08.2013
Hypoxia plays a central role in tumor stem cell genesis and is related to a more malignant tumor phenotype, therapy resistance (e.g. in anti-angiogenic therapies) and radio-insensitivity. Reliable hypoxia imaging would provide crucial metabolic information in the diagnostic work-up of brain tumors. In this study, we applied a novel BOLD-based MRI method for the measurement of relative oxygen extraction fraction (rOEF) in glioma patients and investigated potential benefits and drawbacks. Forty-five glioma patients were examined preoperatively in a pilot study on a 3T MR scanner. rOEF was calculated from quantitative transverse relaxation rates (T2, T2*) and cerebral blood volume (CBV) using a quantitative BOLD approach. rOEF maps were assessed visually and by means of a volume of interest (VOI) analysis. In six cases, MRI-targeted biopsy samples were analyzed using HIF-1a-immunohistochemistry. rOEF maps could be obtained with a diagnostic quality. Focal spots with high rOEF values were observed in the majority of high-grade tumors but in none of the low-grade tumors. VOI analysis revealed potentially hypoxic tumor regions with high rOEF in contrast-enhancing tumor regions as well as in the non-enhancing infiltration zone. Systematic bias was found as a result of non-BOLD susceptibility effects (T2*) and contrast agent leakage affecting CBV. Histological samples demonstrated reasonable correspondence between MRI characteristics and HIF-1a-staining. The presented method of rOEF imaging is a promising tool for the metabolic characterization of human glioma. For the interpretation of rOEF maps, confounding factors must be considered, with a special focus on CBV measurements in the presence of contrast agent leakage. Further validation involving a bigger cohort and extended immuno-histochemical correlation is required.

 

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Deficiency of caveolin-1 in APCmin/+ mice promotes colorectal tumorigenesis

Carcinogenesis, 2013, vol.00 no.00 p. 1 of 10, 2013, doi:10.1093/carcin/bgt142 publiziert am 02.05.2013
Caveolin-1 (Cav1), a scaffold protein of membrane caveolae and coactivator of peroxisome proliferator-activated receptor gamma (PPARg), inhibits oncogenic signaling through Ras and wingless. However, the in vivo role of Cav1 in colorectal cancer (CRC) remained unknown. To test wether loss of Cav1 acceleratest tumorigenesis, we generated a novel mouse model of CRC by crossing C57BL/6 APCmin/+ with B6129 Cav1 knockout (Cav1-/-) mice. APCmin/+ Cav1-/- mice developed large, microinvasive and vascularized interaepithelial adenocarcinomas in the distal colon and rectum with higher incidence than APCmin/+Cav1+/- and APCmin/+Cav1+/+ littermates. Intratumoral gene signatures related to Ras and wingless signaling were elevated, nuclear localization of PPARg protein and expression of PPARg-agonist rosiglitazone prevented tumor formation in mice irrespectively of the Cav1 status and upregulated expression of the Ras-inhibitory protein docking protein-1. Thus, codeficiency of Cav1 and adenomatous polyposis coli facilitated formation of CRC, and activation of PPARg may offer novel strategies for treatment of CRC.

Caveolin-1 Protects B6129 Mice against Helicobacter pylori Gastritis

PLOS Pathogens, 2013, Vol. 9, Issue 4, publiziert am 11.04.2013
Caveolin-1 (Cav1) is a scaffold protein and pathogen receptor in the mucosa of the gastrointestinal tract. Chronic infection of gastric epithelial cells by Helicobacter pylori (H. pylori) is a major risk factor for human gastric cancer (GC) where Cav1 is frequently down-regulated. However, the function of Cav1 in H. pylori infection and pathogenesis of GC remained unknown. We show here that Cav1-deficient mice, infected for 11 months with the CagA-delivery deficient H. pylori strain SS1, developed more severe gastritis and tissue damage, including loss of parietal cells and foveolar hyperplasia, and displayed lower colonisation of the gastric mucosa than wild-type B6129 littermates. Cav1-null mice showed enhanced infiltration of macrophages and B-cells and secretion of chemokines (RANTES) but had reduced levels of CD25+ regulatory T-cells. Cav1-deficient human GC cells (AGS), infected with the CagA-delivery proficient H. pylori strain G27, were more sensitive to CagA-related cytoskeletal stress morphologies (“humming bird”) compared to AGS cells stably transfected with Cav1 (AGS/Cav1). Infection of AGS/Cav1 cells triggered the recruitment of p120 RhoGTPase-activating protein/deleted in liver cancer-1 (p120RhoGAP/DLC1) to Cav1 and counteracted CagA-induced cytoskeletal rearrangements. In human GC cell lines (MKN45, N87) and mouse stomach tissue, H. pylori down-regulated endogenous expression of Cav1 independently of CagA. Mechanistically, H. pylori activated sterol-responsive element-binding protein-1 (SREBP1) to repress transcription of the human Cav1 gene from sterol-responsive elements (SREs) in the proximal Cav1 promoter. These data suggested a protective role of Cav1 against H. pylori-induced inflammation and tissue damage. We propose that H. pylori exploits down-regulation of Cav1 to subvert the host's immune response and to promote signalling of its virulence factors in host cells.

Aldehyde dehydrogenase 1A1- a new mediator of resistance to temozolomide in glioblastoma

Neuro-Oncology, 2012, doi: 10.1093/neuonc/nos270, 13 publiziert am 09.09.2013
Implementation of chemotherapy with the drug temozolomide increased the overall survival of patients with glioblastoma multiforme (GBM; WHO grade IV), in particular when the O6-methylguanine DNA methyltransferase (MGMT) promoter is epigenetically silenced. Nevertheless, the prognosis remains poor, and relapse in GBM occurs regularly. This clinical behavior seems to be due to the existence of a therapy-resistant subpopulation of cells that induce tumor regrowth. The objective of this work was to analyze the role of aldehyde dehydrogenase (ALDH) 1A1 in mediating temozolomide resistance and its value as a predictor of clinical outcome in GBM patients. Nine GBM cell lines were treated with temozolomide alone or in combination with 4-diethylaminobenzaldehyde (DEAB), an inhibitor of ALDH1A1, or with ALDH1A1 short hairpin (sh)RNA. ALDH1A1 expression and MGMT status of 70 primary GBM patients were correlated with median survival. ALDH1A1 overexpression predicted emozolomide resistance in vitro. Sensitivity of ALDH1A1 positive/MGMT-positive cells to temozolomide could be restored by inhibition of ALDH1A1 by DEAB or by knockdown with shRNA, as indicated by increased cytotoxicity, reduced clonogenicity, and accumulation in the G2/M cell-cycle phase. The prognosis of patients with a high level of ALDH1A1 expression was poor compared with that of patients with low levels (P < .0001). ALDH1A1 is a new mediator for resistance of GBM to temozolomide and a reliable predictor of clinical outcome and may serve as a potential target to improve treatment of human GBM.

T2* Mapping with Background Gradient Correction Using Different Excitation Pulse Shapes

American Journal of Neuroradiology (AJNR 2013 34: E65-E68), 2012, DOI 10.3174/ajnr.A3021 publiziert am 26.07.2012
Background gradients induced by magnetic susceptibility variations near air-filled cavities in the brain cause signal-intensity loss in gradient-echo images and shorten T2* considerably. With a correction method in which the exponential decay is restored with section-profile-dependent correction factors, parts of the signal intensity can be recovered. While uncorrected T2* values drop by 20% at a gradient strength of 75 µT/m, with correction and exponential excitation pulses, this boundary is pushed to 220µT/m.

 

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Non-targeted effects of photon and particle irradiation and the interaction with the immune system.

Front Oncol., 2012, 2(80), publiziert am 24.07.2012
 

Ionizing irradiation is an important clinical approach to treat solid tumors. Modern radiation technologies aim to selectively kill tumor cells and protect the surrounding normal tissue. The standard paradigm for radiation effects in cellular systems involves damage of the DNA including DNA double-strand breaks, which are considered as most effective in destroying tumor cells. Due to their enhanced physical and radiobiological properties, high-linear energy transfer radiation qualities are of special interest in tumor therapy. Future radiation therapy strategies aim to utilize carbon ions to effectively treat highly aggressive tumors. More recently, evidence is emerging for non-DNA targeted effects of radiation, including mutations, chromosomal aberrations, and changes in gene expression, which can occur in cells that were not directly exposed to radiation. Radiation oncologists are only gradually beginning to appreciate the clinical relevance of radiation-induced bystander effects, genomic instability, and abscopal effects. Since these effects are sensed by the immune system, a combination of immunotherapy and irradiation presents a new therapeutic opportunity in the future.



Immunotherapeutic Targeting of Membrane Hsp70-Expressing Tumors

PLoS One., 2012, 7(7), e41341 publiziert am 19.07.2012
 

BACKGROUND:

We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner.

 

METHODOLOGY/PRINCIPAL FINDINGS:

Optical imaging of uptake kinetics revealed co-localization of grB with recycling endosomes (Rab9/11) as early as 5 min after internalization, with late endosomes (Rab7) after 30 min, and the lysosomal compartment (LAMP1/2) after 60 to 120 min. Active caspase-3-mediated apoptosis was induced in mouse CT26 monolayer cells and 3D tumor spheroids, but not in normal mouse endothelial cells. Granzyme B selectively reduced the proportion of membrane Hsp70-positive cells in CT26 tumor spheroids. Consecutive i.v. injections of recombinant human grB into mice bearing membrane Hsp70-positive CT26 tumors resulted in significant tumor suppression, and a detailed inspection of normal mouse organs revealed that the administration of anti-tumoral concentrations of grB elicited no clinicopathological changes.

 

CONCLUSIONS/SIGNIFICANCE:

These findings support the future clinical evaluation of human grB as a potential adjuvant therapeutic agent, especially for treating immunosuppressed patients that bear membrane Hsp70-positive tumors.



Radiation, inflammation, and immune responses in cancer.

Front Oncol., 2012, 2:58, publiziert am 04.06.2012
 
Chronic inflammation has emerged as one of the hallmarks of cancer. Inflammation also plays a pivotal role in modulating radiation responsiveness of tumors. As discussed in this review, ionizing radiation (IR) leads to activation of several transcription factors modulating the expression of numerous mediators in tumor cells and cells of the microenvironment promoting cancer development. Novel therapeutic approaches thus aim to interfere with the activity or expression of these factors, either in single-agent or combinatorial treatment or as supplements of the existing therapeutic concepts. Among them, NF-κB, STAT-3, and HIF-1 play a crucial role in radiation-induced inflammatory responses embedded in a complex inflammatory network. A great variety of classical or novel drugs including nutraceuticals such as plant phytochemicals have the capacity to interfere with the inflammatory network in cancer and are considered as putative radiosensitizers. Thus, targeting the inflammatory signaling pathways induced by IR offers the opportunity to improve the clinical outcome of radiation therapy by enhancing radiosensitivity and decreasing putative metabolic effects. Since inflammation and sex steroids also impact tumorigenesis, a therapeutic approach targeting glucocorticoid receptors and radiation-induced production of tumorigenic factors might be effective in sensitizing certain tumors to IR.

Bile Acids Down Regulate Caveolin-1 in Esophageal Epithelial Celis through Sterol Responsive Element-Binding Protein

Mol Endocrinol., 2012, 26(5), 819-32 publiziert am 01.05.2012
 

Bile acids are synthesized from cholesterol and are major risk factors for Barrett adenocarcinoma (BAC) of the esophagus. Caveolin-1 (Cav1), a scaffold protein of membrane caveolae, is transcriptionally regulated by cholesterol via sterol-responsive element-binding protein-1 (SREBP1). Cav1 protects squamous epithelia by controlling cell growth and stabilizing cell junctions and matrix adhesion. Cav1 is frequently down-regulated in human cancers; however, the molecular mechanisms that lead to this event are unknown. We show that the basal layer of the nonneoplastic human esophageal squamous epithelium expressed Cav1 mainly at intercellular junctions. In contrast, Cav1 was lost in 95% of tissue specimens from BAC patients (n = 100). A strong cytoplasmic expression of Cav1 correlated with poor survival in a small subgroup (n = 5) of BAC patients, and stable expression of an oncogenic Cav1 variant (Cav1-P132L) in the human BAC cell line OE19 promoted proliferation. Cav1 was also detectable in immortalized human squamous epithelial, Barrett esophagus (CPC), and squamous cell carcinoma cells (OE21), but was low in BAC cell lines (OE19, OE33). Mechanistically, bile acids down-regulated Cav1 expression by inhibition of the proteolytic cleavage of 125-kDa pre-SREBP1 from the endoplasmic reticulum/Golgi apparatus and nuclear translocation of active 68-kDa SREBP1. This block in SREBP1's posttranslational processing impaired transcriptional activation of SREBP1 response elements in the proximal human Cav1 promoter. Cav1 was also down-regulated in esophagi from C57BL/6 mice on a diet enriched with 1% (wt/wt) chenodeoxycholic acid. Mice deficient for Cav1 or the nuclear bile acid receptor farnesoid X receptor showed hyperplasia and hyperkeratosis of the basal cell layer of esophageal epithelia, respectively. These data indicate that bile acid-mediated down-regulation of Cav1 marks early changes in the squamous epithelium, which may contribute to onset of Barrett esophagus metaplasia and progression to BAC.



Mesenchymal stem cells and glioma cells form a structural as well as a functional syncytium in vitro

Exp Neurol., 2012, 234(1), 208-19 publiziert am 01.03.2012
 

The interaction of human mesenchymal stem cells (hMSCs) and tumor cells has been investigated in various contexts. HMSCs are considered as cellular treatment vectors based on their capacity to migrate towards a malignant lesion. However, concerns about unpredictable behavior of transplanted hMSCs are accumulating. In malignant gliomas, the recruitment mechanism is driven by glioma-secreted factors which lead to accumulation of both, tissue specific stem cells as well as bone marrow derived hMSCs within the tumor. The aim of the present work was to study specific cellular interactions between hMSCs and glioma cells in vitro. We show, that glioma cells as well as hMSCs differentially express connexins, and that they interact via gap-junctional coupling. Besides this so-called functional syncytium formation, we also provide evidence of cell fusion events (structural syncytium). These complex cellular interactions led to an enhanced migration and altered proliferation of both, tumor and mesenchymal stem cell types in vitro. The presented work shows that glioma cells display signs of functional as well as structural syncytium formation with hMSCs in vitro. The described cellular phenomena provide new insight into the complexity of interaction patterns between tumor cells and host cells. Based on these findings, further studies are warranted to define the impact of a functional or structural syncytium formation on malignant tumors and cell based therapies in vivo.

 

Copyright © 2011 Elsevier Inc. All rights reserved.



Cryptate mediated nucleophilic 18F-fluorination without azeotropic drying

Nuklearmedizin., 2012, 50(1), 1-8 publiziert am 16.02.2012
 
The radiosynthesis of the vast majority of 18F-labeled tracers rely on azeotropic drying of [18F]fluoride and subsequent cryptate mediated introduction of [18F]fluoride by nucleophilic substitution. The aim of this study was to develop a method for simplification of this process, based on preparation of reactive [K+⊂2.2.2]18F- by solvent drying of [18F]fluoride adsorbed onto an anion exchange resin. Methods: Aqueous [18F]fluoride (0.5-1 ml) obtained from the 18O(p,n)18F nuclear reaction was trapped on a strong anion-exchange (SAX) cartridge. After washing the cartridge with dry CH3CN, [18F]fluoride was eluted with an anhydrous solution of [K+⊂2.2.2]OH- in CH3CN and directly used for nucleophilic fluorination reactions. Results: [18F]Fluoride from target water was quantitatively retained by the SAX cartridge, and water-free [18F]fluoride recovered in an overall yield of 92±5% (n = 10). [18F]Fluoride obtained by this procedure led to radiochemical yields of 70-90% for [18F]FDG, [18F]FET, [18F]FLT, [18F]FAZA and [18F]Fallypride. Conclusion: SAX-resin adsorbed [18F]fluoride can be dried with non-aqueous solvents and eluted with [K+⊂2.2.2]OH- in CH3CN. The reactivity of [K+⊂2.2.2]F- generated by the new method is comparable to that of [18F]fluoride obtained by azeotropic drying. The described procedure facilitates the automated production of 18F-radiopharmaceuticals in general, and may also simplify the use of microfluidic devices for 18F-radiotracer production.

Basal HIF-1α expression levels are not predictive for radiosensitivity of human cancer cell lines

Strahlenther Onkol., 2012, publiziert am 12.02.2012
 
BACKGROUND AND PURPOSE:

High levels of hypoxia inducible factor (HIF)-1α in tumors are reported to be associated with tumor progression and resistance to therapy. To examine the impact of HIF-1α on radioresistance under normoxia, the sensitivity towards irradiation was measured in human tumor cell lines that differ significantly in their basal HIF-1α levels.

 

MATERIAL AND METHODS:

HIF-1α levels were quantified in lysates of H1339, EPLC-272H, A549, SAS, XF354, FaDu, BHY, and CX- tumor cell lines by ELISA. Protein levels of HIF-1α, HIF-2α, carbonic anhydrase IX (CA IX), and GAPDH were assessed by Western blot analysis. Knock-down experiments were performed using HIF-1α siRNA. Clonogenic survival after irradiation was determined by the colony forming assay.

 

RESULTS:

According to their basal HIF-1α status, the tumor cell lines were divided into low (SAS, XF354, FaDu, A549, CX-), intermediate (EPLC-272H, BHY), and high (H1339) HIF-1α expressors. The functionality of the high basal HIF-1α expression in H1339 cells was proven by reduced CA IX expression after knocking-down HIF-1α. Linear regression analysis revealed no correlation between basal HIF-1α levels and the survival fraction at either 2 or 4 Gy in all tumor cell lines investigated.

 

CONCLUSION:

Our data suggest that basal HIF-1α levels in human tumor cell lines do not predict their radiosensitivity under normoxia.



Krüppel-like factor 8 (KLF8) is expressed in gliomas of different WHO grades and is essential for tumor cell proliferation

PLoS One., 2012, 7(1), e30429 publiziert am 19.01.2012
 
Krüppel-like factor 8 (KLF8) has only recently been identified to be involved in tumor cell proliferation and invasion of several different tumor entities like renal cell carcinoma, hepatocellular carcinoma and breast cancer. In the present study, we show for the first time the expression of KLF8 in gliomas of different WHO grades and its functional impact on glioma cell proliferation. In order to get information about KLF8-mRNA regulation qPCR was performed and did not reveal any significant difference in samples (n = 10 each) of non-neoplastic brain (NNB), low-grade gliomas (LGG, WHO°II) and glioblastomas (GBM, WHO°IV). Immunohistochemistry of tissue samples (n = 7 LGG, 11 AA and 12 GBM) did not show any significant difference in the fraction of KLF8-immunopositive cells of all analyzed cells in LGG (87%), AA (80%) or GBM (89%). Tissue samples from cerebral breast cancer metastasis, meningiomas but also non-neoplastic brain demonstrated comparable relative cell counts as well. Moreover, there was no correlation between KLF8 expression and the expression pattern of the assumed proliferation marker Ki67, which showed high variability between different tumor grade (9% (LGG), 6% (AA) and 15% (GBM) of Ki67-immunopositive cells). Densitometric analysis of Western blotting revealed that the relative amount of KLF8-protein did also not differ between the highly aggressive and proliferative GBM (1.05) compared to LGG (0.93; p<0.05, studens t-test). As demonstrated for some other non-glial cancer entities, KLF8-knockdown by shRNA in U87-MG cells confirmed its functional relevance, leading to an almost complete loss of tumor cell proliferation. Selective blocking of KLF8 might represent a novel anti-proliferative treatment strategy for malignant gliomas. Yet, its simultaneous expression in non-proliferating tissues could hamper this approach.

Chronic inflammation in cancer development

frontiers in immunology, 2012, doi: 10.3389, fimmu.201100098 publiziert am 12.01.2012
 
Chronic inflammatory mediators exert pleiotropic effects in the development of cancer. On the one hand, inflammation favors carcinogenesis, malignant transformation, tumor growth, invasion, and metastatic spread; on the other hand inflammation can stimulate immune effector mechanisms that might limit tumor growth. The linkbetween cancer and inflammation depends on intrinsic and extrinsic pathways. Both pathways result in the activation of transcription factors such as NF-KB, STAT-3, and HIF-1 and in accumulation of tumorigenic factors in tumor and microenvironment. STAT-3 and NF-KB interact at multiple levels and thereby boost tumor-associated inflammation which can suppress anti-tumor immune responses. These factors also promote tumor growth, progression, and metastatic spread. IL-1, IL-6, TNF, and PGHS-2 are key mediators of an inflammatory milieu by modulating the expression of tumor-promoting factors. In this review we concentrate on the crucial role of pro-inflammatory mediators in inflammation-driven carcinogenesis and outline molecular mechanisms of IL-1 signaling in tumors. In addition, we elucidate the dual roles of stress proteins äs danger Signals in the development of anti-cancer immunity and anti-apoptotic functions.

TFAP2E–DKK4 and Chemoresistance in Colorectal Cancer

N Engl J Med., 2012, 366(1), 44-53 publiziert am 05.01.2012
 

Background:

Chemotherapy for advanced colorectal cancer leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential downstream target of TFAP2E and has been implicated in chemotherapy resistance. We aimed to further evaluate the role of TFAP2E and DKK4 as predictors of the response of colorectal cancer to chemotherapy.

 

Methods:

We analyzed the expression, methylation, and function of TFAP2E in colorectal-cancer cell lines in vitro and in patients with colorectal cancer. We examined an initial cohort of 74 patients, followed by four cohorts of patients (total, 220) undergoing chemotherapy or chemoradiation.

 

Results:

TFAP2E was hypermethylated in 38 of 74 patients (51%) in the initial cohort. Hypermethylation was associated with decreased expression of TFAP2E in primary and metastatic colorectal-cancer specimens and cell lines. Colorectal-cancer cell lines overexpressing DKK4 showed increased chemoresistance to fluorouracil but not irinotecan or oxaliplatin. In the four other patient cohorts, TFAP2E hypermethylation was significantly associated with nonresponse to chemotherapy (P<0.001). Conversely, the probability of response among patients with hypomethylation was approximately six times that in the entire population (overall estimated risk ratio, 5.74; 95% confidence interval, 3.36 to 9.79). Epigenetic alterations of TFAP2E were independent of mutations in key regulatory cancer genes, microsatellite instability, and other genes that affect fluorouracil metabolism.

 

Conclusions:

TFAP2E hypermethylation is associated with clinical nonresponsiveness to chemotherapy in colorectal cancer. Functional assays confirm that TFAP2E-dependent resistance is mediated through DKK4. In patients who have colorectal cancer with TFAP2E hypermethylation, targeting of DKK4 may be an option to overcome TFAP2E-mediated drug resistance. (Funded by Deutsche Forschungsgemeinschaft and others.).



MALDI imaging identifies prognostic seven-protein signature of novel tissue markers in intestinal-type gastric cancer

Am J Pathol., 2011, 179(6), 2720-9 publiziert am 18.10.2011
 

Proteomics-based approaches allow us to investigate the biology of cancer beyond genomic initiatives. We used histology-based matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry to identify proteins that predict disease outcome in gastric cancer after surgical resection. A total of 181 intestinal-type primary resected gastric cancer tissues from two independent patient cohorts were analyzed. Protein profiles of the discovery cohort (n = 63) were directly obtained from tumor tissue sections by MALDI imaging. A seven-protein signature was associated with an unfavorable overall survival independent of major clinical covariates. The prognostic significance of three individual proteins identified (CRIP1, HNP-1, and S100-A6) was validated immunohistochemically on tissue microarrays of an independent validation cohort (n = 118). Whereas HNP-1 and S100-A6 were found to further subdivide early-stage (Union Internationale Contre le Cancer [UICC]-I) and late-stage (UICC II and III) cancer patients into different prognostic groups, CRIP1, a protein previously unknown in gastric cancer, was confirmed as a novel and independent prognostic factor for all patients in the validation cohort. The protein pattern described here serves as a new independent indicator of patient survival complementing the previously known clinical parameters in terms of prognostic relevance. These results show that this tissue-based proteomic approach may provide clinically relevant information that might be beneficial in improving risk stratification for gastric cancer patients.

Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.



A novel expression and purification system for the production of enzymatic and biologically active human granzyme B

J Immunol Methods., 2011, 371(1-2), 8-17 publiziert am 31.08.2011
 
The serine protease granzyme B (grB) has previously been shown to induce perforin-independent apoptosis in membrane Hsp70 positive tumor cells in a number of in vitro experimental systems. Ongoing studies that are investigating the in vivo relevance of these findings by assessing the therapeutic potential of grB in a human xenograft tumor mouse model required the development of an expression system for the production of high yields of enzymatic and biologically active human grB. In order to maintain potentially important posttranslational modifications that occur in mammalian cells, human embryonal kidney cells (HEK293) were stably transfected with human grB. The HEK293 host cells were protected from apoptotic cell death by fusing an inactivation site coupled to a (His)(6) tag to the gene sequence of GrB. Inactive grB which was actively released from HEK293 cells by insertion of a Igκ leader sequence was purified on a nickel column utilizing the (His)(6) tag. After enterokinase digestion and heparin affinity chromatography, high yields of enzymatic and biologically active human grB were obtained. The perforin-independent interaction of grB with membrane Hsp70 positive tumor cells appeared to be associated with mammalian glycosylation and mediated by the oligosaccharide moiety of neuraminic acid (NANA).

 

Copyright © 2011. Published by Elsevier B.V.



(18)F-FDG PET-guided salvage neoadjuvant radiochemotherapy of adenocarcinoma of the esophagogastric junction: the MUNICON II trial

J Nucl Med., 2011, 52(8), 1189-96 publiziert am 01.08.2011
 
Previous studies demonstrated that chemotherapy-induced changes in tumor glucose metabolism measured with (18)F-FDG PET identify patients who benefit from preoperative chemotherapy and those who do not. The prognosis for chemotherapy metabolic nonresponders is poorer than for metabolic responders. Therefore, we initiated this prospective trial to improve the clinical outcome of metabolic nonresponders using a salvage neoadjuvant radiochemotherapy.

 

METHODS:

Fifty-six patients with locally advanced adenocarcinomas of the esophagogastric junction were included. Tumor glucose uptake was assessed by (18)F-FDG PET before chemotherapy and 14 d after initiation of chemotherapy. PET nonresponders received salvage neoadjuvant radiochemotherapy, whereas metabolic responders received neoadjuvant chemotherapy for 3 mo before surgery.

 

RESULTS:

Thirty-three patients were metabolic responders, and 23 were nonresponders. Resection was performed on 54 patients. R0 resection rate was 82% (95% confidence interval [CI], 66%-91%) in metabolic responders and 70% (95% CI, 49%-84%) in metabolic nonresponders (P = 0.51). Major histologic remissions were observed in 12 metabolic responders (36%; 95% CI, 22%-53%) and 6 nonresponders (26%; 95% CI, 13%-46%). One-year progression-free rate was 74% ± 8% in PET responders and 57% ± 10% in metabolic nonresponders (log rank test, P = 0.035). One-year overall survival was comparable between the groups (∼80%), and 2-y overall survival was estimated to be 71% ± 8% in metabolic responders and 42% ± 11% in PET nonresponders (hazard ratio, 1.9; 95% CI, 0.87-4.24; P = 0.10).

 

CONCLUSION:

This prospective study showed the feasibility of a PET-guided treatment algorithm. However, by comparing the groups of nonresponding patients in the current trial and the previous published MUNICON (Metabolic response evalUatioN for Individualisation of neoadjuvant Chemotherapy in Esophageal and esophagogastric adeNocarcinoma) I trial, increased histopathologic response was observed after salvage radiochemotherapy, but the primary endpoint of the study to increase the R0 resection rate was not met. The prognosis of the subgroup of PET nonresponders remains poor, indicating their different tumor biology.



Bevacizumab can induce reactivity to VEGF-C and -D in human brain and tumour derived endothelial cells

J Neurooncol., 2011, 104(1), 103-12 publiziert am 01.08.2011
 
Though clinical trials demonstrated effectiveness of the anti-VEGF antibody bevacizumab (Avastin) in adjuvant therapies for some solid tumours, there are rather few experimental data about cellular effects of bevacizumab on tumour cells and tumour associated endothelial cells. Recent reports demonstrate resistance mechanisms and secondary re-angiogenesis after a transient normalization of tumour vessels. Therefore we investigated the influence of bevacizumab on human glioma cells and human brain derived as well as tumour derived endothelial cells focussing on the role of VEGF-C and -D as potential alternative pro-angiogenic factors. Bevacizumab treatment showed no influence on proliferation after short term exposure (1-5 days) but slowed down endothelial cell proliferation by 25-30% after 14 days treatment. There was no significant induction of apoptosis after short or long term exposure. Tube formation capabilities were significantly impaired by bevacizumab with a continuing effect after 14 days of treatment even after omitting the antibody. VEGF-C and -D had no effect on endothelial cells in untreated or short term treatment groups. However, cells developed responsiveness to these factors in terms of increased proliferation and tube formation after 14 days bevacizumab treatment. Furthermore, bevacizumab induced expression of VEGF-C and -D in glioma cells. Treatment with bevacizumab may induce alterations in human brain and tumour endothelial cells leading to escape mechanisms from anti-VEGF therapy. VEGF-C and -D thus might act as alternative pro-angiogenic factors during anti-VEGF therapy.

Detection of irradiation-induced, membrane heat shock protein 70 (Hsp70) in mouse tumors using Hsp70 Fab fragment

Radiother Oncol., 2011, 99(3), 313-6 publiziert am 23.06.2011
 
BACKGROUND AND PURPOSE:

The major stress-inducible heat shock protein 70 (Hsp70) is frequently overexpressed in highly aggressive tumors, and elevated intracellular Hsp70 levels mediate protection against apoptosis. Following therapeutic intervention, such as ionizing irradiation, translocation of cytosolic Hsp70 to the plasma membrane is selectively increased in tumor cells and therefore, membrane Hsp70 might serve as a therapy-inducible, tumor-specific target structure.

 

MATERIALS AND METHODS:

Based on the IgG1 mouse monoclonal antibody (mAb) cmHsp70.1, we produced the Hsp70-specific recombinant Fab fragment (Hsp70 Fab), as an imaging tool for the detection of membrane Hsp70 positive tumor cells in vitro and in vivo.

 

RESULTS:

The binding characteristics of Hsp70 Fab towards mouse colon (CT26) and pancreatic (1048) carcinoma cells at 4 °C were comparable to that of cmHsp70.1 mAb, as determined by flow cytometry. Following a temperature shift to 37 °C, Hsp70 Fab rapidly translocates into subcellular vesicles of mouse tumor cells. Furthermore, in tumor-bearing mice Cy5.5-conjugated Hsp70 Fab, but not unrelated IN-1 control Fab fragment (IN-1 ctrl Fab), gradually accumulates in CT26 tumors between 12 and 55 h after i.v. injection.

 

CONCLUSIONS:

In summary, the Hsp70 Fab provides an innovative, low immunogenic tool for imaging of membrane Hsp70 positive tumors, in vivo.

 

Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.



Targeting membrane heat-shock protein 70 (Hsp70) on tumors by cmHsp70.1 antibody

Proc Natl Acad Sci U S A, 2011, 108(2), 733-8 publiziert am 11.01.2011
 
Immunization of mice with a 14-mer peptide TKDNNLLGRFELSG, termed “TKD,” comprising amino acids 450–461 (aa450–461) in the C terminus of inducible Hsp70, resulted in the generation of an IgG1 mouse mAb cmHsp70.1. The epitope recognized by cmHsp70.1mAb, which has been confirmed to be located in the TKD sequence by SPOT analysis, is frequently detectable on the cell surface of human and mouse tumors, but not on isogenic cells and normal tissues, and membrane Hsp70 might thus serve as a tumor-specific target structure. As shown for human tumors, Hsp70 is associated with cholesterol-rich microdomains in the plasma membrane of mouse tumors.

Herein, we show that the cmHsp70.1 mAb can selectively induce antibody-dependent cellular cytotoxicity (ADCC) of membrane Hsp70+ mouse tumor cells by unstimulated mouse spleen cells. Tumor killing could be further enhanced by activating the effector cells with TKD and IL-2. Three consecutive injections of the cmHsp70.1 mAb into mice bearing CT26 tumors significantly inhibited tumor growth and enhanced the overall survival. These effects were associated with infiltrations of NK cells, macrophages, and granulocytes. The Hsp70 specificity of the ADCC response was confirmed by preventing the antitumor response in tumor-bearing mice by coinjecting the cognate TKD peptide with the cmHsp70.1 mAb, and by blocking the binding of cmHsp70.1 mAb to CT26 tumor cells using either TKD peptide or the C-terminal substrate-binding domain of Hsp70.



Distinguishing integral and receptor-bound heat shock protein 70 (Hsp70) on the cell surface by Hsp70 specific antibodies

Cell Stress Chaperones., 2010, publiziert am 17.12.2010
 
Cell Stress & Chaperones journal has become a major outlet for papers and review articles about anti-heat shock protein (HSP) antibodies. In the last decade it became evident that apart from their intracellular localization, members of the heat shock protein 90 (HSP90, HSPC) and HSP70 (HSPA) family are also found on the cell surface. In this review we will focus on Hsp70 (HSPA1A) the major stress-inducible member of the human HSP70 family. Depending on the cell type, the membrane association of Hsp70 comes in two forms. In tumor cells Hsp70 appears to be integrated within the plasma membrane, whereas in non-malignantly transformed (herein termed normal) cells Hsp70 is associated with cell surface receptors. This observation raises the question whether or not these two surface forms of Hsp70 in tumor and normal cells can be distinguished using Hsp70 specific antibodies. Presently a number of Hsp70 specific antibodies are commercially available. These antibodies were generated by immunizing mice either with recombinant or HeLa-derived human Hsp70 protein, parts of the Hsp70 protein or with synthetic peptides. This review aims to characterize the binding of different anti-human Hsp70 antibodies and their capacity to distinguish between integrated and receptor-bound Hsp70 in tumor and normal cells.

Irradiation-Induced Up-Regulation of HLA-E on Macrovascular Endothelial Cells Confers Protection against Killing by Activated Natural Killer Cells

PLoS One., 2010, 5, (12):e15339 publiziert am 16.12.2010
 
Background

Apart from the platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31), endoglin (CD105) and a positive factor VIII-related antigen staining, human primary and immortalized macro- and microvascular endothelial cells (ECs) differ in their cell surface expression of activating and inhibitory ligands for natural killer (NK) cells. Here we comparatively study the effects of irradiation on the phenotype of ECs and their interaction with resting and activated NKcells.

 

Methodology/Principal Findings

Primary macrovascular human umbilical vein endothelial cells (HUVECs) only express UL16 binding protein 2 (ULBP2) and the major histocompatibility complex (MHC) class I chain-related protein MIC-A (MIC-A) as activating signals for NK cells, whereas the corresponding immortalized EA.hy926 EC cell line additionally present ULBP3, membrane heat shock protein 70 (Hsp70), intercellular adhesion molecule ICAM-1 (CD54) and HLA-E. Apart from MIC-B, the immortalized human microvascular endothelial cell line HMEC, resembles the phenotype of EA.hy926. Surprisingly, primary HUVECs are more sensitive to Hsp70 peptide (TKD) plus IL-2 (TKD/IL-2)-activated NK cells than their immortalized ECcounterparts. This finding is most likely due to the absence of the inhibitory ligand HLA-E, since the activating ligands are shared among the ECs. The co-culture of HUVECs with activated NK cells induces ICAM-1 (CD54) and HLA-E expression on the former which drops to the initial low levels (below 5%) when NK cells are removed. Sublethal irradiation of HUVECs induces similar but less pronounced effects on HUVECs. Along with these findings, irradiation also induces HLA-E expression on macrovascular ECs and this correlates with an increased resistance to killing by activated NK cells. Irradiation had no effect on HLA-E expression on microvascular ECs and the sensitivity of these cells to NK cells remained unaffected.

 

Conclusion/Significance

These data emphasize that an irradiation-induced, transient up-regulation of HLA-E on macrovascular ECs might confer protection against NK cell-mediated vascular injury.



TNF-a respecifies human mesenchymal stem cells to a neural fate and promotes migration toward experimental glioma

Cell Death and Differentiation advance online publication, 2010, publiziert am 03.12.2010
 
Bone marrow-derived human mesenchymal stem cells (hMSCs) have become valuable candidates for cell-based therapeutical applications including neuroregenerative and anti-tumor strategies. Yet, the molecular mechanisms that control hMSC trans-differentiation to neural cells and hMSC tropism toward glioma remain unclear. Here, we demonstrate that hMSCs incubated with 50 ng/ml tumor necrosis factor alpha (TNF-a) acquired astroglial cell morphology without affecting proliferation, which was increased at 5 ng/ml. TNF-a (50 ng/ml) upregulated expression of numerous genes important for neural cell growth and function including LIF (leukemia inhibitory factor), BMP2 (bone morphogenetic protein 2), SOX2 (SRY box 2), and GFAP (glial fibrillary acidic protein), whereas NES (human nestin) transcription ceased suggesting a premature neural phenotype in TNF-a-differentiated hMSCs. Studies on intracellular mitogen-activated protein kinase (MAPK) signaling revealed that inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) activity abolished the TNF-a-mediated regulation of neural genes in hMSCs. In addition, TNF-a significantly enhanced expression of the chemokine receptor CXCR4 (CXC motive chemokine receptor 4), which facilitated the chemotactic invasiveness of hMSCs toward stromal cell-derived factor 1 (SDF-1) alpha. TNF-a-pretreated hMSCs not only exhibited an increased ability to infiltrate glioma cell spheroids dependent on matrix metalloproteinase activity in vitro, but they also showed a potentiated tropism toward intracranial malignant gliomas in an in vivo mouse model. Taken together, our results provide evidence that culture-expansion of hMSCs in the presence of TNF-a triggers neural gene expression and functional capacities, which could improve the use of hMSCs in the treatment of neurological disorders including malignant gliomas.

 

Cell Death and Differentiation advance online publication, 3 December 2010; doi:10.1038/cdd.2010.154



Aldehyde dehydrogenase 1 positive glioblastoma cells show brain tumor stem cell capacity

Neuro Oncol., 2010, publiziert am 13.07.2010
 
Glioblastoma (GBM) is the most aggressive primary brain tumor and is resistant to all therapeutic regimens. Relapse occurs regularly and might be caused by a poorly characterized tumor stem cell (TSC) subpopulation escaping therapy. We suggest aldehyde dehydrogenase 1 (ALDH1) as a novel stem cell marker in human GBM. Using the neurosphere formation assay as a functional method to identify brain TSCs, we show that high protein levels of ALDH1 facilitate neurosphere formation in established GBM cell lines. Even single ALDH1 positive cells give rise to colonies and neurospheres. Consequently, the inhibition of ALDH1 in vitro decreases both the number of neurospheres and their size. Cell lines without expression of ALDH1 do not form tumor spheroids under the same culturing conditions. High levels of ALDH1 seem to keep tumor cells in an undifferentiated, stem cell-like state indicated by the low expression of beta-III-tubulin. In contrast, ALDH1 inhibition induces premature cellular differentiation and reduces clonogenic capacity. Primary cell cultures obtained from fresh tumor samples approve the established GBM cell line results.

Classifiation of HER2/neu Status in Gastric Cancer Using a Breast-Cancer Derived Proteome Classfier

Journal of Proteome Research, 2010, publiziert am 08.06.2010
 
HER2-testing in breast and gastric cancers is mandatory for the treatment with trastuzumab. We hypothesized that imaging mass spectrometry (IMS) of breast cancers may be useful for generating a classifier that may determine HER2-status in other cancer entities irrespective of primary tumor site. A total of 107 breast (n 48) and gastric (n 59) cryo tissue samples was analyzed by IMS (HER2 was present in 29 cases). The obtained proteomic profiles were used to create HER2 prediction models using different classification algorithms. A breast cancer proteome derived classifier, with HER2 present in 15 cases, correctly predicted HER2-status in gastric cancers with a sensitivity of 65% and a specificity of 92%. To create a universal classifier for HER2-status, breast and nonbreast cancer samples were combined, which increased sensitivity to 78%, and specificity was 88%. Our proof of principle study provides evidence that HER2-status can be identified on a proteomic level across different cancer types suggesting that HER2 overexpression may constitute a unique molecular event independent of the tumor site. Furthermore, these results indicate that IMS may be useful for the determination of potential drugable targets, as it offers a quicker, cheaper, and more objective analysis than the standard HER2-testing procedures immunohistochemistry and fluorescence in situ hybridization.

In vivo imaging of CT26 mouse tumors by using cmHsp70.1 monoclonal antibody

Journal of Cellular and Molecular Medicine - Decision on Manuscript ID JCMM, 2010, publiziert am 15.03.2010
 
The major stress-inducible heat shock protein 70 (Hsp70) is frequently present on the cell surface of human tumors, but not on normal cells. Herein, the binding characteristics of the cmHsp70.1 mouse monoclonal antibody (mAb) were evaluated in vitro and in a syngeneic tumor mouse model. More than 50% of the CT26 mouse colon carcinoma cells express Hsp70 on their cell surface at 4°C. After a temperature shift to 37°C, the cmHsp70.1-FITC mAb translocates into early endosomes and lysosomes Intraoperative and near-infrared fluorescence (NIRF) imaging revealed an enrichment of Cy5.5-conjugated mAb cmHsp70.1, but not an identically labelled IgG1 isotype-matched control, in i.p. and s.c. located CT26 tumors, as soon as 30min after i.v. injection into the tail vein. Due to the rapid turnover rate of membrane-bound Hsp70, the fluorescence-labelled cmHsp70.1 mAb became endocytosed and accumulated in the tumor, reaching a maximum after 24h and remained detectable at least up to 96h after a single i.v. injection. The tumor-selective internalization of mAb cmHsp70.1 at the physiological temperature of 37°C might enable a targeted uptake of toxins or radionuclides into Hsp70 membrane-positive tumors. The anti-tumoral activity of the cmHsp70.1 mAb is further supported by its capacity to mediate antibody dependent cytotoxicity (ADCC).

Performance of Epigenetic Markers SEPT9 and ALX4 in Plasma for Detection of Colorectal Precancerous Lesions

PLoS One, 2010, 5, 2 publiziert am 05.02.2010
 
Background: Screening for colorectal cancer (CRC) has shown to reduce cancer-related mortality, however, acceptance and compliance to current programmes are poor. Developing new, more acceptable non-invasive tests for the detection of cancerous and precancerous colorectal lesions would not only allow preselection of individuals for colonoscopy, but may also prevent cancer by removal of precancerous lesions.

 

Methods: Plasma from 128 individuals (cohort I – exploratory study: 73 cases / 55 controls ) was used to test the performance of a single marker, SEPT9, using a real-time quantitative PCR assay. To validate performance of SEPT9, plasma of 76 individuals (cohort II – validation study: 54 cases / 22 controls) was assessed. Additionally, improvement of predictive capability considering SEPT9 and additionally ALX4 methylation was investigated within these patients.

 

Results: In both cohorts combined, methylation of SEPT9 was observed in 9% of controls (3/33), 29% of patients with colorectal precancerous lesions (27/94) and 73% of colorectal cancer patients (24/33). The presence of both SEPT9 and ALX4 markers was analysed in cohort II and was observed in 5% of controls (1/22) and 37% of patients with polyps (18/49). Interestingly, also 3/5 (60%) patients with colorectal cancer were tested positive by the two marker panel in plasma.

 

Conclusions: While these data confirm the detection rate of SEPT9 as a biomarker for colorectal cancer, they also show that methylated DNA from advanced precancerous colorectal lesions can be detected using a panel of two DNA methylation markers, ALX4 and SEPT9. If confirmed in larger studies these data indicate that screening for colorectal precancerous lesions with a blood-based test may be as feasible as screening for invasive cancer.



Monte Carlo Simulationen zur Bestimmung der Dosiserhöhung auf zellulärer Ebene durch tumorgerichtete Gold-Nanopartikel bei der Röntgenbestrahlung

publiziert am 21.09.2018
Aus Zellkulturexperimenten ist bekannt, dass Gold-Nanopartikel den Effekt von Strahlentherapie verstärken können. Jüngste Ergebnisse legen nahe, dass die Konjugation von Gold-Nanopartikeln mit dem Antikörper cmHsp70.1 die Aufnahmerate in Hsp70-positiven Tumorzellen deutlich erhöhen können. Diese Hypothese soll in naher Zukunft durch Experimente in vivo getestet werden. Die zu erwartende Dosiserhöhung der Röntgenstrahlung soll quantitativ simuliert werden.

 

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