Juergen Schlegel

Label-Free Live-Cell Imaging with Confocal Raman Microscopy

Confocal Raman spectroscopy is a noninvasive alternative to established cell imaging methods because it does not require chemical fixation, the use of fluorescent markers, or genetic engineering. In particular, single live-cell, high-resolution imaging by confocal Raman microscopy is desirable because it allows further experiments concerning the individually investigated cells. However, to derive meaningful images from the spectroscopic data, one must identify cell components within the dataset. Using immunofluorescence images as a reference, we derive Raman spectral signatures by means of information measures to identify cell components such as the nucleus, the endoplasmic reticulum, the Golgi apparatus, and mitochondria. The extracted signatures allow us to generate representations equivalent to conventional (immuno)fluorescence images with more than three cell components at a time, exploiting the Raman spectral information alone.

Ultrasmall supraparamagnetic iron oxide-enhanced magnetic resonance imaging of antigen-induced arthritis: a comparative study between SHU 555 C, ferumoxtran-10, and ferumoxytol.

Objectives:We sought to compare the ability of 3 ultrasmall superparamagnetic iron oxides (USPIOs) to detect and characterize antigen-induced arthritis with MR imaging. Materials and Methods:A monoarthritis was induced in the right knee of 18 rats. The left knee served as a normal control. Knees underwent magnetic resonance (MR) imaging before, up to 2 hours, and 24 hours after injection (p.i.) of 200 μmol Fe/kg SHU 555 C (n= 6), ferumoxtran-10 (n = 6), or ferumoxytol (n = 6), using T2-2D-SE 100/20,40,60,80/90 (TR/TE/flipangle), T2*-3D-spoiled gradient recalled (SPGR) 100/15/38, and T1-3D-SPGR 50/1,7/60 sequences. Results:Quantitative signal to noise ratio and ΔSI data of arthritic knees on T1- and T2*-weighted MR images showed no significant differences between the 3 USPIOs (P > 0.05). At 2 hours p.i., SNR and ΔSI data were significantly increased from baseline on T1-weighted images and significantly decreased on T2*-weighted images (P < 0.001). At 24 hours p.i., the T1-enhancement returned to baseline, whereas the T2*-enhancement remained significantly elevated (P < 0.001). Immunostains demonstrated an USPIO compartmentalization in macrophages in the arthritic synovium. Conclusions:Based on the relatively small number of animals in our study group, inflammation in antigen-induced arthritis can be equally detected and characterized with any of the three USPIOs evaluated.

Inhibition of radiation induced migration of human head and neck squamous cell carcinoma cells by blocking of EGF receptor pathways

BackgroundRecently it has been shown that radiation induces migration of glioma cells and facilitates a further spread of tumor cells locally and systemically. The aim of this study was to evaluate whether radiotherapy induces migration in head and neck squamous cell carcinoma (HNSCC). A further aim was to investigate the effects of blocking the epidermal growth factor receptor (EGFR) and its downstream pathways (Raf/MEK/ERK, PI3K/Akt) on tumor cell migration in vitro.MethodsMigration of tumor cells was assessed via a wound healing assay and proliferation by a MTT colorimeritric assay using 3 HNSCC cell lines (BHY, CAL-27, HN). The cells were treated with increasing doses of irradiation (2 Gy, 5 Gy, 8 Gy) in the presence or absence of EGF, EGFR-antagonist (AG1478) or inhibitors of the downstream pathways PI3K (LY294002), mTOR (rapamycin) and MEK1 (PD98059). Biochemical activation of EGFR and the downstream markers Akt and ERK were examined by Western blot analysis.ResultsIn absence of stimulation or inhibition, increasing doses of irradiation induced a dose-dependent enhancement of migrating cells (p < 0.05 for the 3 HNSCC cell lines) and a decrease of cell proliferation (p < 0.05 for the 3 HNSCC cell lines). The inhibition of EGFR or the downstream pathways reduced cell migration significantly (almost all p < 0.05 for the 3 HNSCC cell lines). Stimulation of HNSCC cells with EGF caused a significant increase in migration (p < 0.05 for the 3 HNSCC cell lines). After irradiation alone a pronounced activation of EGFR was observed by Western blot analysis.ConclusionOur results demonstrate that the EGFR is involved in radiation induced migration of HNSCC cells. Therefore EGFR or the downstream pathways might be a target for the treatment of HNSCC to improve the efficacy of radiotherapy.

MRI of arthritis: Comparison of ultrasmall superparamagnetic iron oxide vs. Gd-DTPA

To compare the ability of the ultrasmall superparamagnetic iron oxide (USPIO) SHU555C vs. gadopentetate dimeglumine (Gd‐DTPA) to detect antigen‐induced monoarthritis with MRI.

A missense mutation in the WD40 domain of murine Lyst is linked to severe progressive Purkinje cell degeneration

Disturbance of intracellular trafficking plays a major role in several neurodegenerative disorders including Alzheimer or Parkinson’s disease. The Chediak–Higashi syndrome (CHS), a life-threatening autosomal recessive disease with frequent mutations in the LYST gene, and its animal model, the beige mouse, are both characterized by lysosomal defects with accumulation of giant lysosomes. Clinically they manifest as hypopigmentation, abnormal bleeding and increased susceptibility to infection with various degrees of involvement of the nervous system. In the course of a recessive N-ethyl-N-nitrosurea (ENU) mutagenesis screen, we identified the first murine missense mutation in the lysosomal trafficking regulator gene (LystIng3618) located at a highly conserved position in the WD40 protein domain. Nearly all described human Lyst alleles lead to protein truncation and fatal childhood CHS. Only four different missense mutations have been reported in patients with adolescent or adult forms of CHS involving the nervous system. Interestingly, the LystIng3618 model presents with a predominant neurodegenerative phenotype with progressive degeneration and loss of Purkinje cells and lacks severe impairment of the immune system. Therefore, the LystIng3618 allele could represent a new model for adult CHS with neurological impairment. It could also provide an important tool to elucidate the role of neuronal lysosomal trafficking in the pathophysiology of neurodegeneration.

Prognostic Value of O-(2-[18F]-Fluoroethyl)-L-Tyrosine-Positron Emission Tomography Imaging for Histopathologic Characteristics and Progression-Free Survival in Patients with Low-Grade Glioma

OBJECTIVE O-(2-[18F]-fluoroethyl)-L-tyrosine positron emission tomography ((18)F-FET-PET) imaging is applied for tumor grading, prognostic stratification, and diagnosis of tumor recurrence, especially in high-grade gliomas. Experience with (18)F-FET-PET imaging in low-grade gliomas is limited. Therefore, the objective of the present study was to assess (18)F-FET-PET tracer uptake in low-grade gliomas and to investigate possible correlations with contrast enhancement in magnetic resonance imaging (MRI) and histopathology. METHODS A total of 65 patients (29 female, 36 male, median age 38 years) with newly diagnosed or recurrent low-grade gliomas for whom preoperative MRI and (18)F-FET-PET imaging were available were included. Tumor entity, tumor location, as well as histopathology (isocitrate dehydrogenase [IDH] 1/2 mutation, Ki67, p53, oligodendroglial differentiation, 1p19q codeletion), and progression-free survival were assessed. (18)F-FET-PET images were acquired and fused to MRI (T2-weighted fluid-attenuated inversion recovery) and tumor volume was measured in areas with a tumor-to-background ratio >1.3, >1.6, and >2.0 and in MRI. RESULTS PET tracer uptake was observed in 78.5% of all World Health Organization Grade I and II tumors. (18)F-FET uptake showed a high negative predictive value for oligodendroglial components and for 1p19q codeletion. No further significant correlation between histologic features, progression-free survival, or IDH1/2 mutation status and tracer uptake was observed. CONCLUSIONS We found that 78.5% of low-grade gliomas do show elevated tracer uptake in (18)F-FET-PET imaging. Low-grade glioma without tracer uptake exclude oligodendroglial differentiation and 1p19q codeletion. Further differentiation between molecular subtypes is not possible with static (18)F-FET-PET. No correlation of progression-free survival to tracer uptake and IDH1/2-mutation status was observed.

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

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.

Diffusion tensor image features predict IDH genotype in newly diagnosed WHO grade II/III gliomas.

We hypothesized that machine learning analysis based on texture information from the preoperative MRI can predict IDH mutational status in newly diagnosed WHO grade II and III gliomas. This retrospective study included in total 79 consecutive patients with a newly diagnosed WHO grade II or III glioma. Local binary pattern texture features were generated from preoperative B0 and fractional anisotropy (FA) diffusion tensor imaging. Using a training set of 59 patients, a single hidden layer neural network was then trained on the texture features to predict IDH status. The model was validated based on the prediction accuracy calculated in a previously unseen set of 20 gliomas. Prediction accuracy of the generated model was 92% (54/59 cases; AUC = 0.921) in the training and 95% (19/20; AUC = 0.952) in the validation cohort. The ten most important features were comprised of tumor size and both B0 and FA texture information, underlining the joint contribution of imaging data to classification. Machine learning analysis of DTI texture information and tumor size reliably predicts IDH status in preoperative MRI of gliomas. Such information may increasingly support individualized surgical strategies, supplement pathological analysis and highlight the potential of radiogenomics.

Epstein-Barr virus infection is strictly associated with the metastatic spread of sinonasal squamous-cell carcinomas

BACKGROUND Sinonasal squamous-cell carcinomas (SNSCC) are relatively rare. Thus, data regarding the rate of lymph node metastases are inconsistent in contrast with well-known high metastasis rates in squamous-cell carcinomas of the head and neck (HNSCC) (oral cavity, pharynx and larynx). Hence, the indication for elective neck dissection is difficult in SNSCC. The aim of this study was to assess common genetic alterations and EBV and HPV status as a function of metastasis in SNSCC and HNSCC. METHODS We retrospectively analyzed 44 SNSCC and 65 HNSCC for TP53, EGFR, KRAS, PIK3CA and BRAF mutations using a high-resolution melting analysis followed by Sanger sequencing. EBV and HPV detection was performed using in situ hybridization for virus encoded RNA. Tumor-associated p16(INK4a) expression was visualized by immunohistochemistry and correlated with HPV infection. The mutation data, EBV and HPV status were statistically compared with the clinical data in SNSCC and HNSCC. RESULTS TP53 mutations were exclusively associated with shorter survival in SNSCC (p=0.048). All the other markers had no effect on the metastasis rate and survival. In total, 20 of 44 SNSCC were EBV-positive. Only these EBV positive tumors developed lymph node or distant metastases (p=0.008). LMP1 was positive in 14/44 patients. When combining both methods significance for a correlation between EBV/LMP1 positive patients and metastases was even higher (p=0.001). CONCLUSION In SNSCC, the presence of EBV is strictly associated with metastasis. We recommend an elective neck dissection in patients with EBV-positive SNSCC.

Nachweis der Akkumulation von intravenös injizierten, Eisenoxid- oder FDG-18 markierten zytotoxischen T-Lymphozyten in HER2/neu+ NIH3T3 Tumoren mittels Magnetresonanztomographie und Autoradiographie

Ziele: Nachweis der Akkumulation von intravenos injizierten, Eisenoxid- oder FDG-18 markierten zytotoxischen T-Lymphozyten (NK-92) in HER2/neu positiven NIH3T3 Tumoren mittels Magnetresonanztomographie (MRT) und Autoradiographie. Methode: NK-92 Zellen, die nicht gegen HER2/neu gerichtet waren, und genetisch modifizierte NK-92-scFv(FRP5)-zeta Zellen, die einen fur das HER2/neu Antigen spezifischen chimaren Antigenrezeptor exprimieren, wurden mit Ferucarbotran oder mit FDG-18 markiert. Die Effektivitat der Markierung wurde mittels Bildgebung, Berliner Blau Farbung und Spektrometrie bewertet und optimiert. Dann wurden 15 Balb/c Mausen HER2/neu positive NIH-3T3 Tumoren subkutan in die rechte Milchleiste implantiert. Als die Tumoren einen Durchmesser von etwa 1cm erreicht hatten, wurden 6 Mausen jeweils 5×106 Ferucarbotran-markierte NK-92 Zellen (n=3) oder NK-92-scFv(FRP5)-zeta Zellen (n=3) intravenos injiziert. Die Tumor-Akkumulation dieser Zellen wurde an einem klinischen 1.5 T MRT-Scanner mit T1- und T2*-FFE-Sequenzen vor, 12 und 24h nach Zellinjektion (p.i.) untersucht. Zusatzlich wurden 9 Mausen 5×106 FDG-18 markierte NK-92 Zellen (n=2) oder NK-92-scFv(FRP5)-zeta Zellen (n=7) injiziert. Die Tumor-Akkumulation der FDG-18 markierten Zellen wurde mittels Autoradiographie ex vivo 1h und 2h p.i. dokumentiert. Alle Ergebnisse wurden mit Histopathologien der Tumoren verglichen. Ergebnis: Nach Injektion der NK-92-scFv(FRP5)-zeta Zellen, die gegen das HER2/neu Antigen gerichtet waren, zeigte die MRT einen progressiven, ausgepragten Signalabfall und die Autoradiographie einen deutlich erhohten FDG-18 Tracer Uptake in den HER2/neu positiven Tumoren. Dagegen verursachte eine Injektion der NK-92 Kontrollzellen, die nicht gegen das HER2/neu Antigen gerichtet waren, keine Veranderung der MRT-Signalintensitat und keinen nachweisbaren FDG-18 Uptake des Tumorgewebes. Die Anti-CD 57 Immunhistochemie bestatigte eine Akkumulation der NK-92-scFv(FRP5)-zeta Zellen, aber keine Akkumulation der NK-92 Zellen im Tumorgewebe. Schlussfolgerung: NK-92 Zellen konnen effizient mit klinisch zugelassenen Eisenoxiden und radioaktiven Trauern markiert und die Akkumulation dieser markierten Zellen in Tumoren mittels Bildgebung nachgewiesen werden. Die hier vorgestellten Techniken sind prinzipiell direkt klinisch anwendbar und konnten verwendet werden, um NK-Zellbasierte Immuntherapien in Patienten nicht-invasiv mit bildgebenden Methoden zu dokumentieren und kontrollieren. Korrespondierender Autor: Meier R Technische Universitat Munchen, Radiologie, Schmellerstr. 12, 80337 Munchen E-Mail: reinhardt.meier@googlemail.com