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Artigo em Inglês | MEDLINE | ID: mdl-32668069

RESUMO

The large-scale and unlabeled molecular characterization of single cells inside their natural tissue habitat remains a major challenge in molecular biology. We present a method that integrates morphometric image analysis to delineate and classify individual cells with their single cell-specific molecular profiles. This approach provides new means to study spatial biological processes such as cancer field effects and the relationship between morphometric and molecular features.

3.
Nat Commun ; 11(1): 3068, 2020 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-32555155

RESUMO

Surgical adhesions are bands of scar tissues that abnormally conjoin organ surfaces. Adhesions are a major cause of post-operative and dialysis-related complications, yet their patho-mechanism remains elusive, and prevention agents in clinical trials have thus far failed to achieve efficacy. Here, we uncover the adhesion initiation mechanism by coating beads with human mesothelial cells that normally line organ surfaces, and viewing them under adhesion stimuli. We document expansive membrane protrusions from mesothelia that tether beads with massive accompanying adherence forces. Membrane protrusions precede matrix deposition, and can transmit adhesion stimuli to healthy surfaces. We identify cytoskeletal effectors and calcium signaling as molecular triggers that initiate surgical adhesions. A single, localized dose targeting these early germinal events completely prevented adhesions in a preclinical mouse model, and in human assays. Our findings classifies the adhesion pathology as originating from mesothelial membrane bridges and offer a radically new therapeutic approach to treat adhesions.

4.
Mol Metab ; 36: 100953, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32278304

RESUMO

BACKGROUND: Imaging mass spectrometry enables in situ label-free detection of thousands of metabolites from intact tissue samples. However, automated steps for multi-omics analyses and interpretation of histological images have not yet been implemented in mass spectrometry data analysis workflows. The characterization of molecular properties within cellular and histological features is done via time-consuming, non-objective, and irreproducible definitions of regions of interest, which are often accompanied by a loss of spatial resolution due to mass spectra averaging. METHODS: We developed a new imaging pipeline called Spatial Correlation Image Analysis (SPACiAL), which is a computational multimodal workflow designed to combine molecular imaging data with multiplex immunohistochemistry (IHC). SPACiAL allows comprehensive and spatially resolved in situ correlation analyses on a cellular resolution. To demonstrate the method, matrix-assisted laser desorption-ionization (MALDI) Fourier-transform ion cyclotron resonance (FTICR) imaging mass spectrometry of metabolites and multiplex IHC staining were performed on the very same tissue section of mouse pancreatic islets and on human gastric cancer tissue specimens. The SPACiAL pipeline was used to perform an automatic, semantic-based, functional tissue annotation of histological and cellular features to identify metabolic profiles. Spatial correlation networks were generated to analyze metabolic heterogeneity associated with cellular features. RESULTS: To demonstrate the new method, the SPACiAL pipeline was used to identify metabolic signatures of alpha and beta cells within islets of Langerhans, which are cell types that are not distinguishable via morphology alone. The semantic-based, functional tissue annotation allows an unprecedented analysis of metabolic heterogeneity via the generation of spatial correlation networks. Additionally, we demonstrated intra- and intertumoral metabolic heterogeneity within HER2/neu-positive and -negative gastric tumor cells. CONCLUSIONS: We developed the SPACiAL workflow to provide IHC-guided in situ metabolomics on intact tissue sections. Diminishing the workload by automated recognition of histological and functional features, the pipeline allows comprehensive analyses of metabolic heterogeneity. The multimodality of immunohistochemical staining and extensive molecular information from imaging mass spectrometry has the advantage of increasing both the efficiency and precision for spatially resolved analyses of specific cell types. The SPACiAL method is a stepping stone for the objective analysis of high-throughput, multi-omics data from clinical research and practice that is required for diagnostics, biomarker discovery, or therapy response prediction.

5.
Horm Metab Res ; 52(6): 435-447, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32349131

RESUMO

The adrenal gland integrates catecholamine-producing neuroendocrine cells and steroid-producing cells with mesenchymal origin in a structured manner under one capsule and is a key regulator for vital bioactivity. In addition to adrenal-specific disease, dysregulation of adrenal hormones is associated with systemic effects, leading to undesirable metabolic and cardiovascular consequences. Mass spectrometry imaging (MSI) technique can simultaneously measure a broad range of biomolecules, including metabolites and hormones, which has enabled the study of tissue metabolic and hormone alterations in adrenal and adrenal-related diseases. Furthermore, this technique coupled with labeled immunohistochemistry staining has enabled the study of the pathophysiological adaptation of the adrenal gland under normal and abnormal conditions at different molecular levels. This review discusses the recent applications of in situ MSI in the adrenal gland. For example, the combination of formalin-fixed paraffin-embedded tissue microarray and MSI to tissues from patient cohorts has facilitated the discovery of clinically relevant prognostic biomolecules and generated promising hypotheses for new sights into physiology and pathophysiology of adrenal gland. MSI also has enabled the discovery of clinically significant tissue molecular (i. e., biomarker) and pathway changes in adrenal disease, particularly in adrenal tumors. In addition, MSI has advanced the ability to optimally identify and detect adrenal gland specific molecules. Thus, as a novel analytical methodology, MSI has provided unprecedented capabilities for in situ tissue study.

6.
Cell Death Dis ; 11(3): 192, 2020 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184394

RESUMO

Conditions of impaired adrenal function and tissue destruction, such as in Addison's disease, and treatment resistance of adrenocortical carcinoma (ACC) necessitate improved understanding of the pathophysiology of adrenal cell death. Due to relevant oxidative processes in the adrenal cortex, our study investigated the role of ferroptosis, an iron-dependent cell death mechanism and found high adrenocortical expression of glutathione peroxidase 4 (GPX4) and long-chain-fatty-acid CoA ligase 4 (ACSL4) genes, key factors in the initiation of ferroptosis. By applying MALDI mass spectrometry imaging to normal and neoplastic adrenocortical tissue, we detected high abundance of arachidonic and adrenic acid, two long chain polyunsaturated fatty acids which undergo peroxidation during ferroptosis. In three available adrenal cortex cell models (H295R, CU-ACC1 and CU-ACC-2) a high susceptibility to GPX4 inhibition with RSL3 was documented with EC50 values of 5.7 × 10-8, 8.1 × 10-7 and 2.1 × 10-8 M, respectively, while all non-steroidogenic cells were significantly less sensitive. Complete block of GPX4 activity by RSL3 led to ferroptosis which was completely reversed in adrenal cortex cells by inhibition of steroidogenesis with ketoconazole but not by blocking the final step of cortisol synthesis with metyrapone. Mitotane, the only approved drug for ACC did not induce ferroptosis, despite strong induction of lipid peroxidation in ACC cells. Together, this report is the first to demonstrate extraordinary sensitivity of adrenal cortex cells to ferroptosis dependent on their active steroid synthetic pathways. Mitotane does not induce this form of cell death in ACC cells.

7.
J Cachexia Sarcopenia Muscle ; 11(1): 226-240, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31965747

RESUMO

BACKGROUND: Cachexia is the direct cause of at least 20% of cancer-associated deaths. Muscle wasting in skeletal muscle results in weakness, immobility, and death secondary to impaired respiratory muscle function. Muscle proteins are massively degraded in cachexia; nevertheless, the molecular mechanisms related to this process are poorly understood. Previous studies have reported conflicting results regarding the amino acid abundances in cachectic skeletal muscle tissues. There is a clear need to identify the molecular processes of muscle metabolism in the context of cachexia, especially how different types of molecules are involved in the muscle wasting process. METHODS: New in situ -omics techniques were used to produce a more comprehensive picture of amino acid metabolism in cachectic muscles by determining the quantities of amino acids, proteins, and cellular metabolites. Using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging, we determined the in situ concentrations of amino acids and proteins, as well as energy and other cellular metabolites, in skeletal muscle tissues from genetic mouse cancer models (n = 21) and from patients with cancer (n = 6). Combined results from three individual MALDI mass spectrometry imaging methods were obtained and interpreted. Immunohistochemistry staining for mitochondrial proteins and myosin heavy chain expression, digital image analysis, and transmission electron microscopy complemented the MALDI mass spectrometry imaging results. RESULTS: Metabolic derangements in cachectic mouse muscle tissues were detected, with significantly increased quantities of lysine, arginine, proline, and tyrosine (P = 0.0037, P = 0.0048, P = 0.0430, and P = 0.0357, respectively) and significantly reduced quantities of glutamate and aspartate (P = 0.0008 and P = 0.0124). Human skeletal muscle tissues revealed similar tendencies. A majority of altered amino acids were released by the breakdown of proteins involved in oxidative phosphorylation. Decreased energy charge was observed in cachectic muscle tissues (P = 0.0101), which was related to the breakdown of specific proteins. Additionally, expression of the cationic amino acid transporter CAT1 was significantly decreased in the mitochondria of cachectic mouse muscles (P = 0.0133); this decrease may play an important role in the alterations of cationic amino acid metabolism and decreased quantity of glutamate observed in cachexia. CONCLUSIONS: Our results suggest that mitochondrial dysfunction has a substantial influence on amino acid metabolism in cachectic skeletal muscles, which appears to be triggered by diminished CAT1 expression, as well as the degradation of mitochondrial proteins. These findings provide new insights into the pathobiochemistry of muscle wasting.

8.
Hypertension ; 75(3): 634-644, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31957522

RESUMO

Aldosterone-producing adenomas (APAs) are one of the main causes of primary aldosteronism and the most prevalent surgically correctable form of hypertension. Aldosterone-producing cell clusters (APCCs) comprise tight nests of zona glomerulosa cells, strongly positive for CYP11B2 (aldosterone synthase) in immunohistochemistry. APCCs have been suggested as possible precursors of APAs because they frequently carry driver mutations for constitutive aldosterone production, and a few adrenal lesions with histopathologic features of both APCCs and APAs have been identified. Our objective was to investigate the metabolic phenotypes of APCCs (n=27) compared with APAs (n=6) using in situ matrix-assisted laser desorption/ionization mass spectrometry imaging of formalin-fixed paraffin-embedded adrenals from patients with unilateral primary aldosteronism. Specific distribution patterns of metabolites were associated with APCCs and classified 2 separate APCC subgroups (subgroups 1 and 2) indistinguishable by CYP11B2 immunohistochemistry. Metabolic profiles of APCCs in subgroup 1 were tightly clustered and distinct from subgroup 2 and APAs. Multiple APCCs from the same adrenal displayed metabolic profiles of the same subgroup. Metabolites of APCC subgroup 2 were highly similar to the APA group and indicated enhanced metabolic pathways favoring cell proliferation compared with APCC subgroup 1. In conclusion, we demonstrate specific subgroups of APCCs with strikingly divergent distribution patterns of metabolites. One subgroup displays a metabolic phenotype convergent with APAs and may represent the progression of APCCs to APAs.

9.
Sci Rep ; 10(1): 79, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31919465

RESUMO

Molecular imaging of atherosclerosis by Magnetic Resonance Imaging (MRI) has been impaired by a lack of validation of the specific substrate responsible for the molecular imaging signal. We therefore aimed to investigate the additive value of mass spectrometry imaging (MSI) of atherosclerosis-affine Gadofluorine P for molecular MRI of atherosclerotic plaques. Atherosclerotic Ldlr-/- mice were investigated by high-field MRI (7 T) at different time points following injection of atherosclerosis-affine Gadofluorine P as well as at different stages of atherosclerosis formation (4, 8, 16 and 20 weeks of HFD). At each imaging time point mice were immediately sacrificed after imaging and aortas were excised for mass spectrometry imaging: Matrix Assisted Laser Desorption Ionization (MALDI) Imaging and Laser Ablation - Inductively Coupled Plasma - Mass Spectrometry (LA-ICP-MS) imaging. Mass spectrometry imaging allowed to visualize the localization and measure the concentration of the MR imaging probe Gadofluorine P in plaque tissue ex vivo with high spatial resolution and thus adds novel and more target specific information to molecular MR imaging of atherosclerosis.

10.
Radiat Oncol ; 15(1): 7, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906998

RESUMO

BACKGROUND: Definitive chemoradiotherapy (dCRT) is a standard treatment for patients with locally advanced head and neck cancer. There is a clinical need for a stratification of this prognostically heterogeneous group of tumors in order to optimize treatment of individual patients. We retrospectively reviewed all patients with head and neck squamous cell carcinoma (HNSCC) of the oral cavity, oropharynx, hypopharynx, or larynx, treated with dCRT from 09/2008 until 03/2016 at the Department of Radiation Oncology, LMU Munich. Here we report the clinical results of the cohort which represent the basis for biomarker discovery and molecular genetic research within the framework of a clinical cooperation group. METHODS: Patient data were collected and analyzed for outcome and treatment failures with regard to previously described and established risk factors. RESULTS: We identified 184 patients with a median follow-up of 65 months and a median age of 64 years. Patients received dCRT with a median dose of 70 Gy and simultaneous chemotherapy in 90.2% of cases, mostly mitomycin C / 5-FU in concordance with the ARO 95-06 trial. The actuarial 3-year overall survival (OS), local, locoregional and distant failure rates were 42.7, 29.8, 34.0 and 23.4%, respectively. Human papillomavirus-associated oropharynx cancer (HPVOPC) and smaller gross tumor volume were associated with significantly improved locoregional tumor control rate, disease-free survival (DFS) and OS in multivariate analysis. Additionally, lower hemoglobin levels were significantly associated with impaired DFS und OS in univariate analysis. The extent of lymph node involvement was associated with distant failure, DFS and OS. Moreover, 92 patients (50%) of our cohort have been treated in concordance with the ARO 95-06 study, corroborating the results of this study. CONCLUSION: Our cohort is a large unselected monocentric cohort of HNSCC patients treated with dCRT. Tumor control rates and survival rates compare favorably with the results of previously published reports. The clinical data, together with the available tumor samples from biopsies, will allow translational research based on molecular genetic analyses.

11.
Haematologica ; 105(4): 937-950, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31248967

RESUMO

Glutathione peroxidase 4 (GPX4) is unique as it is the only enzyme that can prevent detrimental lipid peroxidation in vivo by reducing lipid peroxides to the respective alcohols thereby stabilizing oxidation products of unsaturated fatty acids. During reticulocyte maturation, lipid peroxidation mediated by 15-lipoxygenase in humans and rabbits and by 12/15-lipoxygenase (ALOX15) in mice was considered the initiating event for the elimination of mitochondria but is now known to occur through mitophagy. Yet, genetic ablation of the Alox15 gene in mice failed to provide evidence for this hypothesis. We designed a different genetic approach to tackle this open conundrum. Since either other lipoxygenases or non-enzymatic autooxidative mechanisms may compensate for the loss of Alox15, we asked whether ablation of Gpx4 in the hematopoietic system would result in the perturbation of reticulocyte maturation. Quantitative assessment of erythropoiesis indices in the blood, bone marrow (BM) and spleen of chimeric mice with Gpx4 ablated in hematopoietic cells revealed anemia with an increase in the fraction of erythroid precursor cells and reticulocytes. Additional dietary vitamin E depletion strongly aggravated the anemic phenotype. Despite strong extramedullary erythropoiesis reticulocytes failed to mature and accumulated large autophagosomes with engulfed mitochondria. Gpx4-deficiency in hematopoietic cells led to systemic hepatic iron overload and simultaneous severe iron demand in the erythroid system. Despite extremely high erythropoietin and erythroferrone levels in the plasma, hepcidin expression remained unchanged. Conclusively, perturbed reticulocyte maturation in response to Gpx4 loss in hematopoietic cells thus causes ineffective erythropoiesis, a phenotype partially masked by dietary vitamin E supplementation.

12.
Sci Rep ; 9(1): 19483, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31862894

RESUMO

Dual-specificity phosphatase 8 (Dusp8) acts as physiological inhibitor for the MAPKs Jnk, Erk and p38 which are involved in regulating multiple CNS processes. While Dusp8 expression levels are high in limbic areas such as the hippocampus, the functional role of Dusp8 in hippocampus morphology, MAPK-signaling, neurogenesis and apoptosis as well as in behavior are still unclear. It is of particular interest whether human carriers of a DUSP8 allelic variant show similar hippocampal alterations to mice. Addressing these questions using Dusp8 WT and KO mouse littermates, we found that KOs suffered from mildly impaired spatial learning, increased locomotor activity and elevated anxiety. Cell proliferation, apoptosis and p38 and Jnk phosphorylation were unaffected, but phospho-Erk levels were higher in hippocampi of the KOs. Consistent with a decreased hippocampus size in Dusp8 KO mice, we found reduced volumes of the hippocampal subregions subiculum and CA4 in humans carrying the DUSP8 allelic variant SNP rs2334499:C > T. Overall, aberrations in morphology and behavior in Dusp8 KO mice and a decrease in hippocampal volume of SNP rs2334499:C > T carriers point to a novel, translationally relevant role of Dusp8 in hippocampus function that warrants further studies on the role of Dusp8 within the limbic network.

13.
Nature ; 576(7786): 287-292, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31776510

RESUMO

Mammals form scars to quickly seal wounds and ensure survival by an incompletely understood mechanism1-5. Here we show that skin scars originate from prefabricated matrix in the subcutaneous fascia. Fate mapping and live imaging revealed that fascia fibroblasts rise to the skin surface after wounding, dragging their surrounding extracellular jelly-like matrix, including embedded blood vessels, macrophages and peripheral nerves, to form the provisional matrix. Genetic ablation of fascia fibroblasts prevented matrix from homing into wounds and resulted in defective scars, whereas placing an impermeable film beneath the skin-preventing fascia fibroblasts from migrating upwards-led to chronic open wounds. Thus, fascia contains a specialized prefabricated kit of sentry fibroblasts, embedded within a movable sealant, that preassemble together diverse cell types and matrix components needed to heal wounds. Our findings suggest that chronic and excessive skin wounds may be attributed to the mobility of the fascia matrix.


Assuntos
Fáscia/patologia , Cicatrização , Animais , Biomarcadores/análise , Movimento Celular , Fáscia/transplante , Fibroblastos , Queloide , Camundongos Endogâmicos C57BL
14.
Small ; 15(49): e1904112, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31639283

RESUMO

Targeted delivery of nanomedicine/nanoparticles (NM/NPs) to the site of disease (e.g., the tumor or lung injury) is of vital importance for improved therapeutic efficacy. Multimodal imaging platforms provide powerful tools for monitoring delivery and tissue distribution of drugs and NM/NPs. This study introduces a preclinical imaging platform combining X-ray (two modes) and fluorescence imaging (three modes) techniques for time-resolved in vivo and spatially resolved ex vivo visualization of mouse lungs during pulmonary NP delivery. Liquid mixtures of iodine (contrast agent for X-ray) and/or (nano)particles (X-ray absorbing and/or fluorescent) are delivered to different regions of the lung via intratracheal instillation, nasal aspiration, and ventilator-assisted aerosol inhalation. It is demonstrated that in vivo propagation-based phase-contrast X-ray imaging elucidates the dynamic process of pulmonary NP delivery, while ex vivo fluorescence imaging (e.g., tissue-cleared light sheet fluorescence microscopy) reveals the quantitative 3D drug/particle distribution throughout the entire lung with cellular resolution. The novel and complementary information from this imaging platform unveils the dynamics and mechanisms of pulmonary NM/NP delivery and deposition for each of the delivery routes, which provides guidance on optimizing pulmonary delivery techniques and novel-designed NM for targeting and efficacy.

15.
Clin Chem ; 65(10): 1276-1286, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31492715

RESUMO

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare tumor with variable prognosis even within the same tumor stage. Cancer-related sex hormones and their sulfated metabolites in body fluids can be used as tumor markers. The role of steroid sulfation in ACC has not yet been studied. MALDI mass spectrometry imaging (MALDI-MSI) is a novel tool for tissue-based chemical phenotyping. METHODS: We performed phenotyping of formalin-fixed, paraffin-embedded tissue samples from 72 ACC by MALDI-MSI at a metabolomics level. RESULTS: Tumoral steroid hormone metabolites-estradiol sulfate [hazard ratio (HR) 0.26; 95% CI, 0.10-0.69; P = 0.005] and estrone 3-sulfate (HR 0.22; 95% CI, 0.07-0.63; P = 0.003)-were significantly associated with prognosis in Kaplan-Meier analyses and after multivariable adjustment for age, tumor stage, and sex (HR 0.29; 95% CI, 0.11-0.79; P = 0.015 and HR 0.30; 95% CI, 0.10-0.91; P = 0.033, respectively). Expression of sulfotransferase SULT2A1 was associated with prognosis to a similar extent and was validated to be a prognostic factor in two published data sets. We discovered the presence of estradiol-17ß 3,17-disulfate (E2S2) in a subset of tumors with particularly poor overall survival. Electron microscopy revealed novel membrane-delimited organelles in only these tumors. By applying cluster analyses of metabolomic data, 3 sulfation-related phenotypes exhibited specific metabolic features unrelated to steroid metabolism. CONCLUSIONS: MALDI-MSI provides novel insights into the pathophysiology of ACC. Steroid hormone sulfation may be used for prognostication and treatment stratification. Sulfation-related metabolic reprogramming may be of relevance also in conditions beyond the rare ACC and can be directly investigated by the use of MALDI-MSI.

16.
PLoS One ; 14(9): e0221454, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31483811

RESUMO

Side effects caused by radiation are a limiting factor to the amount of dose that can be applied to a tumor volume. A novel method to reduce side effects in radiotherapy is the use of spatial fractionation, in which a pattern of sub-millimeter beams (minibeams) is applied to spare healthy tissue. In order to determine the skin reactions in dependence of single beam sizes, which are relevant for spatially fractionated radiotherapy approaches, single pencil beams of submillimeter to 6 millimeter size were applied in BALB/c mice ears at a Small Animal Radiation Research Platform (SARRP) with a plateau dose of 60 Gy. Radiation toxicities in the ears were observed for 25 days after irradiation. Severe radiation responses were found for beams ≥ 3 mm diameter. The larger the beam diameter the stronger the observed reactions. No ear swelling and barely reddening or desquamation were found for the smallest beam sizes (0.5 and 1 mm). The findings were confirmed by histological sections. Submillimeter beams are preferred in minibeam therapy to obtain optimized tissue sparing. The gradual increase of radiation toxicity with beam size shows that also larger beams are capable of healthy tissue sparing in spatial fractionation.


Assuntos
Orelha/efeitos da radiação , Raios gama/efeitos adversos , Pele/patologia , Animais , Orelha/fisiologia , Eritema/etiologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Dosímetros de Radiação , Pele/metabolismo , Pele/efeitos da radiação
17.
JCI Insight ; 4(17)2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31484828

RESUMO

Recent genetic examinations and multisteroid profiles have provided the basis for subclassification of aldosterone-producing adenomas (APAs). The objective of the current study was to produce a comprehensive, high-resolution mass spectrometry imaging (MSI) map of APAs in relation to morphometry, immunohistochemical profiles, mutational status, and clinical outcome. The study cohort comprised 136 patients with unilateral primary aldosteronism. Matrix-assisted laser desorption/ionization-Fourier transform-ion cyclotron resonance MSI was conducted, and metabolite profiles were analyzed with genotype/phenotype information, including digital image analysis from morphometry and IHC of steroidogenic enzymes. Distinct molecular signatures between KCNJ5- and CACNA1D-mutated APAs with significant differences of 137 metabolites, including metabolites of purine metabolism and steroidogenesis, were observed. Intratumor concentration of 18-oxocortisol and 18-hydroxycortisol were inversely correlated with the staining intensity of CYP11B1. Lower staining intensity of CYP11B1 and higher levels of 18-oxocortisol were associated with a higher probability of complete clinical success after surgery. The present study demonstrates distinct metabolomic profiles of APAs in relation to tumor genotype. In addition, we reveal an inverse correlation between cortisol derivatives and CYP11B1 and the impact of 18-oxocortisol and CYP11B1 on clinical outcome, which provides unprecedented insights into the pathophysiology, clinical features, and steroidogenesis of APAs.

18.
Artigo em Inglês | MEDLINE | ID: mdl-31379752

RESUMO

Background: Adrenocortical carcinoma (ACC) is a rare tumor entity with restricted therapeutic opportunities. HSP90 (Heat Shock Protein 90) chaperone activity is fundamental for cell survival and contributes to different oncogenic signaling pathways. Indeed, agents targeting HSP90 function have shown therapeutic efficacy in several cancer types. We have examined the expression of HSP90 in different adrenal tumors and evaluated the use of HSP90 inhibitors in vitro as possible therapy for ACC. Methods: Immunohistochemical expression of HSP90 isoforms was investigated in different adrenocortical tumors and associated with clinical features. Additionally, a panel of N-terminal (17-allylamino-17-demethoxygeldanamycin (17-AAG), luminespib, and ganetespib) and C-terminal (novobiocin and silibinin) HSP90 inhibitors were tested on various ACC cell lines. Results: Within adrenocortical tumors, ACC samples exhibited the highest expression of HSP90ß. Within a cohort of ACC patients, HSP90ß expression levels were inversely correlated with recurrence-free and overall survival. In functional assays, among five different compounds tested luminespib and ganetespib induced a significant decrease in cell viability in single as well as in combined treatments with compounds of the clinically used EDP-M scheme (etoposide, doxorubicin, cisplatin, mitotane). Inhibition of cell viability correlated furthermore with a decrease in proliferation, in cell migration and an increase in apoptosis. Moreover, analysis of cancer pathways indicated a modulation of the ERK1/2-and AKT-pathways by luminespib and ganetespib treatment. Conclusions: Our findings emphasize HSP90 as a marker with prognostic impact and promising target with N-terminal HSP90 inhibitors as drugs with potential therapeutic efficacy toward ACC.

19.
Lab Invest ; 99(10): 1535-1546, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31148595

RESUMO

Multimodal tissue analyses that combine two or more detection technologies provide synergistic value compared to single methods and are employed increasingly in the field of tissue-based diagnostics and research. Here, we report a technical pipeline that describes a combined approach of HER2/CEP17 fluorescence in situ hybridization (FISH) analysis with MALDI imaging on the very same section of formalin-fixed and paraffin-embedded (FFPE) tissue. FFPE biopsies and a tissue microarray of human gastroesophageal adenocarcinoma were analyzed by MALDI imaging. Subsequently, the very same section was hybridized by HER2/CEP17 FISH. We found that tissue morphology of both, the biopsies and the tissue microarray, was unaffected by MALDI imaging and the HER2 and CEP17 FISH signals were analyzable. In comparison with FISH analysis of samples without MALDI imaging, we observed no difference in terms of fluorescence signal intensity and gene copy number. Our combined approach revealed adenosine monophosphate, measured by MALDI imaging, as a prognostic marker. HER2 amplification, which was detected by FISH, is a stratifier between good and poor patient prognosis. By integrating both stratification parameters on the basis of our combined approach, we were able to strikingly improve the prognostic effect. Combining molecules detected by MALDI imaging with the gene copy number detected by HER2/CEP17 FISH, we found a synergistic effect, which enhances patient prognosis. This study shows that our combined approach allows the detection of genetic and metabolic properties from one very same FFPE tissue section, which are specific for HER2 and hence suitable for prognosis. Furthermore, this synergism might be useful for response prediction in tumors.

20.
ACS Nano ; 13(7): 8114-8123, 2019 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-31194509

RESUMO

Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of Quasibacillus thermotolerans (Qt), which in combination with the recently characterized encapsulin system from Myxococcus xanthus (Mx) enables multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with T = 4 icosahedral symmetry and a diameter of ∼43 nm harboring two putative pore regions at the 5-fold and 3-fold axes. We also found that upon heterologous expression in mammalian cells, the native cargo is autotargeted to the inner surface of the shell and exhibits ferroxidase activity leading to efficient intraluminal iron biomineralization, which enhances cellular TEM contrast. We furthermore demonstrate that the two differently sized encapsulins of Qt and Mx do not intermix and can be robustly differentiated by conventional TEM via a deep learning classifier to enable automated multiplexed EM gene reporter imaging.

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