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BACKGROUND: Non-viral manufacturing of CAR T cells via the Sleeping Beauty transposon is cost effective and reduces the risk of insertional mutagenesis from viral transduction. However, the current gold standard methodology requires ex vivo numerical expansion of these cells on artificial antigen-presenting cells (AaPCs) for 4 weeks to generate CAR T cells of presumed sufficient quantity and function for clinical applications. METHOD: We engineered EGFRvIII-specific CAR T cells and monitored phenotypic changes throughout their ex vivo manufacturing. To reduce the culture time required to generate the CAR T-cell population, we selected for T cells in peripheral blood mononuclear cells prior to CAR modification (to eliminate the competing NK cell population). RESULTS: While we found increased expression of exhaustion markers (such as PD-1, PD-L1, TIM-3, and LAG-3) after 2 weeks in culture, whose levels continued to rise over time, we were able to generate a CAR+ T-cell population with comparable CAR expression and cell numbers in 2 weeks, thereby reducing manufacturing time by 50%, with lower expression of immune exhaustion markers. The CAR T cells manufactured at 2 weeks showed superior therapeutic efficacy in mice bearing established orthotopic EGFRvIII+ U87 gliomas. CONCLUSION: These findings demonstrate a novel, rapid method to generate CAR T cells by non-viral modification that results in CAR T cells superior in phenotype and function and further emphasizes that careful monitoring of CAR T-cell phenotype prior to infusion is critical for generating an optimal CAR T-cell product with full antitumor potential.
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Receptores ErbB/imunologia , Glioma , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/imunologia , Transfecção/métodos , Animais , Antígenos de Neoplasias/imunologia , Humanos , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.
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Apoptose/fisiologia , Compostos de Bifenilo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Leucemia Mieloide Aguda , Nitrofenóis , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Sulfonamidas , Animais , Compostos de Bifenilo/metabolismo , Compostos de Bifenilo/uso terapêutico , Linhagem Celular , Dimerização , Células-Tronco Hematopoéticas/fisiologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Nitrofenóis/metabolismo , Nitrofenóis/uso terapêutico , Piperazinas/metabolismo , Piperazinas/uso terapêutico , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sulfonamidas/metabolismo , Sulfonamidas/uso terapêutico , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Early brain damage (EBI) following subarachnoid hemorrhage (SAH) is a long-lasting condition with a high occurrence. However, treatment options are restricted. Wu-zhu-yu Decoction (WZYD) can treat headaches and vomiting, which are similar to the early symptoms of subarachnoid hemorrhage (SAH). However, it is yet unknown if WZYD can reduce EBI following SAH and its underlying mechanisms. AIM OF THE STUDY: This study aimed to investigate whether WZYD protects against EBI following SAH by inhibiting oxidative stress through activating nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling via Sirtuin 6 (SIRT6)-mediated histone H3 lysine 56 (H3K56) deacetylation. MATERIALS AND METHODS: In the current investigation, the principal components of WZYD were identified using high-performance liquid chromatography-diode array detection (HPLC-DAD). The SAH model in rats using the internal carotid artery plug puncture approach and the SAH model in primary neurons using hemoglobin incubation were developed. WZYD with different doses (137 mg kg-1, 274 mg kg-1, 548 mg kg-1) and the positive drug-Nimodipine (40 mg kg-1) were intragastrically administered in SAH model rats, respectively. The PC12 cells were cultured with corresponding medicated for 24h. In our investigation, neurological scores, brain water content, Evans blue leakage, Nissl staining, TUNEL staining, oxidative stress, expression of apoptosis-related proteins, and Nrf2/HO-1 signaling were evaluated. The interaction between SIRT6 and Nrf2 was detected by co-immunoprecipitation. SIRT6 knockdown was used to confirm its role in WZYD's neuroprotection. RESULTS: The WZYD treatment dramatically reduced cerebral hemorrhage and edema, and enhanced neurological results in EBI following SAH rats. WZYD administration inhibited neuronal apoptosis via reducing the expression levels of Cleaved cysteinyl aspartate specific proteinase-3(Cleaved Caspase-3), cysteinyl aspartate specific proteinase-3(caspase-3), and Bcl-2, Associated X Protein (Bax) and increasing the expression of B-cell lymphoma-2(Bal2). It also decreased reactive oxygen species and malondialdehyde levels and increased Nrf2 and HO-1 expression in the rat brain after SAH. In vitro, WZYD attenuated hemoglobin-induced cytotoxicity, oxidative stress and apoptosis in primary neurons. Mechanistically, WZYD enhanced SIRT6 expression and H3K56 deacetylation, activated Nrf2/HO-1 signaling, and promoted the interaction between SIRT6 and Nrf2. Knockdown of SIRT6 abolished WZYD-induced neuroprotection. CONCLUSIONS: WZYD attenuates EBI after SAH by activating Nrf2/HO-1 signaling through SIRT6-mediated H3K56 deacetylation, suggesting its therapeutic potential for SAH treatment.
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Lesões Encefálicas , Fármacos Neuroprotetores , Sirtuínas , Hemorragia Subaracnóidea , Ratos , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Caspase 3 , Ratos Sprague-Dawley , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/tratamento farmacológico , Hemorragia Subaracnóidea/metabolismo , Ácido Aspártico/farmacologia , Ácido Aspártico/uso terapêutico , Lesões Encefálicas/tratamento farmacológico , Apoptose , Hemoglobinas/farmacologia , Hemoglobinas/uso terapêutico , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêuticoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Migraine, a chronic and intricate disorder, manifests as recurrent episodic headaches accompanied by various neurological symptoms. Wuzhuyu Decoction (WZYD) is a traditional Chinese medical formula with promising effects in treating migraines; however, its underlying mechanisms have not yet been clarified. AIM OF STUDY: The study aimed to evaluate WZYD's effectiveness in migraine treatment and investigate the potential mechanism of WZYD's effects on migraine and oxidative stress. MATERIALS AND METHODS: Behavior tests and immunofluorescence assay for the intensity of migraine markers to assess the migraine-relieving effect of WZYD after chronic migraine model induced by nitroglycerin in mice. The impacts of WZYD on oxidative stress-related markers, including reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), nuclear factor erythroid 2-related factor 2 (NRF2), heme oxygenase 1 (HO1), and NAD (P)H quinone oxidoreductase 1 (NQO1) in brain tissue were examined. In addition, protein expression or mRNA levels of the MZF1/PGK1 were detected using Western blot or PCR, respectively. Finally, the MZF1 overexpression vector was constructed to the higher level of MZF1. The MZF1/PGK1 signaling pathway expression was evaluated by markers of oxidative stress including NRF2 and others in this series of experiments. RESULTS: Through murine model experimentation, we observed that WZYD effectively alleviates migraine symptoms, signifying its therapeutic efficacy. Mechanistically, WZYD emerges as a potent activator of the NRF2, acting as a robust defense against oxidative stress. In vitro investigations demonstrated that WZYD combats oxidative stress and curbs cell apoptosis induced by these detrimental conditions. Furthermore, by suppressing the transcriptional expression of PGK1, an influential player in the NRF2 pathway, WZYD effectively activates NRF2 signaling. Intriguingly, we have identified MZF1 as the mediator orchestrating the regulation of the PGK1/NRF2 pathway by WZYD. CONCLUSION: The study confirms the effectiveness of WZYD in alleviating migraine symptoms. Mechanistically, WZYD activated the NRF2 signaling pathway; moreover, the action of WZYD involved the down-regulation of PGK1 mediated by MZF1, which promoted the activation of the NRF2 pathway. This study advances our understanding of the intricate mechanisms driving WZYD's efficacy, paving the way for novel treatments in migraine management.
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Antioxidantes , Transtornos de Enxaqueca , Camundongos , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Antioxidantes/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Nitroglicerina , Elementos de Resposta Antioxidante , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transtornos de Enxaqueca/induzido quimicamente , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/genéticaRESUMO
Subarachnoid hemorrhage (SAH), a specific subtype of cerebrovascular accident, is characterized by the extravasation of blood into the interstice between the brain and its enveloping delicate tissues. This pathophysiological phenomenon can precipitate an early brain injury (EBI), which is characterized by inflammation and neuronal death. Rutaecarpine (Rut), a flavonoid compound discovered in various plants, has been shown to have protective effects against SAH-induced cerebral insult in rodent models. In our study, we used a rodent SAH model to evaluate the effect of Rut on EBI and investigated the effect of Rut on the inflammatory response and its regulation of SIRT6 expression in vitro. We found that Rut exerts a protective effect on EBI in SAH rats, which is partly due to its ability to inhibit the inflammatory response. Notably, Rut up-regulated Sirtuin 6 (SIRT6) expression, leading to an increase in H3K9 deacetylation and inhibition of nuclear factor-kappa B (NF-[Formula: see text]B) transcriptional activation, thereby mediating the inflammatory response. In addition, further data showed that SIRT6 was proven to mediate the regulation of Rut on the microglial inflammatory response. These findings highlight the importance of SIRT6 in the regulation of inflammation and suggest a potential mechanism for the protective effect of Rut on EBI. In summary, Rut may have the potential to prevent and treat SAH-induced brain injury by interacting with SIRT6. Our findings may provide a new therapeutic strategy for the treatment of SAH-induced EBI.
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Alcaloides Indólicos , NF-kappa B , Quinazolinas , Ratos Sprague-Dawley , Sirtuínas , Hemorragia Subaracnóidea , Animais , Hemorragia Subaracnóidea/tratamento farmacológico , Hemorragia Subaracnóidea/complicações , Sirtuínas/metabolismo , Sirtuínas/genética , Alcaloides Indólicos/farmacologia , NF-kappa B/metabolismo , Masculino , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Modelos Animais de Doenças , Lesões Encefálicas/etiologia , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/metabolismo , Ratos , Inflamação/tratamento farmacológico , Inflamação/etiologia , Fitoterapia , Transdução de Sinais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , QuinazolinonasRESUMO
BACKGROUND: Due to its widespread prevalence, migraine is a common neurovascular condition that has a major impact on people's health and quality of life. Rutaecarpine (RUT) is one of the main effective components of Evodia rutaecarpa, which has a wide range of biological activities. However, the exact mechanism by which RUT improves migraine remain unknown. PURPOSE: The purpose of this study was to investigate whether RUT improves migraine by inhibiting oxidative stress via activating the Nrf2 antioxidant system through the PTEN/PGK1 signaling pathway. METHODS: In vivo, a mouse model of chronic migraine (CM) was established by repeated intraperitoneal injection of nitroglycerin (NTG). After treatment with RUT and Sumatriptan, behavioral tests were performed, followed by measurements of oxidative stress-related indicators in the trigeminal nucleus caudalis, expression of proteins associated with the Nrf2 antioxidant system, and the PTEN/PGK1 pathway. In vitro, PC12 cells were stimulated by 100 µM H2O2 for 24 h to induce oxidative stress, which was then treated with RUT. Furthermore, the role of PTEN in antioxidant stress of RUT was elucidated by knockout of the PTEN gene. RESULTS: The results showed that RUT treatment improved NTG-induced migraine in mice by inhibiting oxidative stress. Importantly, RUT inhibited oxidative stress in NTG-induced mice or H2O2-induced PC12 cells via activating the Nrf2 antioxidant system by inhibiting PGK1 activity through PTEN. These results provide evidence that RUT improves migraine by activation of the Nrf2 antioxidant system through the PTEN/PGK1 pathway and provide new insights into the potential use of RUT as an effective drug development candidate for migraine.
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Transtornos de Enxaqueca , Nitroglicerina , Ratos , Camundongos , Animais , Nitroglicerina/farmacologia , Antioxidantes/uso terapêutico , Fator 2 Relacionado a NF-E2/metabolismo , Peróxido de Hidrogênio/farmacologia , Qualidade de Vida , Transdução de Sinais , Transtornos de Enxaqueca/induzido quimicamente , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/metabolismoRESUMO
BACKGROUND: Traumatic brain injury (TBI) is a leading cause of death and disability worldwide as well as a risk factor for neurodegenerative diseases later in life. Evodiamine (Evo), a compound derived from Evodia rutaecarpa, is known to possess pharmacological activities. However, whether Evo confers protection after TBI remains unknown. OBJECTIVE: To study whether Evo protects against TBI through inhibiting oxidative stress via the phosphoglycerate kinase 1 (PGK1)/nuclear factor erythroid 2-related factor 2 (NRF2) pathway. MATERIALS AND METHODS: In vivo, adult male C57BL/6J mice were subjected to controlled cortical impact (impact velocity: 6 m/s; penetration depth: 2 mm) to establish a murine model of TBI. Evodiamine was administrated at 24 h, 30 min prior to TBI and 2, 24, 48, 72 h post TBI. In vitro, pheochromacytoma 12 (PC12) cells were pretreated with Evo for 24 h, then exposed to 300 µM H2O2 stimulation for another 24 h to induce oxidative stress. Furthermore, transfection of PGK1 overexpressing vectors or PGK1 siRNAs was performed to decipher the role of PGK1 in Evo-produced effect in TBI. RESULTS: Treatment with Evo alleviated TBI-induced neurological dysfunction, BBB breakdown, histopathological changes in H&E staining, and increased apoptosis. Importantly, Evo enhanced catalase (CAT) and superoxide dismutase (SOD) activities, and reduced reactive oxygen species (ROS) generation through PGK1 inhibition-induced activation of the NRF2/heme oxygenase-1 (HO-1) signaling in TBI mice or H2O2-exposed PC12 cells. Of note, the protective effect of Evo in the in vitro TBI was similar to that of PGK1 siRNAs; overexpression of PGK1 compromised Evo-produced protection in H2O2-stimulated PC12 cells. DISCUSSION AND CONCLUSIONS: Taken together, we demonstrated that Evo improved the outcomes after TBI by targeting the PGK1/NRF2 signaling-regulated oxidative stress. Evo may represent a potential therapy to promote recovery from TBI.
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Lesões Encefálicas Traumáticas , Fator 2 Relacionado a NF-E2 , Animais , Apoptose , Lesões Encefálicas Traumáticas/metabolismo , Heme Oxigenase-1/metabolismo , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Fosfoglicerato Quinase , Quinazolinas , RatosRESUMO
The oxidative stress response caused by traumatic brain injury (TBI) leads to secondary damage in the form of tissue damage and cell death. Nuclear transcription-related factor 2 (NRF2) is a key factor in the body against oxidative stress and has an important role in combating oxidative damage in TBI neurons. In the present study, we investigated whether rutaecarpine could activate the PGK1/KEAP1/NRF2 pathway to antagonize oxidative damage in TBI neurons. We performed controlled cortical impact (CCI) surgery on mice and taken H2O2 treatment on PC12 cells to construct TBI models. The results of western blot showed that the expression of PGK1, KEAP and NRF2 was regulated and accompanied by altered levels of oxidative stress, and the use of rutaecarpine in the TBI model mice significantly improved cognitive dysfunction, increased antioxidant capacity and reduced apoptosis in brain tissue. Similar antioxidant damage results were obtained using rutaecarpine in a PC12 cell model. Furthermore, through the use of the protein synthesis inhibitor CHX and the proteasome synthesis inhibitor MG-132, rutaecarpine was found to promote the expreesions of PGK1 and NRF2 by accelerating PGK1 ubiquitination to reduce PGK1 expression. Therefore, rutaecarpine may be a promising therapeutic agent for the treatment of TBI-related neuro-oxidative damage.
RESUMO
Traumatic brain injuries (TBI) are the greatest source of death in trauma, and post-traumatic epilepsy (PTE) is one of the common complications of TBI. Oxidative stress and inflammatory responses play an important role in the process of PTE. Many studies have shown that Jujuboside A has powerful antioxidant and anti-inflammatory properties. However, it is not known whether Jujuboside A has an anti-epileptic effect. The influences of Jujuboside A in the experimental FeCl3-induced model of PTE were tested by estimating the grade of seizures and performing behavioral tests. Following that, we detected oxidative stress indicators and inflammatory factors. Additionally, western blotting was used to test the protein levels of signaling molecules in MAPK pathways. In this study, Jujuboside A was found to have improved the recognition deficiency and epilepsy syndromes in the experimental rat model. Moreover, oxidative stress and inflammatory responses induced by FeCl3 injection were relieved by Jujuboside A. In addition, Jujuboside A was found to be capable of reducing the increased expression of p-P38 and p-ERK1/2 caused by iron ions. Collectively, our results demonstrated that Jujuboside A exhibits an antiepileptogenic effect by alleviating oxidative stress and inflammatory responses via the p38 and ERK1/2 pathways.
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SDF-1alpha/CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions. We previously reported that bone marrow-derived stromal cells inhibit chemotherapy-induced apoptosis in acute myeloid leukemia (AML). Here we demonstrate that the CXCR4 inhibitor AMD3465 antagonized stromal-derived factor 1alpha (SDF-1alpha)-induced and stroma-induced chemotaxis and inhibited SDF-1alpha-induced activation of prosurvival signaling pathways in leukemic cells. Further, CXCR4 inhibition partially abrogated the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. Fetal liver tyrosine kinase-3 (FLT3) gene mutations activate CXCR4 signaling, and coculture with stromal cells significantly diminished antileukemia effects of FLT3 inhibitors in cells with mutated FLT3. Notably, CXCR4 inhibition increased the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of the FLT3 inhibitor sorafenib. In vivo studies demonstrated that AMD3465, alone or in combination with granulocyte colony-stimulating factor, induced mobilization of AML cells and progenitor cells into circulation and enhanced antileukemic effects of chemotherapy and sorafenib, resulting in markedly reduced leukemia burden and prolonged survival of the animals. These findings indicate that SDF-1alpha/CXCR4 interactions contribute to the resistance of leukemic cells to signal transduction inhibitor- and chemotherapy-induced apoptosis in systems mimicking the physiologic microenvironment. Disruption of these interactions with CXCR4 inhibitors represents a novel strategy of sensitizing leukemic cells by targeting their protective bone marrow microenvironment.
Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Leucemia Experimental/tratamento farmacológico , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Receptores CXCR4/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Quimiotaxia/efeitos dos fármacos , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Leucemia Experimental/metabolismo , Leucemia Experimental/patologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos BALB C , Mutação/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismo , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
PURPOSE: The blood-brain barrier (BBB) inhibits adequate dosing/penetration of therapeutic agents to malignancies in the brain. Low-intensity pulsed ultrasound (LIPU) is a safe therapeutic method of temporary BBB disruption (BBBD) to enhance chemotherapeutic delivery to the tumor and surrounding brain parenchyma for treatment of glioblastoma. EXPERIMENTAL DESIGN: We investigated if LIPU could enhance therapeutic efficacy of anti-PD-1 in C57BL/6 mice bearing intracranial GL261 gliomas, epidermal growth factor receptor variant III (EGFRvIII) chimeric antigen receptor (CAR) T cells in NSG mice with EGFRvIII-U87 gliomas, and a genetically engineered antigen-presenting cell (APC)-based therapy producing the T-cell attracting chemokine CXCL10 in the GL261-bearing mice. RESULTS: Mice treated with anti-PD-1 and LIPU-induced BBBD had a median survival duration of 58 days compared with 39 days for mice treated with anti-PD-1, and long-term survivors all remained alive after contralateral hemisphere rechallenge. CAR T-cell administration with LIPU-induced BBBD resulted in significant increases in CAR T-cell delivery to the CNS after 24 (P < 0.005) and 72 (P < 0.001) hours and increased median survival by greater than 129%, in comparison with CAR T cells alone. Local deposition of CXCL10-secreting APCs in the glioma microenvironment with LIPU enhanced T-cell glioma infiltration during the therapeutic window (P = 0.004) and markedly enhanced survival (P < 0.05). CONCLUSIONS: LIPU increases immune therapeutic delivery to the tumor microenvironment with an associated increase in survival and is an emerging technique for enhancing novel therapies in the brain.
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Barreira Hematoencefálica/efeitos da radiação , Neoplasias Encefálicas/terapia , Glioma/terapia , Imunoterapia , Ondas Ultrassônicas , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Resultado do TratamentoRESUMO
Only a subset of recurrent glioblastoma (rGBM) responds to anti-PD-1 immunotherapy. Previously, we reported enrichment of BRAF/PTPN11 mutations in 30% of rGBM that responded to PD-1 blockade. Given that BRAF and PTPN11 promote MAPK/ERK signaling, we investigated whether activation of this pathway is associated with response to PD-1 inhibitors in rGBM, including patients that do not harbor BRAF/PTPN11 mutations. Here we show that immunohistochemistry for ERK1/2 phosphorylation (p-ERK), a marker of MAPK/ERK pathway activation, is predictive of overall survival following adjuvant PD-1 blockade in two independent rGBM patient cohorts. Single-cell RNA-sequencing and multiplex immunofluorescence analyses revealed that p-ERK was mainly localized in tumor cells and that high-p-ERK GBMs contained tumor-infiltrating myeloid cells and microglia with elevated expression of MHC class II and associated genes. These findings indicate that ERK1/2 activation in rGBM is predictive of response to PD-1 blockade and is associated with a distinct myeloid cell phenotype.
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Glioblastoma , Glioblastoma/tratamento farmacológico , Humanos , Imunoterapia , Sistema de Sinalização das MAP Quinases , Recidiva Local de Neoplasia/tratamento farmacológico , FosforilaçãoRESUMO
PURPOSE: Anti-programmed cell death protein 1 (PD-1) therapy has demonstrated inconsistent therapeutic results in patients with glioblastoma (GBM) including those with profound impairments in CD8 T-cell effector responses. EXPERIMENTAL DESIGN: We ablated the CD8α gene in BL6 mice and intercrossed them with Ntv-a mice to determine how CD8 T cells affect malignant progression in forming endogenous gliomas. Tumor-bearing mice were treated with PD-1 to determine the efficacy of this treatment in the absence of T cells. The tumor microenvironment of treated and control mice was analyzed by IHC and FACS. RESULTS: We observed a survival benefit in immunocompetent mice with endogenously arising intracranial glioblastomas after intravenous administration of anti-PD-1. The therapeutic effect of PD-1 administration persisted in mice even after genetic ablation of the CD8 gene (CD8-/-). CD11b+ and Iba1+ monocytes and macrophages were enriched in the glioma microenvironment of the CD8-/- mice. The macrophages and microglia assumed a proinflammatory M1 response signature in the setting of anti-PD-1 blockade through the elimination of PD-1-expressing macrophages and microglia in the tumor microenvironment. Anti-PD-1 can inhibit the proliferation of and induce apoptosis of microglia through antibody-dependent cellular cytotoxicity, as fluorescently labeled anti-PD-1 was shown to gain direct access to the glioma microenvironment. CONCLUSIONS: Our results show that the therapeutic effect of anti-PD-1 blockade in GBM may be mediated by the innate immune system, rather than by CD8 T cells. Anti-PD-1 immunologically modulates innate immunity in the glioma microenvironment-likely a key mode of activity.
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Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Inibidores de Checkpoint Imunológico/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Macrófagos Associados a Tumor/efeitos dos fármacos , Animais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Glioblastoma/imunologia , Glioblastoma/patologia , Humanos , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunidade Inata/efeitos dos fármacos , Ativação de Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/imunologiaRESUMO
PURPOSE: Cytomegalovirus (CMV) antigens occur in glioblastoma but not in normal brains, making them desirable immunologic targets. PATIENTS AND METHODS: Highly functional autologous polyclonal CMV pp65-specific T cells from patients with glioblastoma were numerically expanded under good manufacturing practice compliant conditions and administered after 3 weeks of lymphodepleting dose-dense temozolomide (100 mg/m2) treatment. The phase I component used a 3+3 design, ascending through four dose levels (5 × 106-1 × 108 cells). Treatment occurred every 6 weeks for four cycles. In vivo persistence and effector function of CMV-specific T cells was determined by dextramer staining and multiparameter flow cytometry in serially sampled peripheral blood and in the tumor microenvironment. RESULTS: We screened 65 patients; 41 were seropositive for CMV; 25 underwent leukapheresis; and 20 completed ≥1 cycle. No dose-limiting toxicities were observed. Radiographic response was complete in 1 patient, partial in 2. Median progression-free survival (PFS) time was 1.3 months [95% confidence interval (CI), 0-8.3 months]; 6-month PFS was 19% (95% CI, 7%-52%); and median overall survival time was 12 months (95% CI, 6 months to not reached). Repeated infusions of CMV-T cells paralleled significant increases in circulating CMV+ CD8+ T cells, but cytokine production showing effector activity was suppressed, especially from T cells obtained directly from glioblastomas. CONCLUSIONS: Adoptive infusion of CMV-specific T cells after lymphodepletion with dose-dense temozolomide was well tolerated. But apparently CMV seropositivity does not guarantee tumor susceptibility to CMV-specific T cells, suggesting heterogeneity in CMV antigen expression. Moreover, effector function of these T cells was attenuated, indicating a requirement for further T-cell modulation to prevent their dysfunction before conducting large-scale clinical studies.
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Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/terapia , Glioblastoma/terapia , Imunoterapia Adotiva/métodos , Proteínas da Matriz Viral/imunologia , Adulto , Citomegalovirus/imunologia , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/mortalidade , Infecções por Citomegalovirus/virologia , Feminino , Glioblastoma/imunologia , Glioblastoma/mortalidade , Glioblastoma/virologia , Humanos , Leucaférese , Depleção Linfocítica/métodos , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Temozolomida/administração & dosagem , Transplante Autólogo/métodos , Microambiente Tumoral/imunologiaRESUMO
Sporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole-genome, exome, transcriptome, and methylation sequencing of 83 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, TP53 and cell-cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA-methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.
Assuntos
Neoplasias Encefálicas/genética , Metilação de DNA/genética , Glioma/genética , Mutação/genética , Animais , Cães , Exoma/genética , Humanos , Isocitrato Desidrogenase/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
We and others have reported that C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) effectively inhibits the growth of multiple cancer cell types. Our previous studies indicated that prolonged CDDO-Me treatment inactivated extracellular signal-regulated kinase signaling in acute myelogenous leukemia cells. Whether treatment with CDDO-Me has an earlier effect on other proteins that are important for either signal transduction or oncogenesis is unknown. Constitutively activated signal transducer and activator of transcription 3 (STAT3) is frequently found in human breast cancer samples. Constitutively activated STAT3 was shown to up-regulate c-Myc in several types of cancer and has a feedback effect on Src and Akt. To examine the effects of CDDO-Me on STAT3 signaling in breast cancer, we used the murine 4T1 breast tumor model, which is largely resistant to chemotherapy. In vitro, after treatment of 4T1 cells with 500 nmol/L CDDO-Me for 2 h, we found (a) inactivation of STAT3, (b) inactivation of Src and Akt, (c) 4-fold reduction of c-Myc mRNA levels, (d) accumulation of cells in G(2)-M cell cycle phase, (e) abrogation of invasive growth of 4T1 cells, and (f) lack of apoptosis induction. In in vivo studies, CDDO-Me completely eliminated 4T1 breast cancer growth and lung metastases induced by 4T1 cells in mice when treatment started 1 day after tumor implantation and significantly inhibited tumor growth when started after 5 days. In vivo studies also indicated that splenic mature dendritic cells were restored after CDDO-Me treatment. In summary, these data suggest that CDDO-Me may have therapeutic potential in breast cancer therapy, in part, through inactivation of STAT3.
Assuntos
Neoplasias Mamárias Experimentais/tratamento farmacológico , Ácido Oleanólico/análogos & derivados , Fator de Transcrição STAT3/metabolismo , Animais , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Vetores Genéticos/genética , Lentivirus/genética , Luciferases de Renilla , Neoplasias Pulmonares/prevenção & controle , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Ácido Oleanólico/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologiaRESUMO
Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene-deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin αvß5 (ITGαvß5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. Systemic administration in mice of 4-1BB-OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.
Assuntos
Neoplasias Encefálicas/imunologia , Regulação Neoplásica da Expressão Gênica/imunologia , Glioblastoma/imunologia , Imunidade Inata , Macrófagos/imunologia , Proteínas de Neoplasias/imunologia , Osteopontina/imunologia , Animais , Aptâmeros de Nucleotídeos/farmacologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Linfócitos T CD8-Positivos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/genética , Macrófagos/patologia , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Osteopontina/genética , Receptores de Vitronectina/genética , Receptores de Vitronectina/imunologiaRESUMO
Constitutively activated STAT3 is involved in the formation of multiple types of tumors including breast cancer. We examined the effects of Stat3 protein knockdown by RNA interference using a dicistronic lentivirus small hairpin (shRNA) delivery system on the growth of mammary tumors in BALB/c mice induced by the 4T1 cell line. A single exposure of 4T1 cells to shRNA/STAT3 lentivirus transduced 75% of the cells with green fluorescent protein (GFP) within 96 hours. In cells selected for GFP expression, neither Stat3 protein nor phosphotyrosine Stat3 was detected. Tumor formation induced by injecting 4T1 cells into the mammary fat pad was blocked by expression of the shRNA for STAT3 whereas all mice injected with 4T1 cells expressing only GFP efficiently formed tumors. c-Myc expression was reduced 75% in cells expressing greatly reduced levels of Stat3 compared with the GFP control. Of interest, the level of activated Src, which is known to activate Stat3, was virtually eliminated but the level of the Src protein itself remained the same. Importantly, expression of Twist protein, a metastatic regulator, was eliminated in STAT3 knockdown cells. Invasion activity of STAT3 knockdown cells was strongly inhibited. However, the proliferation rate of cells in Stat3 knockdown cells was similar to that of the GFP control; the cell cycle was also not affected. We conclude from these studies that activated Stat3 protein plays a critical role in the induction of breast tumors induced by 4T1 cells by enhancing the expression of several important genes including c-Myc and the metastatic regulator Twist. These studies suggest that stable expression of small interfering RNA for STAT3 has potential as a therapeutic strategy for breast cancer.
Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/prevenção & controle , Interferência de RNA , Transativadores/antagonistas & inibidores , Animais , Processos de Crescimento Celular/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Feminino , Hospedeiro Imunocomprometido , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Conformação de Ácido Nucleico , Fator de Transcrição STAT3 , Transativadores/biossíntese , Transativadores/genéticaRESUMO
The genetic heterogeneity of acute myeloid leukemia (AML) and the variable responses of individual patients to therapy suggest that different AML genotypes may influence the bone marrow (BM) microenvironment in different ways. We performed gene expression profiling of bone marrow mesenchymal stromal cells (BM-MSC) isolated from normal C57BL/6 mice or mice inoculated with syngeneic murine leukemia cells carrying different human AML genotypes, developed in mice with Trp53 wild-type or nullgenetic backgrounds. We identified a set of genes whose expression in BM-MSC was modulated by all four AML genotypes tested. In addition, there were sets of differentially-expressed genes in AML-exposed BM-MSC that were unique to the particular AML genotype or Trp53 status. Our findings support the hypothesis that leukemia cells alter the transcriptome of surrounding BM stromal cells, in both common and genotype-specific ways. These changes are likely to be advantageous to AML cells, affecting disease progression and response to chemotherapy, and suggest opportunities for stroma-targeting therapy, including those based on AML genotype.
RESUMO
Background: Previously we showed therapeutic efficacy of unprotected miR-124 in preclinical murine models of glioblastoma, including in heterogeneous genetically engineered murine models by exploiting the immune system and thereby negating the need for direct tumor delivery. Although these data were promising, to implement clinical trials, we required a scalable formulation that afforded protection against circulatory RNases. Methods: We devised lipid nanoparticles that encapsulate and protect the miRs from degradation and provide enhanced delivery into the immune cell compartment and tested in vivo antitumor effects. Results: Treatment with nanoparticle-encapsulated miR-124, LUNAR-301, demonstrated a median survival exceeding 70 days, with an associated reversal of tumor-mediated immunosuppression and induction of immune memory. In both canine and murine models, the safety profile of LUNAR-301 was favorable. Conclusions: For the first time, we show that nanoparticles can direct a therapeutic response by targeting intracellular immune pathways. Although shown in the context of gliomas, this therapeutic approach would be applicable to other malignancies.