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1.
Br J Cancer ; 131(2): 258-270, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38834745

RESUMO

BACKGROUND: Diffuse invasion remains a primary cause of treatment failure in pediatric high-grade glioma (pHGG). Identifying cellular driver(s) of pHGG invasion is needed for anti-invasion therapies. METHODS: Ten highly invasive patient-derived orthotopic xenograft (PDOX) models of pHGG were subjected to isolation of matching pairs of invasive (HGGINV) and tumor core (HGGTC) cells. RESULTS: pHGGINV cells were intrinsically more invasive than their matching pHGGTC cells. CSC profiling revealed co-positivity of CD133 and CD57 and identified CD57+CD133- cells as the most abundant CSCs in the invasive front. In addition to discovering a new order of self-renewal capacities, i.e., CD57+CD133- > CD57+CD133+ > CD57-CD133+ > CD57-CD133- cells, we showed that CSC hierarchy was impacted by their spatial locations, and the highest self-renewal capacities were found in CD57+CD133- cells in the HGGINV front (HGGINV/CD57+CD133- cells) mediated by NANOG and SHH over-expression. Direct implantation of CD57+ (CD57+/CD133- and CD57+/CD133+) cells into mouse brains reconstituted diffusely invasion, while depleting CD57+ cells (i.e., CD57-CD133+) abrogated pHGG invasion. CONCLUSION: We revealed significantly increased invasive capacities in HGGINV cells, confirmed CD57 as a novel glioma stem cell marker, identified CD57+CD133- and CD57+CD133+ cells as a new cellular driver of pHGG invasion and suggested a new dual-mode hierarchy of HGG stem cells.


Assuntos
Antígeno AC133 , Neoplasias Encefálicas , Antígenos CD57 , Glioma , Invasividade Neoplásica , Células-Tronco Neoplásicas , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/metabolismo , Humanos , Animais , Glioma/patologia , Glioma/imunologia , Glioma/metabolismo , Camundongos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/metabolismo , Antígenos CD57/metabolismo , Criança , Antígeno AC133/metabolismo
2.
Int J Mol Sci ; 25(2)2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-38256041

RESUMO

The link between mitochondria and major depressive disorder (MDD) is increasingly evident, underscored both by mitochondria's involvement in many mechanisms identified in depression and the high prevalence of MDD in individuals with mitochondrial disorders. Mitochondrial functions and energy metabolism are increasingly considered to be involved in MDD's pathogenesis. This study focused on cellular and mitochondrial (dys)function in two atypical cases: an antidepressant non-responding MDD patient ("Non-R") and another with an unexplained mitochondrial disorder ("Mito"). Skin biopsies from these patients and controls were used to generate various cell types, including astrocytes and neurons, and cellular and mitochondrial functions were analyzed. Similarities were observed between the Mito patient and a broader MDD cohort, including decreased respiration and mitochondrial function. Conversely, the Non-R patient exhibited increased respiratory rates, mitochondrial calcium, and resting membrane potential. In conclusion, the Non-R patient's data offered a new perspective on MDD, suggesting a detrimental imbalance in mitochondrial and cellular processes, rather than simply reduced functions. Meanwhile, the Mito patient's data revealed the extensive effects of mitochondrial dysfunctions on cellular functions, potentially highlighting new MDD-associated impairments. Together, these case studies enhance our comprehension of MDD.


Assuntos
Caricaceae , Transtorno Depressivo Maior , Humanos , Astrócitos , Depressão , Mitocôndrias , Neurônios , Fibroblastos , Mitomicina
3.
J Transl Med ; 21(1): 444, 2023 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-37415222

RESUMO

BACKGROUND: Animal models representing different molecular subtypes of glioblastoma multiforme (GBM) is desired for developing new therapies. SVV-001 is an oncolytic virus selectively targeting cancer cells. It's capacity of passing through the blood brain barrier makes is an attractive novel approach for GBM. MATERIALS AND METHODS: 23 patient tumor samples were implanted into the brains of NOD/SCID mice (1 × 105 cells/mouse). Tumor histology, gene expression (RNAseq), and growth rate of the developed patient-derived orthotopic xenograft (PDOX) models were compared with the originating patient tumors during serial subtransplantations. Anti-tumor activities of SVV-001 were examined in vivo; and therapeutic efficacy validated in vivo via single i.v. injection (1 × 1011 viral particle) with or without fractionated (2 Gy/day x 5 days) radiation followed by analysis of animal survival times, viral infection, and DNA damage. RESULTS: PDOX formation was confirmed in 17/23 (73.9%) GBMs while maintaining key histopathological features and diffuse invasion of the patient tumors. Using differentially expressed genes, we subclassified PDOX models into proneural, classic and mesenchymal groups. Animal survival times were inversely correlated with the implanted tumor cells. SVV-001 was active in vitro by killing primary monolayer culture (4/13 models), 3D neurospheres (7/13 models) and glioma stem cells. In 2/2 models, SVV-001 infected PDOX cells in vivo without harming normal brain cells and significantly prolonged survival times in 2/2 models. When combined with radiation, SVV-001 enhanced DNA damages and further prolonged animal survival times. CONCLUSION: A panel of 17 clinically relevant and molecularly annotated PDOX modes of GBM is developed, and SVV-001 exhibited strong anti-tumor activities in vitro and in vivo.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Terapia Viral Oncolítica , Vírus Oncolíticos , Humanos , Animais , Camundongos , Glioblastoma/radioterapia , Glioblastoma/metabolismo , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Animais de Doenças , Linhagem Celular Tumoral
4.
Proc Natl Acad Sci U S A ; 117(43): 26766-26772, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33051299

RESUMO

Archaea swim using the archaellum (archaeal flagellum), a reversible rotary motor consisting of a torque-generating motor and a helical filament, which acts as a propeller. Unlike the bacterial flagellar motor (BFM), ATP (adenosine-5'-triphosphate) hydrolysis probably drives both motor rotation and filamentous assembly in the archaellum. However, direct evidence is still lacking due to the lack of a versatile model system. Here, we present a membrane-permeabilized ghost system that enables the manipulation of intracellular contents, analogous to the triton model in eukaryotic flagella and gliding Mycoplasma We observed high nucleotide selectivity for ATP driving motor rotation, negative cooperativity in ATP hydrolysis, and the energetic requirement for at least 12 ATP molecules to be hydrolyzed per revolution of the motor. The response regulator CheY increased motor switching from counterclockwise (CCW) to clockwise (CW) rotation. Finally, we constructed the torque-speed curve at various [ATP]s and discuss rotary models in which the archaellum has characteristics of both the BFM and F1-ATPase. Because archaea share similar cell division and chemotaxis machinery with other domains of life, our ghost model will be an important tool for the exploration of the universality, diversity, and evolution of biomolecular machinery.


Assuntos
Membrana Celular , Quimiotaxia/fisiologia , Haloferax volcanii , Modelos Biológicos , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Flagelos/química , Flagelos/metabolismo , Haloferax volcanii/citologia , Haloferax volcanii/metabolismo , Cinética , Proteínas Quimiotáticas Aceptoras de Metil/química , Proteínas Quimiotáticas Aceptoras de Metil/metabolismo , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo
5.
Lab Invest ; 102(2): 185-193, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34802040

RESUMO

Brain tumors are the leading cause of cancer-related death in children. Tazemetostat is an FDA-approved enhancer of zeste homolog (EZH2) inhibitor. To determine its role in difficult-to-treat pediatric brain tumors, we examined EZH2 levels in a panel of 22 PDOX models and confirmed EZH2 mRNA over-expression in 9 GBM (34.6 ± 12.7-fold) and 11 medulloblastoma models (6.2 ± 1.7 in group 3, 6.0 ± 2.4 in group 4) accompanied by elevated H3K27me3 expression. Therapeutic efficacy was evaluated in 4 models (1 GBM, 2 medulloblastomas and 1 ATRT) via systematically administered tazemetostat (250 and 400 mg/kg, gavaged, twice daily) alone and in combination with cisplatin (5 mg/kg, i.p., twice) and/or radiation (2 Gy/day × 5 days). Compared with the untreated controls, tazemetostat significantly (Pcorrected < 0.05) prolonged survival times in IC-L1115ATRT (101% at 400 mg/kg) and IC-2305GBM (32% at 250 mg/kg, 45% at 400 mg/kg) in a dose-dependent manner. The addition of tazemetostat with radiation was evaluated in 3 models, with only one [IC-1078MB (group 4)] showing a substantial, though not statistically significant, prolongation in survival compared to radiation treatment alone. Combining tazemetostat (250 mg/kg) with cisplatin was not superior to cisplatin alone in any model. Analysis of in vivo drug resistance detected predominance of EZH2-negative cells in the remnant PDOX tumors accompanied by decreased H3K27me2 and H3K27me3 expressions. These data supported the use of tazemetostat in a subset of pediatric brain tumors and suggests that EZH2-negative tumor cells may have caused therapy resistance and should be prioritized for the search of new therapeutic targets.


Assuntos
Neoplasias Encefálicas/terapia , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Adolescente , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzamidas/administração & dosagem , Benzamidas/farmacologia , Compostos de Bifenilo/administração & dosagem , Compostos de Bifenilo/farmacologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Quimiorradioterapia , Criança , Cisplatino/administração & dosagem , Terapia Combinada/métodos , Avaliação Pré-Clínica de Medicamentos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Inibidores Enzimáticos/administração & dosagem , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Lactente , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Morfolinas/administração & dosagem , Morfolinas/farmacologia , Piridonas/administração & dosagem , Piridonas/farmacologia , Dosagem Radioterapêutica
6.
Bioprocess Biosyst Eng ; 40(10): 1519-1527, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28656375

RESUMO

The monitoring of microbiological processes using Raman spectroscopy has gained in importance over the past few years. Commercial Raman spectroscopic equipment consists of a laser, spectrometer, and fiberoptic immersion probe in direct contact with the fermentation medium. To avoid possible sterilization problems and biofilm formation on the probe tip, a large-aperture Raman probe was developed. The design of the probe enables non-contact in-line measurements through glass vessels or inspection glasses of bioreactors and chemical reactors. The practical applicability of the probe was tested during yeast fermentations by monitoring the consumption of substrate glucose and the formation of ethanol as the product. Multiple linear regression models were applied to evaluate the Raman spectra. Reference values were determined by high-performance liquid chromatography. The relative errors of prediction for glucose and ethanol were 5 and 3%, respectively. The presented Raman probe allows simple adaption to a wide range of processes in the chemical, pharmaceutical, and biotechnological industries.


Assuntos
Etanol/metabolismo , Fermentação , Glucose/análise , Saccharomyces cerevisiae/crescimento & desenvolvimento , Análise Espectral Raman/métodos , Glucose/metabolismo
7.
Anal Chem ; 88(19): 9368-9374, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27603732

RESUMO

In process analytics, the applicability of Raman spectroscopy is restricted by high excitation intensities or the long integration times required. In this work, a novel Raman system was developed to minimize photon flux losses. It allows specific reduction of spectral resolution to enable the use of Raman spectroscopy for real-time analytics when strongly increased sensitivity is required. The performance potential of the optical setup was demonstrated in two exemplary applications: First, a fast exothermic reaction (Michael addition) was monitored with backscattering fiber optics under strongly attenuated laser power (7 mW). Second, high-speed scanning of a segmented multiphase flow (water/toluene) with submicroliter droplets was achieved by aligning the focus of a coaxial Raman probe with long focal length directly into a perfluoroalkoxy (PFA) capillary. With an acquisition rate of 333 Raman spectra per second, chemical information was obtained separately for both of the rapidly alternating phases. The experiment with reduced laser power demonstrates that the technique described in this paper is applicable in chemical production processes, especially in hazardous environments. Further potential uses can be envisioned in medical or biological applications with limited power input. The realization of high-speed measurements shows new possibilities for analysis of heterogeneous phase systems and of fast reactions or processes.

8.
J Neurooncol ; 128(1): 47-56, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26956263

RESUMO

Metastatic intracranial germinoma is difficult to treat. Although the proto-oncogene KIT is recognized as one of the most frequent genetic abnormalities in CNS germinoma, the development of new target therapeutic agents for CNS germinoma is hampered by the lack of clinically-relevant animal models that replicate the mutated or over-expressed KIT. CNS germinoma tumor cells from five pediatric patients were directly implanted into the brains of Rag2/severe combined immune deficiency mice. Once established, the xenograft tumors were sub-transplanted in vivo in mouse brains. Characterization of xenograft tumors were performed through histologic and immunohistochemical staining, and KIT mutation analysed with quantitative pyro-sequencing. Expression of putative cancer stem cell markers (CD133, CD15, CD24, CD44, CD49f) was analyzed through flow cytometry. Two patient-derived orthotopic xenograft (PDOX) models (IC-6999GCT and IC-9302GCT) were established from metastatic germinoma and serially sub-transplanted five times in mouse brains. Similar to the original patient tumors, they both exhibited faint expression (+) of PLAP, no expression (-) of ß-HCG and strong (+++) expression of KIT. KIT mutation (D816H), however, was only found in IC-9320GCT. This mutation was maintained during the five in vivo tumor passages with an increased mutant allele frequency compared to the patient tumor. Expression of putative cancer stem cell markers CD49f and CD15 was also detected in a small population of tumor cells in both models. This new pair of PDOX models replicated the key biological features of pediatric intracranial germinoma and should facilitate the biological and pre-clinical studies for metastatic intracranial germinomas.


Assuntos
Neoplasias Encefálicas/genética , Germinoma/genética , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-kit/genética , Adolescente , Animais , Biomarcadores Tumorais/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Criança , Feminino , Germinoma/metabolismo , Germinoma/patologia , Xenoenxertos , Humanos , Imuno-Histoquímica , Lactente , Masculino , Camundongos SCID , Metástase Neoplásica , Células-Tronco Neoplásicas , Proto-Oncogene Mas , Análise de Sequência de DNA , Análise de Sobrevida
9.
Blood ; 121(23): 4729-39, 2013 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-23599269

RESUMO

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL-Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target.


Assuntos
Apoptose , Linfócitos B/patologia , Membrana Celular/metabolismo , Proteína Ligante Fas/metabolismo , Linfoma de Células B/patologia , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos B/metabolismo , Western Blotting , Proliferação de Células , Citometria de Fluxo , Humanos , Imunoprecipitação , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fosfoproteínas/genética , Ligação Proteica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Células Tumorais Cultivadas , Nucleolina
10.
Br J Haematol ; 167(2): 207-13, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25040450

RESUMO

(90) Y-ibritumomab-tiuxetan ((90) YIT) was used as a first-line therapy for patients with early-stage follicular lymphoma (FL) or marginal zone B-cell lymphoma (MZL). Thirty-one patients were treated, with an overall 3-month response rate of 100% (68% complete response, 29% unconfirmed complete response and 3% partial response). At a median follow-up of 56 months, ten patients (32%) had disease relapse or progression. The progression-free rates at 3 and 5 years were lower in males, patients with FL, stage II disease and non-bulky disease, although they did not reach statistical significance. Grade 3-4 neutropenia, thrombocytopenia and anaemia were 61%, 35%, and 3%, respectively. (90) YIT was well tolerated, including in those patients over 60 years old, and achieved high response rates in patients with early-stage low-grade B-cell lymphomas. Bulky disease did not adversely affect tumour response.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfoma de Zona Marginal Tipo Células B/radioterapia , Linfoma Folicular/radioterapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/efeitos adversos , Progressão da Doença , Feminino , Seguimentos , Doenças Hematológicas/etiologia , Humanos , Linfoma de Zona Marginal Tipo Células B/patologia , Linfoma Folicular/patologia , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Lesões por Radiação/etiologia , Radioimunoterapia/efeitos adversos , Radioimunoterapia/métodos , Análise de Sobrevida , Resultado do Tratamento , Radioisótopos de Ítrio/efeitos adversos , Radioisótopos de Ítrio/uso terapêutico
11.
Acta Biochim Biophys Sin (Shanghai) ; 46(9): 774-81, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25079679

RESUMO

Non-steroid anti-inflammatory drugs (NSAIDs) are generally used in the treatment of inflammation and pain through cyclooxygenase (COX) inhibition. Mounting evidence has indicated additional COX-independent targets for NSAIDs including acid-sensing ion channels (ASICs) 1a and 3. However, detailed function and mechanism of ASICs still remain largely elusive. In this study, the impact of NSAIDs on ASICs in nucleus pulposus cells of the human intervertebral disk was investigated. Nucleus pulposus cells were isolated and cultured from protruded disk tissues of 40 patients. It was shown that ASIC1a and ASIC3 were expressed and functional in these cells by analyzing proton-gated currents after ASIC inhibition. We further investigated the neuroprotective capacity of ibuprofen (a COX inhibitor), psalmotoxin-1 (PcTX1, a tarantula toxin specific for homomeric ASIC1a), and amiloride (a classic inhibitor of the epithelial sodium channel ENaC/DEG family to which ASICs belong). PcTX1-containing venom has been shown to be comparable with amiloride in its neuroprotective features in rodent models of ischemia. Taken together, our data showed that amiloride, PcTX1, and ibuprofen decreased ASIC protein expression and thereby exerted protective effects from ASIC inhibition-mediated cell damage.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Disco Intervertebral/efeitos dos fármacos , Sequência de Bases , Primers do DNA , Humanos , Ibuprofeno/farmacologia , Disco Intervertebral/metabolismo , Reação em Cadeia da Polimerase
12.
Front Immunol ; 15: 1388769, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38726003

RESUMO

Background: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Methods: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. Results: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Conclusion: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.


Assuntos
Vesículas Extracelulares , Glioblastoma , MicroRNAs , Organoides , Microambiente Tumoral , Humanos , Glioblastoma/imunologia , Glioblastoma/patologia , Glioblastoma/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Organoides/imunologia , MicroRNAs/genética , Microambiente Tumoral/imunologia , Transdução de Sinais , Células Tumorais Cultivadas , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Linhagem Celular Tumoral , Técnicas de Cultura de Células em Três Dimensões/métodos
13.
Cancers (Basel) ; 16(9)2024 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-38730671

RESUMO

Background: Despite multimodality therapies, the prognosis of patients with malignant brain tumors remains extremely poor. One of the major obstacles that hinders development of effective therapies is the limited availability of clinically relevant and biologically accurate (CRBA) mouse models. Methods: We have developed a freehand surgical technique that allows for rapid and safe injection of fresh human brain tumor specimens directly into the matching locations (cerebrum, cerebellum, or brainstem) in the brains of SCID mice. Results: Using this technique, we successfully developed 188 PDOX models from 408 brain tumor patient samples (both high-and low-grade) with a success rate of 72.3% in high-grade glioma, 64.2% in medulloblastoma, 50% in ATRT, 33.8% in ependymoma, and 11.6% in low-grade gliomas. Detailed characterization confirmed their replication of the histopathological and genetic abnormalities of the original patient tumors. Conclusions: The protocol is easy to follow, without a sterotactic frame, in order to generate large cohorts of tumor-bearing mice to meet the needs of biological studies and preclinical drug testing.

14.
Microlife ; 4: uqad027, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37305433

RESUMO

Second messengers transfer signals from changing intra- and extracellular conditions to a cellular response. Over the last few decades, several nucleotide-based second messengers have been identified and characterized in especially bacteria and eukaryotes. Also in archaea, several nucleotide-based second messengers have been identified. This review will summarize our understanding of nucleotide-based second messengers in archaea. For some of the nucleotide-based second messengers, like cyclic di-AMP and cyclic oligoadenylates, their roles in archaea have become clear. Cyclic di-AMP plays a similar role in osmoregulation in euryarchaea as in bacteria, and cyclic oligoadenylates are important in the Type III CRISPR-Cas response to activate CRISPR ancillary proteins involved in antiviral defense. Other putative nucleotide-based second messengers, like 3',5'- and 2',3'-cyclic mononucleotides and adenine dinucleotides, have been identified in archaea, but their synthesis and degradation pathways, as well as their functions as secondary messengers, still remain to be demonstrated. In contrast, 3'-3'-cGAMP has not yet been identified in archaea, but the enzymes required to synthesize 3'-3'-cGAMP have been found in several euryarchaeotes. Finally, the widely distributed bacterial second messengers, cyclic diguanosine monophosphate and guanosine (penta-)/tetraphosphate, do not appear to be present in archaea.

15.
Cells ; 12(14)2023 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-37508520

RESUMO

Conventional 2D cultures are commonly used in cancer research though they come with limitations such as the lack of microenvironment or reduced cell heterogeneity. In this study, we investigated in what respect a scaffold-based (Matrigel™) 3D culture technique can ameliorate the limitations of 2D cultures. NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in around 36% of 3D models, indicating the reoccurrence of an immune suppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. We also investigated the 3D tumor organoids in a co-culture setting with tumor-infiltrating lymphocytes (TILs). Of note, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release, as well as live cell staining and proliferation analysis, pointed toward an elevated resistance of 3D models. In conclusion, we found that the scaffold-based (Matrigel™) 3D culture technique affects the transcriptional profile in a subset of GBM models. Thus, these models allow for depicting clinically relevant aspects of tumor-immune interaction, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.


Assuntos
Glioblastoma , Humanos , Glioblastoma/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Imunossupressores/uso terapêutico , Fenótipo , Microambiente Tumoral
16.
Acta Neuropathol Commun ; 11(1): 147, 2023 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-37697350

RESUMO

TSPO is a promising novel tracer target for positron-emission tomography (PET) imaging of brain tumors. However, due to the heterogeneity of cell populations that contribute to the TSPO-PET signal, imaging interpretation may be challenging. We therefore evaluated TSPO enrichment/expression in connection with its underlying histopathological and molecular features in gliomas. We analyzed TSPO expression and its regulatory mechanisms in large in silico datasets and by performing direct bisulfite sequencing of the TSPO promotor. In glioblastoma tissue samples of our TSPO-PET imaging study cohort, we dissected the association of TSPO tracer enrichment and protein labeling with the expression of cell lineage markers by immunohistochemistry and fluorescence multiplex stains. Furthermore, we identified relevant TSPO-associated signaling pathways by RNA sequencing.We found that TSPO expression is associated with prognostically unfavorable glioma phenotypes and that TSPO promotor hypermethylation is linked to IDH mutation. Careful histological analysis revealed that TSPO immunohistochemistry correlates with the TSPO-PET signal and that TSPO is expressed by diverse cell populations. While tumor core areas are the major contributor to the overall TSPO signal, TSPO signals in the tumor rim are mainly driven by CD68-positive microglia/macrophages. Molecularly, high TSPO expression marks prognostically unfavorable glioblastoma cell subpopulations characterized by an enrichment of mesenchymal gene sets and higher amounts of tumor-associated macrophages.In conclusion, our study improves the understanding of TSPO as an imaging marker in gliomas by unveiling IDH-dependent differences in TSPO expression/regulation, regional heterogeneity of the TSPO PET signal and functional implications of TSPO in terms of tumor immune cell interactions.


Assuntos
Glioblastoma , Glioma , Células-Tronco Mesenquimais , Humanos , Glioblastoma/diagnóstico por imagem , Glioblastoma/genética , Macrófagos Associados a Tumor , Macrófagos , Receptores de GABA/genética
17.
Acta Neuropathol Commun ; 11(1): 75, 2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-37158962

RESUMO

Glioblastoma (GB) IDH-wildtype is the most malignant primary brain tumor. It is particularly resistant to current immunotherapies. Translocator protein 18 kDa (TSPO) is upregulated in GB and correlates with malignancy and poor prognosis, but also with increased immune infiltration. Here, we studied the role of TSPO in the regulation of immune resistance of human GB cells. The role of TSPO in tumor immune resistance was experimentally determined in primary brain tumor initiating cells (BTICs) and cell lines through genetic manipulation of TSPO expression and subsequent cocultures with antigen specific cytotoxic T cells and autologous tumor-infiltrating T cells. Death inducing intrinsic and extrinsic apoptotic pathways affected by TSPO were investigated. TSPO-regulated genes mediating apoptosis resistance in BTICs were identified through gene expression analysis and subsequent functional analyses. TSPO transcription in primary GB cells correlated with CD8+ T cell infiltration, cytotoxic activity of T cell infiltrate, expression of TNFR and IFNGR and with the activity of their downstream signalling pathways, as well as with the expression of TRAIL receptors. Coculture of BTICs with tumor reactive cytotoxic T cells or with T cell-derived factors induced TSPO up-regulation through T cell derived TNFα and IFNγ. Silencing of TSPO sensitized BTICs against T cell-mediated cytotoxicity. TSPO selectively protected BTICs against TRAIL-induced apoptosis by regulating apoptosis pathways. TSPO also regulated the expression of multiple genes associated with resistance against apoptosis. We conclude that TSPO expression in GB is induced through T cell-derived cytokines TNFα and IFNγ and that TSPO expression protects GB cells against cytotoxic T cell attack through TRAIL. Our data thereby provide an indication that therapeutic targeting of TSPO may be a suitable approach to sensitize GB to immune cell-mediated cytotoxicity by circumventing tumor intrinsic TRAIL resistance.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/genética , Fator de Necrose Tumoral alfa , Encéfalo , Linfócitos T CD8-Positivos , Neoplasias Encefálicas/genética , Receptores de GABA/genética
18.
Transl Oncol ; 18: 101368, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35182954

RESUMO

Clinical outcomes in patients with WHO grade II/III astrocytoma, oligodendroglioma or secondary glioblastoma remain poor. Isocitrate dehydrogenase 1 (IDH1) is mutated in > 70% of these tumors, making it an attractive therapeutic target. To determine the efficacy of our newly developed mutant IDH1 inhibitor, SYC-435 (1-hydroxypyridin-2-one), we treated orthotopic glioma xenograft model (IC-BT142AOA) carrying R132H mutation and our newly established orthotopic patient-derived xenograft (PDX) model of recurrent anaplastic oligoastrocytoma (IC-V0914AOA) bearing R132C mutation. In addition to suppressing IDH1 mutant cell proliferation in vitro, SYC-435 (15 mg/kg, daily x 28 days) synergistically prolonged animal survival times with standard therapies (Temozolomide + fractionated radiation) mediated by reduction of H3K4/H3K9 methylation and expression of mitochondrial DNA (mtDNA)-encoded molecules. Furthermore, RNA-seq of the remnant tumors identified genes (MYO1F, CTC1 and BCL9) and pathways (base excision repair, TCA cycle II, sirtuin signaling, protein kinase A, eukaryotic initiation factor 2 and α-adrenergic signaling) as mediators of therapy resistance. Our data demonstrated the efficacy SYC-435 in targeting IDH1 mutant gliomas when combined with standard therapy and identified a novel set of genes that should be prioritized for future studies to overcome SYC-435 resistance.

19.
Nat Commun ; 13(1): 6689, 2022 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-36335125

RESUMO

Recurrence is frequent in pediatric ependymoma (EPN). Our longitudinal integrated analysis of 30 patient-matched repeated relapses (3.67 ± 1.76 times) over 13 years (5.8 ± 3.8) reveals stable molecular subtypes (RELA and PFA) and convergent DNA methylation reprogramming during serial relapses accompanied by increased orthotopic patient derived xenograft (PDX) (13/27) formation in the late recurrences. A set of differentially methylated CpGs (DMCs) and DNA methylation regions (DMRs) are found to persist in primary and relapse tumors (potential driver DMCs) and are acquired exclusively in the relapses (potential booster DMCs). Integrating with RNAseq reveals differentially expressed genes regulated by potential driver DMRs (CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and potential booster DMRs (PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). DMCs predicators of relapse are also identified in the primary tumors. This study provides a high-resolution epigenetic roadmap of serial EPN relapses and 13 orthotopic PDX models to facilitate biological and preclinical studies.


Assuntos
Ependimoma , Simportadores , Humanos , Criança , Ependimoma/genética , Ependimoma/patologia , Metilação de DNA/genética , Recidiva , Epigênese Genética , Simportadores/genética
20.
Front Microbiol ; 12: 779012, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34880846

RESUMO

Research on nucleotide-based second messengers began in 1956 with the discovery of cyclic adenosine monophosphate (3',5'-cAMP) by Earl Wilbur Sutherland and his co-workers. Since then, a broad variety of different signaling molecules composed of nucleotides has been discovered. These molecules fulfill crucial tasks in the context of intracellular signal transduction. The vast majority of the currently available knowledge about nucleotide-based second messengers originates from model organisms belonging either to the domain of eukaryotes or to the domain of bacteria, while the archaeal domain is significantly underrepresented in the field of nucleotide-based second messenger research. For several well-stablished eukaryotic and/or bacterial nucleotide-based second messengers, it is currently not clear whether these signaling molecules are present in archaea. In order to shed some light on this issue, this study analyzed cell extracts of two major archaeal model organisms, the euryarchaeon Haloferax volcanii and the crenarchaeon Sulfolobus acidocaldarius, using a modern mass spectrometry method to detect a broad variety of currently known nucleotide-based second messengers. The nucleotides 3',5'-cAMP, cyclic guanosine monophosphate (3',5'-cGMP), 5'-phosphoadenylyl-3',5'-adenosine (5'-pApA), diadenosine tetraphosphate (Ap4A) as well as the 2',3'-cyclic isomers of all four RNA building blocks (2',3'-cNMPs) were present in both species. In addition, H. volcanii cell extracts also contain cyclic cytosine monophosphate (3',5'-cCMP), cyclic uridine monophosphate (3',5'-cUMP) and cyclic diadenosine monophosphate (3',5'-c-di-AMP). The widely distributed bacterial second messengers cyclic diguanosine monophosphate (3',5'-c-di-GMP) and guanosine (penta-)/tetraphosphate [(p)ppGpp] could not be detected. In summary, this study gives a comprehensive overview on the presence of a large set of currently established or putative nucleotide-based second messengers in an eury- and a crenarchaeal model organism.

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