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Stem cell transplant recipients (SCTR) are imperiled to increased risks after SARS-CoV2 infection, supporting the need for effective vaccination strategies for this vulnerable group. With respect to pediatric patients, data on immunogenicity of SARS-CoV2 mRNA-based vaccination is limited. We therefore comprehensively examined specific humoral, B- and T cell responses in a cohort of 2-19 year old SCTR after the second and third vaccine dose. Only after booster vaccination, transplant recipients reached similar levels of vaccine-specific IgG, IgA and neutralizing antibodies against omicron variant as age-matched controls. Although frequencies of SARS-CoV2 specific B cells increased after the third dose, they were still fourfold reduced in patients compared to controls. Overall, the majority of individuals enrolled mounted SARS-CoV2 Spike protein-specific CD4+ T helper cell responses with patients showing significantly higher portions than controls after the third dose. With respect to functionality, however, SCTR were characterized by reduced frequencies of specific interferon gamma producing CD4+ T cells, along with an increase in IL-2 producers. In summary, our data identify distinct quantitative and qualitative impairments within the SARS-CoV2 vaccination specific B- and CD4+ T cell compartments. More importantly, humoral analyses highlight the need for a booster vaccination of SCTR particularly for development of neutralizing antibodies.
Assuntos
COVID-19 , RNA Viral , Humanos , Criança , Pré-Escolar , Adolescente , Adulto Jovem , Adulto , Transplantados , COVID-19/prevenção & controle , SARS-CoV-2 , Vacinas Sintéticas , Anticorpos Neutralizantes , Transplante de Células-Tronco , Vacinas de mRNARESUMO
Circular RNAs (circRNAs) are a regulatory RNA class. While cancer-driving functions have been identified for single circRNAs, how they modulate gene expression in cancer is not well understood. We investigate circRNA expression in the pediatric malignancy, neuroblastoma, through deep whole-transcriptome sequencing in 104 primary neuroblastomas covering all risk groups. We demonstrate that MYCN amplification, which defines a subset of high-risk cases, causes globally suppressed circRNA biogenesis directly dependent on the DHX9 RNA helicase. We detect similar mechanisms in shaping circRNA expression in the pediatric cancer medulloblastoma implying a general MYCN effect. Comparisons to other cancers identify 25 circRNAs that are specifically upregulated in neuroblastoma, including circARID1A. Transcribed from the ARID1A tumor suppressor gene, circARID1A promotes cell growth and survival, mediated by direct interaction with the KHSRP RNA-binding protein. Our study highlights the importance of MYCN regulating circRNAs in cancer and identifies molecular mechanisms, which explain their contribution to neuroblastoma pathogenesis.
Assuntos
Neuroblastoma , RNA Circular , Criança , Humanos , RNA Circular/genética , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Linhagem Celular Tumoral , RNA/genética , RNA/metabolismo , Neuroblastoma/metabolismo , Regulação Neoplásica da Expressão GênicaRESUMO
BACKGROUND: Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. METHODS: Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. RESULTS: Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. CONCLUSIONS: Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.
Assuntos
Neuroblastoma , Medicina de Precisão , Quinase do Linfoma Anaplásico/genética , Linhagem Celular Tumoral , Criança , Humanos , Mutação , Neuroblastoma/tratamento farmacológico , Neuroblastoma/genética , Neuroblastoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de SinaisRESUMO
Chimeric antigen receptor (CAR) T cell therapy has emerged as a promising treatment strategy, however, therapeutic success against solid tumors such as neuroblastoma remains modest. Recurrence of antigen-poor tumor variants often ultimately results in treatment failure. Using antigen-independent killing mechanisms such as the FAS receptor (FAS)-FAS ligand (FASL) axis through epigenetic manipulation may be a way to counteract the escape achieved by antigen downregulation. Analysis of public RNA-sequencing data from primary neuroblastomas revealed that a particular epigenetic modifier, the histone lysine demethylase 1A (KDM1A), correlated negatively with FAS expression. KDM1A is known to interact with TP53 to repress TP53-mediated transcriptional activation of genes, including FAS. We showed that pharmacologically blocking KDM1A activity in neuroblastoma cells with the small molecule inhibitor, SP-2509, increased FAS cell-surface expression in a strictly TP53-dependent manner. FAS upregulation sensitized neuroblastoma cells to FAS-FASL-dependent killing and augmented L1CAM-directed CAR T cell therapy against antigen-poor or even antigen-negative tumor cells in vitro. The improved therapeutic response was abrogated when the FAS-FASL interaction was abolished with an antagonistic FAS antibody. Our results show that KDM1A inhibition unleashes an antigen-independent killing mechanism via the FAS-FASL axis to make tumor cell variants that partially or totally suppress antigen expression susceptible to CAR T cell therapy.
RESUMO
Chimeric antigen receptor (CAR) T cell performance against solid tumors in mouse models and clinical trials is often less effective than predicted by CAR construct selection in two-dimensional (2D) cocultures. Three-dimensional (3D) solid tumor architecture is likely to be crucial for CAR T cell efficacy. We used a three-dimensional (3D) bioprinting approach for large-scale generation of highly reproducible 3D human tumor models for the test case, neuroblastoma, and compared these to 2D cocultures for evaluation of CAR T cells targeting the L1 cell adhesion molecule, L1CAM. CAR T cells infiltrated the model, and both CAR T and tumor cells were viable for long-term experiments and could be isolated as single-cell suspensions for whole-cell assays quantifying CAR T cell activation, effector function and tumor cell cytotoxicity. L1CAM-specific CAR T cell activation by neuroblastoma cells was stronger in the 3D model than in 2D cocultures, but neuroblastoma cell lysis was lower. The bioprinted 3D neuroblastoma model is highly reproducible and allows detection and quantification of CAR T cell tumor infiltration, representing a superior in vitro analysis tool for preclinical CAR T cell characterization likely to better select CAR T cells for in vivo performance than 2D cocultures.
Assuntos
Bioimpressão , Imunoterapia Adotiva , Neuroblastoma/terapia , Impressão Tridimensional , Receptores de Antígenos Quiméricos/genética , Linfócitos T/transplante , Linhagem Celular Tumoral , Técnicas de Cocultura , Citotoxicidade Imunológica , Humanos , Ativação Linfocitária , Neuroblastoma/genética , Neuroblastoma/imunologia , Neuroblastoma/patologia , Linfócitos T/imunologia , Fatores de TempoRESUMO
A polyphasic approach was applied to investigate the diversity of microbiota that evolved during cold storage beef ripening. Isolate V4/DAB/S4/2aT with a unique BOX-rep-PCR fingerprint profile revealed more than 99â% nucleotide identities upon pairwise comparisons of 16S rDNA sequences from the type strains Pseudomonas versuta DSM 101070T, Pseudomonas saxonica DSM 108989T, Pseudomonas deceptionensis DSM 26521T and Pseudomonas weihenstephanensis DSM 29166T, placing it within the Pseudomonas fragi / lundensis branch of the genus Pseudomonas. Additional rpoB based comparison revealed P. versuta DSM 101070T as the nearest relative, with 98.5â% nucleotide identity. Calculation of ANIb values of the V4/DAB/S4/2aT draft genome identified P. versuta DSM 101070T with 90.1â%, P. deceptionensis DSM 26521T with 85.1â%, P. fragi DSM 3456T with 84.4â%, Pseudomonas psychrophila DSM 17535T and Pseudomonas bubulae DSM 107389T with 84.2â% similarities each. Pairwise genome-to-genome distance calculations [digital DNA-DNA hybridization (dDDH)] resulted in values of 47.1, 35.1, 34.8, 34.2 and 34.1â%, respectively. A second isolate was detected years later in ground beef and showed ANIb values of 99.3â% and dDDH of 96.1â% relatedness to V4/DAB/S4/2aT. The DNA G+C content was 58.6 mol% for both isolates. The predominant cellular fatty acids of V4/DAB/S4/2aT were C16â:â0, C18â:â1ω7c, C17â:â0 cyclo and a summed feature containing C16â:â1ω7c and/or C15â:â0 iso 2-OH. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, the major respiratory quinone was Q9, with a small portion of Q8. The combined data on genotypic and phenotypic features support the proposal of a novel species, for which the name Pseudomonas paraversuta sp. nov. is proposed. The type strain is V4/DAB/S4/2aT (=DSM 111361T=LMG 31844T) and a second isolate is UBT376 (=DSM 111360=LMG 31845).
Assuntos
Microbiologia de Alimentos , Filogenia , Pseudomonas/classificação , Carne Vermelha/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Bovinos , DNA Bacteriano/genética , Ácidos Graxos/química , Genes Bacterianos , Alemanha , Hibridização de Ácido Nucleico , Fosfolipídeos/química , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/químicaRESUMO
Spacer or co-stimulatory components in chimeric antigen receptor (CAR) design influence CAR T cell effector function. Few preclinical mouse models optimally support CAR candidate pre-selection for clinical development. Here we use a model in which murine CAR T cells can be exploited with human tumor xenografts. This mouse-in-mouse approach avoids limitations caused by species-specific factors crucial for CAR T cell survival, trafficking and function. We compared trafficking, expansion and tumor control for T cells expressing different CAR construct designs targeting two antigens (L1CAM or HER2), structurally identical except for spacer (long or short) or co-stimulatory (4-1BB or CD28) domains to be evaluated. Using monoclonal, murine-derived L1CAM-specific CAR T cells in Rag-/- mice harboring established xenografted tumors from a human neuroblastoma cell line revealed a clear superiority in CAR T cell trafficking using CD28 co-stimulation. L1CAM-targeting short spacer-CD28/ζ CAR T cells expanded the most at the tumor site and induced initial tumor regression. Treating patient-derived neuroblastoma xenografts with human L1CAM-targeting CAR T cells confirmed the superiority of CD28 co-stimulus. CD28 superiority was also demonstrated with HER2-specific CAR T cells (targeting ovarian carcinoma xenografts). Our findings encourage incorporating CD28 signaling into CAR design for adoptive T cell treatment of solid tumors.
RESUMO
During a project focusing on the diversity of meat microbiota associated with beef ripening, a Pseudomonas strain was isolated exhibiting high 16S rRNA gene sequence similarities (>99â%) to Pseudomonas carnis DSM 107652T, P. lactis DSM 29167T, P. paralactis DSM 29164T and P. azotoformans DSM 18862T. Phylogenetic analysis of the complete rpoB gene sequences of the isolate V5/DAB/2/5T indicated a separate branch with about 99.0â% nucleotide identities to the closest relatives P. carnis DSM 107652T, P. lactis DSM 29167T and P. paralactis DSM 29164T, while average nucleotide identities (ANIb) calculated from the draft genomes were 94.8, 94.2 and 90.2â%, respectively. Pairwise genome-to-genome distance calculations (GGDC) resulted in values of 67.7, 63.5 and 45.7â%, respectively, lying below the actual species demarcation line as well. A second isolate, UBT403, was detected some years later by using matrix-assisted laser desorption ionization-time of flight MS of the microbiota of minced beef. The fatty acid profile of V5/DAB/2/5T consisted of C16â:â0, summed feature C 16â:â1 ω7c/iso-C15â:â0 2-OH, C18â:â1 ω7c, C17â:â0 cyclo, C12â:â0, C12â:â0 3-OH, C10â:â0 3-OH and C12â:â0 2-OH. The major cellular lipids were aminopholipids, phospholipids, phosphatidylethanolamine and phosphatidylglycerol; the major quinone was Q9 with a minor proportion of Q8. Based on phenotypic and chemotaxonomic characterizations, the isolates can be considered as representing a novel species, for which the name Pseudomonas paracarnis sp. nov. is proposed. The type strain is V5/DAB/2/5T (=DSM 111363T=LMG 31846T); a second strain is UBT403 (=DSM 111362=LMG 31847).
Assuntos
Microbiologia de Alimentos , Filogenia , Pseudomonas/classificação , Carne Vermelha/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Bovinos , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Fosfolipídeos/química , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
Here we sought metabolic alterations specifically associated with MYCN amplification as nodes to indirectly target the MYCN oncogene. Liquid chromatography-mass spectrometry-based proteomics identified seven proteins consistently correlated with MYCN in proteomes from 49 neuroblastoma biopsies and 13 cell lines. Among these was phosphoglycerate dehydrogenase (PHGDH), the rate-limiting enzyme in de novo serine synthesis. MYCN associated with two regions in the PHGDH promoter, supporting transcriptional PHGDH regulation by MYCN. Pulsed stable isotope-resolved metabolomics utilizing 13 C-glucose labeling demonstrated higher de novo serine synthesis in MYCN-amplified cells compared to cells with diploid MYCN. An independence of MYCN-amplified cells from exogenous serine and glycine was demonstrated by serine and glycine starvation, which attenuated nucleotide pools and proliferation only in cells with diploid MYCN but did not diminish these endpoints in MYCN-amplified cells. Proliferation was attenuated in MYCN-amplified cells by CRISPR/Cas9-mediated PHGDH knockout or treatment with PHGDH small molecule inhibitors without affecting cell viability. PHGDH inhibitors administered as single-agent therapy to NOG mice harboring patient-derived MYCN-amplified neuroblastoma xenografts slowed tumor growth. However, combining a PHGDH inhibitor with the standard-of-care chemotherapy drug, cisplatin, revealed antagonism of chemotherapy efficacy in vivo. Emergence of chemotherapy resistance was confirmed in the genetic PHGDH knockout model in vitro. Altogether, PHGDH knockout or inhibition by small molecules consistently slows proliferation, but stops short of killing the cells, which then establish resistance to classical chemotherapy. Although PHGDH inhibition with small molecules has produced encouraging results in other preclinical cancer models, this approach has limited attractiveness for patients with neuroblastoma.
Assuntos
Amplificação de Genes , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/tratamento farmacológico , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Feminino , Glicina/metabolismo , Humanos , Camundongos , Neuroblastoma/genética , Serina/metabolismoRESUMO
Chimeric antigen receptor (CAR) T cell efficacy against solid tumors is currently limited by several immune escape mechanisms, which may include tumor-derived extracellular vesicles. Advanced neuroblastoma is an aggressive childhood tumor without curative treatment options for most relapsed patients today. We here evaluated the role of tumor-derived extracellular vesicles on the efficacy of CAR T cells targeting the neuroblastoma-specific antigen, CD171. For this purpose, CAR T cell activation, cytokine production, exhaustion, and tumor cell-directed cytotoxicity upon co-culture was evaluated. Tumor-derived extracellular vesicles isolated from SH-SY5Y neuroblastoma cells neither affected CAR T cell activation nor expression of inhibitory markers. Importantly, exposure of CD4+ CD171-specific CAR T cells to tumor-derived extracellular vesicles significantly impaired tumor cytotoxicity of CAR T cells. This effect was independent of neurotrophic receptor tyrosine kinases 1 or 2 (NTRK1, NTRK2) expression, which is known to impact immune responses against neuroblastoma. Our results demonstrate for the first time the impact of tumor-derived extracellular vesicles and non-cell-mediated tumor-suppressive effects on CD4+ CAR T cell efficacy in a preclinical setting. We conclude that these factors should be considered for any CAR T cell-based therapy to make CAR T cell therapy successful against solid tumors.
Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD4-Positivos/imunologia , Vesículas Extracelulares , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos/imunologia , Linhagem Celular Tumoral , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Humanos , Neuroblastoma/imunologiaRESUMO
The immunosuppressive microenvironment in solid tumors is thought to form a barrier to the entry and efficacy of cell-based therapies such as chimeric antigen receptor (CAR) T cells. Combining CAR T cell therapy with checkpoint inhibitors has been demonstrated to oppose immune escape mechanisms in solid tumors and augment antitumor efficacy. We evaluated PD-1/PD-L1 signaling capacity and the impact of an inhibitor of this checkpoint axis in an in vitro system for cancer cell challenge, the coculture of L1CAM-specific CAR T cells with neuroblastoma cell lines. Fluorescence-activated cell sorting-based analyses and luciferase reporter assays were used to assess PD-1/PD-L1 expression on CAR T and tumor cells as well as CAR T cell ability to kill neuroblastoma cells. Coculturing neuroblastoma cell lines with L1CAM-CAR T cells upregulated PD-L1 expression on neuroblastoma cells, confirming adaptive immune resistance. Exposure to neuroblastoma cells also upregulated the expression of the PD-1/PD-L1 axis in CAR T cells. The checkpoint inhibitor, nivolumab, enhanced L1CAM-CAR T cell-directed killing. However, nivolumab-enhanced L1CAM-CAR T cell killing did not strictly correlate with PD-L1 expression on neuroblastoma cells. In fact, checkpoint inhibitor success relied on strong PD-1/PD-L1 axis expression in the CAR T cells, which in turn depended on costimulatory domains within the CAR construct, and more importantly, on the subset of T cells selected for CAR T cell generation. Thus, T cell subset selection for CAR T cell generation and CAR T cell prescreening for PD-1/PD-L1 expression could help determine when combination therapy with checkpoint inhibitors could improve treatment efficacy.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Humanos , Neuroblastoma/metabolismo , Fenótipo , Receptor de Morte Celular Programada 1/metabolismo , Microambiente Tumoral/fisiologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
During investigations of spoilage-associated meat microbiota, Pseudomonas isolates were found in two different laboratories showing highest similarities to Pseudomonas lactis DSM 29167T, Pseudomonas paralactis DSM 29164T and Pseudomonas azotoformans DSM 18862T based on 16S rRNA gene sequence comparisons. Phylogenetic analysis of the complete rpoB gene sequences of isolates B4-1T and SpeckC indicated a separate branch with 99.0 and 99.1â% identity, respectively, to their closest relative (P. lactis DSM 29167T). Further phenotypic and chemotaxonomic characterizations, as well as average nucleotide identity (ANIb) values obtained from the draft genomes, revealed that these isolates could be considered as representing a novel species, with ANIb values of around 94 and 90â% with their closest relatives P. lactis and P. paralactis. Other related species showed ANIb values below 90â%, including Pseudomonas libanensis DSM 17149T, Pseudomonas synxantha DSM 18928T, Pseudomonas orientalis DSM 17489T, Pseudomonas veronii DSM 11331T and P. azotoformans DSM 18862T. Genome-to-genome distance calculations between B4-1T and its closest relative, P. lactis DSM 29167T, showed 62.6â% relatedness. The G+C contents of B4-1T and SpeckC were 59.8 and 59.9 mol%, respectively. The major cellular lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major quinone was Q9. Based on these data, the new species Pseudomonas carnis sp. nov. is proposed, the type strain is B4-1T (=DSM 107652T=LMG 30892T); a second strain is SpeckC (=DSM 107651=LMG 30893).
Assuntos
Microbiologia de Alimentos , Carne/microbiologia , Filogenia , Pseudomonas/classificação , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Genes Bacterianos , Alemanha , Hibridização de Ácido Nucleico , Fosfolipídeos/química , Aves Domésticas , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Suínos , Ubiquinona/químicaRESUMO
Extrachromosomal circularization of DNA is an important genomic feature in cancer. However, the structure, composition and genome-wide frequency of extrachromosomal circular DNA have not yet been profiled extensively. Here, we combine genomic and transcriptomic approaches to describe the landscape of extrachromosomal circular DNA in neuroblastoma, a tumor arising in childhood from primitive cells of the sympathetic nervous system. Our analysis identifies and characterizes a wide catalog of somatically acquired and undescribed extrachromosomal circular DNAs. Moreover, we find that extrachromosomal circular DNAs are an unanticipated major source of somatic rearrangements, contributing to oncogenic remodeling through chimeric circularization and reintegration of circular DNA into the linear genome. Cancer-causing lesions can emerge out of circle-derived rearrangements and are associated with adverse clinical outcome. It is highly probable that circle-derived rearrangements represent an ongoing mutagenic process. Thus, extrachromosomal circular DNAs represent a multihit mutagenic process, with important functional and clinical implications for the origins of genomic remodeling in cancer.
Assuntos
Carcinogênese/patologia , DNA Circular/genética , Herança Extracromossômica/genética , Rearranjo Gênico , Genoma Humano , Neuroblastoma/patologia , Oncogenes/genética , Recombinação Genética , Humanos , Neuroblastoma/genética , Células Tumorais CultivadasRESUMO
Two Pseudomonas isolates derived independently from raw refrigerated processing meat of bovine origin intended for the manufacture of Bologna-type cooked sausage could be distinguished from other known species in subsequent phylogenetic analyses. Comparison of the complete rpoB gene sequences in combination with nearly complete 16S rRNA gene sequences revealed a separate branch within the Pseudomonas fragi group. In further analyses, comprising phenotypic and chemotaxonomic characterization as well as average nucleotide identity (ANI) values obtained from the draft genome assemblies, the two isolates could be distinguished from all so far published closely related species. The closest relative was P. fragi DSM 3456T with ANI values of about 90.2â%. Other close Pseudomonas neighbours were P. psychrophila DSM 17535T (86.5â%), P. deceptionensis DSM 26521T (86.4â%), P. versuta DSM 101070T (83.8â%), P. taetrolens DSM 21104T (83.2â%), P. weihenstephanensis DSM 29166T (82.3â%), P. helleri DSM 29165T (82.7â%) and P. lundensis DSM 6252T (81.9â%). The G+C contents of isolates TH39T and TH26 were both 58.2 mol%. The major cellular lipids of strain TH39T were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol; the major quinone was Q9 with small amounts of Q8. Based on these data, the isolates TH39T and TH26 (=DSM 107389=LMG 30831) represent a novel species within the genus Pseudomonas , for which the name Pseudomonas bubulae sp. nov. is proposed. The type strain is TH39T (=DSM 107390T=LMG 30830T).
Assuntos
Filogenia , Pseudomonas/classificação , Carne Vermelha/microbiologia , Animais , Técnicas de Tipagem Bacteriana , Composição de Bases , Bovinos , DNA Bacteriano/genética , Ácidos Graxos/química , Genes Bacterianos , Hibridização de Ácido Nucleico , Fosfolipídeos/química , Pseudomonas/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/químicaRESUMO
BACKGROUND: Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. METHODS: CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. RESULTS: All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. CONCLUSION: Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.
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Terapia Baseada em Transplante de Células e Tecidos , Gangliosídeos/imunologia , Molécula L1 de Adesão de Célula Nervosa/imunologia , Receptores de Antígenos Quiméricos , Retinoblastoma/terapia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Criança , Pré-Escolar , Citotoxicidade Imunológica , Feminino , Humanos , Lactente , Masculino , Retinoblastoma/imunologia , Retinoblastoma/metabolismo , Estudos Retrospectivos , Linfócitos T/imunologiaRESUMO
Fewer than 50% of patients with high-risk neuroblastoma survive five years after diagnosis with current treatment protocols. Molecular targeted therapies are expected to improve survival. Although MDM2 has been validated as a promising target in preclinical models, no MDM2 inhibitors have yet entered clinical trials for neuroblastoma patients. Toxic side effects, poor bioavailability and low efficacy of the available MDM2 inhibitors that have entered phase I/II trials drive the development of novel MDM2 inhibitors with an improved risk-benefit profile. We investigated the effect of the novel MDM2 small molecular inhibitor, DS-3032b, on viability, proliferation, senescence, migration, cell cycle arrest and apoptosis in a panel of six neuroblastoma cell lines with different TP53 and MYCN genetic backgrounds, and assessed efficacy in a murine subcutaneous model for high-risk neuroblastoma. Re-analysis of existing expression data from 476 primary neuroblastomas showed that high-level MDM2 expression correlated with poor patient survival. DS-3032b treatment enhanced TP53 target gene expression and induced G1 cell cycle arrest, senescence and apoptosis. CRISPR-mediated MDM2 knockout in neuroblastoma cells mimicked DS-3032b treatment. TP53 signaling was selectively activated by DS-3032b in neuroblastoma cells with wildtype TP53, regardless of the presence of MYCN amplification, but was significantly reduced by TP53 mutations or expression of a dominant-negative TP53 mutant. Oral DS-3032b administration inhibited xenograft tumor growth and prolonged mouse survival. Our in vitro and in vivo data demonstrate that DS-3032b reactivates TP53 signaling even in the presence of MYCN amplification in neuroblastoma cells, to reduce proliferative capacity and cause cytotoxicity.