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Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-4, the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy.
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Citidina Desaminase , Neoplasias Pulmonares , Humanos , Citidina Desaminase/deficiência , Citidina Desaminase/efeitos dos fármacos , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Terapia de Alvo Molecular , Mutação , Resistencia a Medicamentos AntineoplásicosRESUMO
The metabolic status of the kidney is a determinant of injury susceptibility and a measure of progression for many disease processes; however, noninvasive modalities to assess kidney metabolism are lacking. In this study, we employed positron emission tomography (PET) and intravital multiphoton microscopy (MPM) to assess cortical and proximal tubule glucose tracer uptake, respectively, following experimental perturbations of kidney metabolism. Applying dynamic image acquisition PET with 2-18fluoro-2-deoxyglucose (18F-FDG) and tracer kinetic modeling, we found that an intracellular compartment in the cortex of the kidney could be distinguished from the blood and urine compartments in animals. Given emerging literature that the tumor suppressor protein p53 is an important regulator of cellular metabolism, we demonstrated that PET imaging was able to discern a threefold increase in cortical 18F-FDG uptake following the pharmacological inhibition of p53 in animals. Intravital MPM with the fluorescent glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) provided increased resolution and corroborated these findings at the level of the proximal tubule. Extending our observation of p53 inhibition on proximal tubule glucose tracer uptake, we demonstrated by intravital MPM that pharmacological inhibition of p53 diminishes mitochondrial potential difference. We provide additional evidence that inhibition of p53 alters key metabolic enzymes regulating glycolysis and increases intermediates of glycolysis. In summary, we provide evidence that PET is a valuable tool for examining kidney metabolism in preclinical and clinical studies, intravital MPM is a powerful adjunct to PET in preclinical studies of metabolism, and p53 inhibition alters basal kidney metabolism.
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Glucose/metabolismo , Rim/diagnóstico por imagem , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Tomografia por Emissão de Pósitrons/métodos , Animais , Desoxiglucose , Radioisótopos de Flúor , Rim/metabolismo , Masculino , Camundongos , Ratos , Ratos Sprague-DawleyRESUMO
Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Industries region of the Elizabeth River, Virginia, have passed polycyclic aromatic hydrocarbon (PAH) resistance to their offspring as evidenced by early life stage testing of developmental toxicity after exposure to specific PAHs. Our study focused on environmentally relevant PAH mixtures in the form of Elizabeth River sediment extract (ERSE). Juvenile (5 month) F1 progeny of pollution-adapted Atlantic Wood (AW) parents and of reference site (King's Creek [KC]) parents were exposed as embryos to ERSE. Liver alterations, including nonneoplastic lesions and microvesicular vacuolation, were observed in both populations. ERSE-exposed KC fish developed significantly more alterations than unexposed KC fish. Interestingly, unexposed AW killifish developed significantly more alterations than unexposed KC individuals, suggesting that AW juveniles are not fully protected from liver disease; rapid growth of juvenile fish may also be an accelerating factor for tumorigenesis. Because recent reports show hepatic tumor formation in adult AW fish, the differing responses from the 2 populations provided a way to determine whether embryo toxicity protection extends to juveniles. Future investigations will analyze older life stages of killifish to determine differences in responses related to chronic disease.
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Adaptação Fisiológica/efeitos dos fármacos , Fundulidae , Fígado/efeitos dos fármacos , Fígado/patologia , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Poluentes Químicos da Água/toxicidade , Envelhecimento , Animais , Animais Recém-Nascidos , Sedimentos Geológicos , RiosRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) remains a global health problem with unique diagnostic and therapeutic challenges, including difficulties in identifying the highest risk patients. Previous work from our lab has established the murine multidrug resistance-2 mouse (MDR2) model of HCC as a reasonable preclinical model that parallels the changes seen in human inflammatory associated HCC. The purpose of this study is to evaluate modalities of PET/CT in MDR2(-/-) mice in order to facilitate therapeutic translational studies from bench to bedside. METHODS: 18F-FDG and 11C-acetate PET/CT was performed on 12 m MDR2(-/-) mice (n = 3/tracer) with HCC and 12 m MDR2(-/+) control mice (n = 3/tracer) without HCC. To compare PET/CT to biological markers of HCC and cellular function, serum alpha-fetoprotein (AFP), lysophosphatidic acid (LPA), cAMP and hepatic tumor necrosis factor α (TNFα) were quantified in 3-12 m MDR2(-/-) (n = 10) mice using commercially available ELISA analysis. To translate results in mice to patients 11C-acetate PET/CT was also performed in 8 patents suspected of HCC recurrence following treatment and currently on the liver transplant wait list. RESULTS: Hepatic18F-FDG metabolism was not significantly increased in MDR2(-/-) mice. In contrast, hepatic 11C-acetate metabolism was significantly elevated in MDR2(-/-) mice when compared to MDR2(-/+) controls. Serum AFP and LPA levels increased in MDR2(-/-) mice contemporaneous with the emergence of HCC. This was accompanied by a significant decrease in serum cAMP levels and an increase in hepatic TNFα. In patients suspected of HCC recurrence there were 5 true positives, 2 true negatives and 1 suspected false 11C-acetate negative. CONCLUSIONS: Hepatic 11C-acetate PET/CT tracks well with HCC in MDR2(-/-) mice and patients with underlying liver disease. Consequently 11C-acetate PET/CT is well suited to study (1) HCC emergence/progression in patients and (2) reduce animal numbers required to study new chemotherapeutics in murine models of HCC.
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Acetatos , Carbono , Carcinoma Hepatocelular/diagnóstico , Modelos Animais de Doenças , Fluordesoxiglucose F18 , Neoplasias Hepáticas/diagnóstico , Tomografia por Emissão de Pósitrons/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Animais , Camundongos , Camundongos Knockout , Imagem Multimodal/métodos , Compostos Radiofarmacêuticos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios X/métodos , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
RIT1 is a rare and understudied oncogene in lung cancer. Despite structural similarity to other RAS GTPase proteins such as KRAS, oncogenic RIT1 activity does not appear to be tightly regulated by nucleotide exchange or hydrolysis. Instead, there is a growing understanding that the protein abundance of RIT1 is important for its regulation and function. We previously identified the deubiquitinase USP9X as a RIT1 dependency in RIT1-mutant cells. Here, we demonstrate that both wild-type and mutant forms of RIT1 are substrates of USP9X. Depletion of USP9X leads to decreased RIT1 protein stability and abundance and resensitizes cells to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in vitro and in vivo. Our work expands upon the current understanding of RIT1 protein regulation and presents USP9X as a key regulator of RIT1-driven oncogenic phenotypes.
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RIT1 is a rare and understudied oncogene in lung cancer. Despite structural similarity to other RAS GTPase proteins such as KRAS, oncogenic RIT1 activity does not appear to be tightly regulated by nucleotide exchange or hydrolysis. Instead, there is a growing understanding that the protein abundance of RIT1 is important for its regulation and function. We previously identified the deubiquitinase USP9X as a RIT1 dependency in RIT1-mutant cells. Here, we demonstrate that both wild-type and mutant forms of RIT1 are substrates of USP9X. Depletion of USP9X leads to decreased RIT1 protein stability and abundance and resensitizes cells to EGFR tyrosine kinase inhibitors. Our work expands upon the current understanding of RIT1 protein regulation and presents USP9X as a key regulator of RIT1-driven oncogenic phenotypes.
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Glial cells, including astrocytes, microglia, and oligodendrocytes, are brain cells that support and dynamically interact with neurons and each other. These intercellular dynamics undergo changes during stress and disease states. In response to most forms of stress, astrocytes will undergo some variation of activation, meaning upregulation in certain proteins expressed and secreted and either upregulations or downregulations to various constitutive and normal functions. While types of activation are many and contingent on the particular disturbance that triggers these changes, there are two main overarching categories that have been delineated thus far: A1 and A2. Named in the convention of microglial activation subtypes, and with the acknowledgement that the types are not completely distinct or completely comprehensive, the A1 subtype is generically associated with toxic and pro-inflammatory factors, and the A2 phenotype is broadly associated with anti-inflammatory and neurogenic factors. The present study served to measure and document dynamic changes in these subtypes at multiple timepoints using an established experimental model of cuprizone toxic demyelination. The authors found increases in proteins associated with both cell types at different timepoints, with protein increases in the A1 marker C3d and the A2 marker Emp1 in the cortex at one week and protein increases in Emp1 in the corpus callosum at three days and four weeks. There were also increases in Emp1 staining specifically colocalized with astrocyte staining in the corpus callosum at the same timepoints as the protein increases, and in the cortex weeks later at four weeks. C3d colocalization with astrocytes also increased most at four weeks. This indicates simultaneous increases of both types of activation as well as the likely existence of astrocytes expressing both markers. The authors also found the increase in two A1 associated proteins (TNF alpha and C3d) did not show a linear relationship in line with findings from other research and indicating a more complex relationship between cuprizone toxicity and astrocyte activation. The increases in TNF alpha and IFN gamma did not occur at timepoints preceding increases in C3d and Emp1, showing that other factors also precipitate the subtypes associated (A1 for C3d and A2 for Emp1). These findings add to the body of research showing the specific early timepoints at which A1 and A2 markers are most increased during the course of cuprizone treatment, including the fact that these increases can be non-linear in the case of Emp1. This provides additional information on optimal times for targeted interventions during the cuprizone model.
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INTRODUCTION: Social determinants are structures and conditions in the biological, physical, built, and social environments that affect health, social and physical functioning, health risk, quality of life, and health outcomes. The adoption of recommended, standard measurement protocols for social determinants of health will advance the science of minority health and health disparities research and provide standard social determinants of health protocols for inclusion in all studies with human participants. METHODS: A PhenX (consensus measures for Phenotypes and eXposures) Working Group of social determinants of health experts was convened from October 2018 to May 2020 and followed a well-established consensus process to identify and recommend social determinants of health measurement protocols. The PhenX Toolkit contains data collection protocols suitable for inclusion in a wide range of research studies. The recommended social determinants of health protocols were shared with the broader scientific community to invite review and feedback before being added to the Toolkit. RESULTS: Nineteen social determinants of health protocols were released in the PhenX Toolkit (https://www.phenxtoolkit.org) in May 2020 to provide measures at the individual and structural levels for built and natural environments, structural racism, economic resources, employment status, occupational health and safety, education, environmental exposures, food environment, health and health care, and sociocultural community context. CONCLUSIONS: Promoting the adoption of well-established social determinants of health protocols can enable consistent data collection and facilitate comparing and combining studies, with the potential to increase their scientific impact.
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Qualidade de Vida , Determinantes Sociais da Saúde , Humanos , Fenótipo , Coleta de Dados , Projetos de PesquisaRESUMO
In recent work, we performed CRISPR/Cas9 screening in RIT1 (Ras-like in all tissues)-mutant cancer cells. We found that RIT1-mutant cells are vulnerable to loss of mitotic regulators, and mutant RIT1 synergizes with YAP1 (yes-associated protein 1) in oncogenesis. These findings can be leveraged to identify targeted therapies for RIT1-mutant cancer.
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CRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1. Here, genome-wide CRISPR screening in KRAS, EGFR, and RIT1-mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1-mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer.
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Neoplasias Pulmonares/genética , Oncogenes/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Receptores ErbB/genética , Feminino , Técnicas de Inativação de Genes , Ensaios de Triagem em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Terapia de Alvo Molecular , Mutação , Células NIH 3T3 , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAP , Proteínas rasRESUMO
Cancer-associated fibroblasts (CAFs) are highly heterogeneous. With the lack of a comprehensive understanding of CAFs' functional distinctions, it remains unclear how cancer treatments could be personalized based on CAFs in a patient's tumor. We have established a living biobank of CAFs derived from biopsies of patients' non-small lung cancer (NSCLC) that encompasses a broad molecular spectrum of CAFs in clinical NSCLC. By functionally interrogating CAF heterogeneity using the same therapeutics received by patients, we identify three functional subtypes: (1) robustly protective of cancers and highly expressing HGF and FGF7; (2) moderately protective of cancers and highly expressing FGF7; and (3) those providing minimal protection. These functional differences among CAFs are governed by their intrinsic TGF-ß signaling, which suppresses HGF and FGF7 expression. This CAF functional classification correlates with patients' clinical response to targeted therapies and also associates with the tumor immune microenvironment, therefore providing an avenue to guide personalized treatment.
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Fibroblastos Associados a Câncer/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Fator 7 de Crescimento de Fibroblastos/genética , Fator de Crescimento de Hepatócito/genética , Neoplasias Pulmonares/patologia , Biópsia , Fibroblastos Associados a Câncer/química , Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Medicina de Precisão , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Regulação para CimaRESUMO
Transmembrane protein 67 (TMEM67) is mutated in Meckel Gruber Syndrome type 3 (MKS3) resulting in a pleiotropic phenotype with hydrocephalus and renal cystic disease in both humans and rodent models. The precise pathogenic mechanisms remain undetermined. Herein it is reported for the first time that a point mutation of TMEM67 leads to a gene dose-dependent hydrocephalic phenotype in the Wistar polycystic kidney (Wpk) rat. Animals with TMEM67 heterozygous mutations manifest slowly progressing hydrocephalus, observed during the postnatal period and continuing into adulthood. These animals have no overt renal phenotype. The TMEM67 homozygous mutant rats have severe ventriculomegaly as well as severe polycystic kidney disease and die during the neonatal period. Protein localization in choroid plexus epithelial cells indicates that aquaporin 1 and claudin-1 both remain normally polarized in all genotypes. The choroid plexus epithelial cells may have selectively enhanced permeability as evidenced by increased Na+, K+ and Cl- in the cerebrospinal fluid of the severely hydrocephalic animals. Collectively, these results suggest that TMEM67 is required for the regulation of choroid plexus epithelial cell fluid and electrolyte homeostasis. The Wpk rat model, orthologous to human MKS3, provides a unique platform to study the development of both severe and mild hydrocephalus.
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Transtornos da Motilidade Ciliar/metabolismo , Encefalocele/metabolismo , Hidrocefalia/metabolismo , Proteínas de Membrana/metabolismo , Doenças Renais Policísticas/metabolismo , Retinose Pigmentar/metabolismo , Animais , Encéfalo/metabolismo , Cloretos/líquido cefalorraquidiano , Plexo Corióideo/metabolismo , Transtornos da Motilidade Ciliar/genética , Encefalocele/genética , Feminino , Hidrocefalia/genética , Proteínas de Membrana/genética , Mutação/genética , Doenças Renais Policísticas/genética , Potássio/líquido cefalorraquidiano , Ratos , Retinose Pigmentar/genética , Sódio/líquido cefalorraquidianoRESUMO
We present a cohort of 41 patients with osimertinib resistance biopsies, including 2 with an acquired CCDC6-RET fusion. Although RET fusions have been identified in resistant EGFR-mutant non-small cell lung cancer (NSCLC), their role in acquired resistance to EGFR inhibitors is not well described. To assess the biological implications of RET fusions in an EGFR-mutant cancer, we expressed CCDC6-RET in PC9 (EGFR del19) and MGH134 (EGFR L858R/T790M) cells and found that CCDC6-RET was sufficient to confer resistance to EGFR tyrosine kinase inhibitors (TKI). The selective RET inhibitors BLU-667 and cabozantinib resensitized CCDC6-RET-expressing cells to EGFR inhibition. Finally, we treated 2 patients with EGFR-mutant NSCLC and RET-mediated resistance with osimertinib and BLU-667. The combination was well tolerated and led to rapid radiographic response in both patients. This study provides proof of concept that RET fusions can mediate acquired resistance to EGFR TKIs and that combined EGFR and RET inhibition with osimertinib/BLU-667 may be a well-tolerated and effective treatment strategy for such patients. SIGNIFICANCE: The role of RET fusions in resistant EGFR-mutant cancers is unknown. We report that RET fusions mediate resistance to EGFR inhibitors and demonstrate that this bypass track can be effectively targeted with a selective RET inhibitor (BLU-667) in the clinic.This article is highlighted in the In This Issue feature, p. 1494.
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Acrilamidas/uso terapêutico , Compostos de Anilina/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Acrilamidas/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Compostos de Anilina/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismoRESUMO
The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of ALK mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4-ALK. Under comparable conditions, N-ethyl-N-nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single ALK mutations. In similar screens with EML4-ALK containing single ALK resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound ALK mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound ALK mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of ALK mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound ALK mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies.Significance: Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound ALK mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance. Cancer Discov; 8(6); 714-29. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.
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Quinase do Linfoma Anaplásico/genética , Resistencia a Medicamentos Antineoplásicos , Lactamas Macrocíclicas/administração & dosagem , Neoplasias Pulmonares/genética , Mutação , Aminopiridinas , Animais , Linhagem Celular Tumoral , Crizotinibe/administração & dosagem , Crizotinibe/farmacologia , Etilnitrosoureia/efeitos adversos , Feminino , Humanos , Lactamas , Lactamas Macrocíclicas/farmacologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Proteínas de Fusão Oncogênica/genética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Pirazóis , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The purinergic receptor subtype 7 (P2X7R) represents a novel molecular target for imaging neuroinflammation via PET. GSK1482160, a potent P2X7R antagonist, has high receptor affinity, high blood-brain barrier penetration, and the ability to be radiolabeled with 11C. We report the initial physical and biologic characterization of this novel ligand. Methods:11C-GSK1482160 was synthesized according to published methods. Cell density studies were performed on human embryonic kidney cell lines expressing human P2X7R (HEK293-hP2X7R) and underwent Western blotting, an immunofluorescence assay, and radioimmunohistochemistry analysis using P2X7R polyclonal antibodies. Receptor density and binding potential were determined by saturation and association-disassociation kinetics, respectively. Peak immune response to lipopolysaccharide treatment in mice was determined in time course studies and analyzed via Iba1 and P2X7R Western blotting and Iba1 immunohistochemistry. Whole-animal biodistribution studies were performed on saline- or lipopolysaccharide-treated mice at 15, 30, and 60 min after radiotracer administration. Dynamic in vivo PET/CT was performed on the mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking, and 2-compartment, 5-parameter tracer kinetic modeling of brain regions was performed. Results: P2X7R changed linearly with concentrations or cell numbers. For high-specific-activity 11C-GSK1482160, receptor density and Kd were 1.15 ± 0.12 nM and 3.03 ± 0.10 pmol/mg, respectively, in HEK293-hP2X7R membranes. Association constant kon, dissociation constant koff, and binding potential (kon/koff) in HEK293-hP2X7R cells were 0.2312 ± 0.01542 min-1â nM-1, 0.2547 ± 0.0155 min-1, and 1.0277 ± 0.207, respectively. Whole-brain Iba1 expression in lipopolysaccharide-treated mice peaked by 72 h on immunohistochemistry, and Western blot analysis of P2X7R for saline- and lipopolysaccharide-treated brain sections showed a respective 1.8- and 1.7-fold increase in signal enhancement at 72 h. Biodistribution of 11C-GSK1482160 in saline- and lipopolysaccharide-treated mice at 72 h was statistically significant across all tissues studied. In vivo dynamic 11C-GSK1482160 PET/CT of mice at 72 h after administration of saline, lipopolysaccharide, or lipopolysaccharide + blocking showed a 3.2-fold increase and 97% blocking by 30 min. The total distribution volumes for multiple cortical regions and the hippocampus showed statistically significant increases and were blocked by an excess of authentic standard GSK1482160. Conclusion: The current study provides compelling data that support the suitability of 11C-GSK1482160 as a radioligand targeting P2X7R, a biomarker of neuroinflammation.
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Encefalite/diagnóstico por imagem , Encefalite/metabolismo , Mediadores da Inflamação/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Ácido Pirrolidonocarboxílico/farmacocinética , Receptores Purinérgicos P2X7/metabolismo , Animais , Biomarcadores/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Radioisótopos de Carbono/farmacocinética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular/métodos , Compostos Radiofarmacêuticos/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Renal sympathetic denervation (RD) is a promising method of neuromodulation for the management of cardiac arrhythmia. OBJECTIVE: We tested the hypothesis that RD is antiarrhythmic in ambulatory dogs because it reduces the stellate ganglion nerve activity (SGNA) by remodeling the stellate ganglion (SG) and brain stem. METHODS: We implanted a radiotransmitter to record SGNA and electrocardiogram in 9 ambulatory dogs for 2 weeks, followed by a second surgery for RD and 2 months SGNA recording. Cell death was probed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. RESULTS: Integrated SGNA at baseline and 1 and 2 months after RD were 14.0 ± 4.0, 9.3 ± 2.8, and 9.6 ± 2.0 µV, respectively (P = .042). The SG from RD but not normal control dogs (n = 5) showed confluent damage. An average of 41% ± 10% and 40% ± 16% of ganglion cells in the left and right SG, respectively, were TUNEL positive in RD dogs compared with 0% in controls dogs (P = .005 for both). The left and right SG from RD dogs had more tyrosine hydroxylase-negative ganglion cells than did the left SG of control dogs (P = .028 and P = .047, respectively). Extensive TUNEL-positive neurons and glial cells were also noted in the medulla, associated with strongly positive glial fibrillary acidic protein staining. The distribution was heterogeneous, with more cell death in the medial than lateral aspects of the medulla. CONCLUSION: Bilateral RD caused significant central and peripheral sympathetic nerve remodeling and reduced SGNA in ambulatory dogs. These findings may in part explain the antiarrhythmic effects of RD.
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Arritmias Cardíacas/cirurgia , Tronco Encefálico/fisiopatologia , Rim/inervação , Gânglio Estrelado/fisiopatologia , Simpatectomia , Sistema Nervoso Simpático/cirurgia , Animais , Cães , Frequência Cardíaca , Plasticidade Neuronal , Monitorização Neurofisiológica/métodos , Simpatectomia/efeitos adversos , Simpatectomia/métodos , Resultado do TratamentoRESUMO
A growing number of studies have investigated combination treatment as an approach to treat bone disease. The goal of this study was to investigate the combination of alendronate and raloxifene with a particular focus on mechanical properties. To achieve this goal we utilized a large animal model, the beagle dog, used previously by our laboratory to study both alendronate and raloxifene monotherapies. Forty-eight skeletally mature female beagles (1-2 years old) received daily oral treatment: saline vehicle (VEH), alendronate (ALN), raloxifene (RAL) or both ALN and RAL. After 6 and 12 months of treatment, all animals underwent assessment of bone material properties using in vivo reference point indentation (RPI) and skeletal hydration using ultra-short echo magnetic resonance imaging (UTE-MRI). End point measures include imaging, histomorphometry, and mechanical properties. Bone formation rate was significantly lower in iliac crest trabecular bone of animals treated with ALN (-71%) and ALN+RAL (-81%) compared to VEH. In vivo assessment of properties by RPI yielded minimal differences between groups while UTE-MRI showed a RAL and RAL+ALN treatment regimens resulted in significantly higher bound water compared to VEH (+23 and +18%, respectively). There was no significant difference among groups for DXA- or CT-based measures lumbar vertebra, or femoral diaphysis. Ribs of RAL-treated animals were smaller and less dense compared to VEH and although mechanical properties were lower the material-level properties were equivalent to normal. In conclusion, we present a suite of data in a beagle dog model treated for one year with clinically-relevant doses of alendronate and raloxifene monotherapies or combination treatment with both agents. Despite the expected effects on bone remodeling, our study did not find the expected benefit of ALN to BMD or structural mechanical properties, and thus the viability of the combination therapy remains unclear.
Assuntos
Alendronato/farmacologia , Conservadores da Densidade Óssea/farmacologia , Densidade Óssea/efeitos dos fármacos , Diáfises/fisiologia , Fêmur/fisiologia , Vértebras Lombares/fisiologia , Cloridrato de Raloxifeno/farmacologia , Alendronato/efeitos adversos , Animais , Remodelação Óssea/efeitos dos fármacos , Diáfises/efeitos dos fármacos , Cães , Quimioterapia Combinada/efeitos adversos , Feminino , Fêmur/efeitos dos fármacos , Vértebras Lombares/efeitos dos fármacos , Imageamento por Ressonância Magnética , Modelos Animais , Osteoporose/tratamento farmacológico , Cloridrato de Raloxifeno/efeitos adversosRESUMO
Personalized cancer therapy is based on a patient's tumor lineage, histopathology, expression analyses, and/or tumor DNA or RNA analysis. Here, we aim to develop an in vitro functional assay of a patient's living cancer cells that could complement these approaches. We present methods for developing cell cultures from tumor biopsies and identify the types of samples and culture conditions associated with higher efficiency of model establishment. Toward the application of patient-derived cell cultures for personalized care, we established an immunofluorescence-based functional assay that quantifies cancer cell responses to targeted therapy in mixed cell cultures. Assaying patient-derived lung cancer cultures with this method showed promise in modeling patient response for diagnostic use. This platform should allow for the development of co-clinical trial studies to prospectively test the value of drug profiling on tumor-biopsy-derived cultures to direct patient care.
Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias/tratamento farmacológico , Medicina de Precisão/métodos , Cultura Primária de Células/métodos , Acrilamidas , Aminopiridinas , Quinase do Linfoma Anaplásico , Compostos de Anilina , Biomarcadores Tumorais/metabolismo , Biópsia , Crizotinibe , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/uso terapêutico , Células Alimentadoras/citologia , Imunofluorescência/métodos , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Queratina-18/genética , Queratina-18/metabolismo , Queratina-8/genética , Queratina-8/metabolismo , Lactamas , Lactamas Macrocíclicas/uso terapêutico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Mutação , Neoplasias/classificação , Neoplasias/genética , Neoplasias/patologia , Piperazinas/uso terapêutico , Pirazóis/uso terapêutico , Piridinas/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Células Tumorais CultivadasRESUMO
Recent advancements in PET instrumentation have made the non-invasive assessment of cardiovascular function in small animals a reality. The majority of small animal PET systems use stationary detector gantries, thus affording high temporal resolution imaging of cardiac function. Systems designed to maximize spatial resolution and detection sensitivity employing rotating gantry designs are suboptimal when high temporal resolution imaging is needed. To overcome this limitation, the current work developed a novel view-sharing data analysis scheme suitable for dynamic cardiac PET imaging using 18F-NaF as the tracer and tracer kinetic model analysis. This scheme was tested in a rat model of cardiovascular function where the relationship between direct transonic flow measures of cardiac output were highly correlated (f(x) = 1.0216x - 24.233, R = 0.9158, p < 0.001) with the new model. Similarly, derived measures of stroke volume were also highly correlated (f(x) = 0.9655x - 0.0428, R = 0.9453, p < 0.001) with the current approach. Administration of xylazine caused a statistically significant increase in stroke volume (0.32 ± 0.07 ml, p = 0.003, n = 4) and a significant decrease in both heart rate (-155 ± 7.1 beats/min, p < 0.001, n = 4) and cardiac output (-75.9 ± 23.0 ml/kg min, p = 0.01, n = 4). These findings suggest that the new sinogram binning and kinetic modeling methods produce reliable cardiac function measures suitable for longitudinal monitoring of cardiovascular function.
RESUMO
Raloxifene positively affects mechanical properties of the bone matrix in part through modification of skeletal-bound water. The goal of this study was to determine if raloxifene-induced alterations in skeletal hydration could be measured in vivo using ultra-short echotime magnetic resonance imaging (UTE-MRI). Twelve skeletally mature female beagle dogs (n = 6/group) were treated for 6 months with oral doses of saline vehicle (VEH, 1 mL/kg/d) or raloxifene (RAL, 0.5 mg/kg/d). After 6 months of treatment, all animals underwent in vivo UTE-MRI of the proximal tibial cortical bone. UTE-MRI signal intensity versus echotime curves were analyzed by fitting a double exponential to determine the short and long relaxation times of water with the bone (dependent estimations of bound and free water, respectively). Raloxifene-treated animals had significantly higher bound water (+14%; p = 0.05) and lower free water (-20%) compared with vehicle-treated animals. These data provide the first evidence that drug-induced changes in skeletal hydration can be noninvasively assessed using UTE-MRI.