RESUMO
Recapitulation of the tumor microenvironment is critical for probing mechanisms involved in cancer, and for evaluating the tumor-killing potential of chemotherapeutic agents, targeted therapies and immunotherapies. Microfluidic devices have emerged as valuable tools for both mechanistic studies and for preclinical evaluation of therapeutic agents, due to their ability to precisely control drug concentrations and gradients of oxygen and other species in a scalable and potentially high throughput manner. Most existing in vitro microfluidic cancer models are comprised of cultured cancer cells embedded in a physiologically relevant matrix, collocated with vascular-like structures. However, the recent emergence of immune checkpoint inhibitors (ICI) as a powerful therapeutic modality against many cancers has created a need for preclinical in vitro models that accommodate interactions between tumors and immune cells, particularly for assessment of unprocessed tumor fragments harvested directly from patient biopsies. Here we report on a microfluidic model, termed EVIDENT (ex vivo immuno-oncology dynamic environment for tumor biopsies), that accommodates up to 12 separate tumor biopsy fragments interacting with flowing tumor-infiltrating lymphocytes (TILs) in a dynamic microenvironment. Flow control is achieved with a single pump in a simple and scalable configuration, and the entire system is constructed using low-sorption materials, addressing two principal concerns with existing microfluidic cancer models. The system sustains tumor fragments for multiple days, and permits real-time, high-resolution imaging of the interaction between autologous TILs and tumor fragments, enabling mapping of TIL-mediated tumor killing and testing of various ICI treatments versus tumor response. Custom image analytic algorithms based on machine learning reported here provide automated and quantitative assessment of experimental results. Initial studies indicate that the system is capable of quantifying temporal levels of TIL infiltration and tumor death, and that the EVIDENT model mimics the known in vivo tumor response to anti-PD-1 ICI treatment of flowing TILs relative to isotype control treatments for syngeneic mouse MC38 tumors.
Assuntos
Técnicas Analíticas Microfluídicas/instrumentação , Modelos Biológicos , Microambiente Tumoral/imunologia , Animais , Carcinoma Pulmonar de Células não Pequenas/química , Carcinoma Pulmonar de Células não Pequenas/imunologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Células Cultivadas , Desenho de Equipamento , Humanos , Processamento de Imagem Assistida por Computador/métodos , Neoplasias Pulmonares/química , Neoplasias Pulmonares/imunologia , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos , Técnicas Analíticas Microfluídicas/métodosRESUMO
The neurotoxic actions of methamphetamine (METH) may be mediated in part by reactive oxygen species (ROS). Methamphetamine administration leads to increases in ROS formation and lipid peroxidation in rodent brain; however, the extent to which proteins may be modified or whether affected brain regions exhibit similar elevations of lipid and protein oxidative markers have not been investigated. In this study we measured concentrations of TBARs, protein carbonyls and monoamines in various mouse brain regions at 4 h and 24 h after the last of four injections of METH (10 mg/kg/injection q 2 h). Substantial increases in TBARs and protein carbonyls were observed in the striatum and hippocampus but not the frontal cortex nor the cerebellum of METH-treated mice. Furthermore, lipid and protein oxidative markers were highly correlated within each brain region. In the hippocampus and striatum elevations in oxidative markers were significantly greater at 24 h than at 4 h. Monoamine levels were maximally reduced within 4 h (striatal dopamine [DA] by 95% and serotonin [5-HT] in striatum, cortex and hippocampus by 60-90%). These decrements persisted for 7 days after METH, indicating effects reflective of nerve terminal damage. Interestingly, NE was only transiently depleted in the brain regions investigated (hippocampus and cortex), suggesting a pharmacological and non-toxic action of METH on the noradrenergic nerve terminals. This study provides the first evidence for concurrent formation of lipid and protein markers of oxidative stress in several brain regions of mice that are severely affected by large neurotoxic doses of METH. Moreover, the differential time course for monoamine depletion and the elevations in oxidative markers indicate that the source of oxidative stress is not derived directly from DA or 5HT oxidation.
Assuntos
Biomarcadores/análise , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Peroxidação de Lipídeos , Metanfetamina/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Monoaminas Biogênicas/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Masculino , Camundongos , Oxirredução , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Serotonina/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/análiseRESUMO
Defects in mitochondrial energy metabolism have been implicated in the pathology of several neurodegenerative disorders. In addition, the reactive metabolites generated from the metabolism and oxidation of the neurotransmitter dopamine (DA) are thought to contribute to the damage to neurons of the basal ganglia. We have previously demonstrated that infusions of the metabolic inhibitor malonate into the striata of mice or rats produce degeneration of DA nerve terminals. In the present studies, we demonstrate that an intrastriatal infusion of malonate induces a substantial increase in DA efflux in awake, behaving mice as measured by in vivo microdialysis. Furthermore, pretreatment of mice with tetrabenazine (TBZ) or the TBZ analogue Ro 4-1284 (Ro-4), compounds that reversibly inhibit the vesicular storage of DA, attenuates the malonate-induced DA efflux as well as the damage to DA nerve terminals. Consistent with these findings, the damage to both DA and GABA neurons in mesencephalic cultures by malonate exposure was attenuated by pretreatment with TBZ or Ro-4. Treatment with these compounds did not affect the formation of free radicals or the inhibition of oxidative phosphorylation resulting from malonate exposure alone. Our data suggest that DA plays an important role in the neurotoxicity produced by malonate. These findings provide direct evidence that inhibition of succinate dehydrogenase causes an increase in extracellular DA levels and indicate that bioenergetic defects may contribute to the pathogenesis of chronic neurodegenerative diseases through a mechanism involving DA.
Assuntos
Corpo Estriado/citologia , Dopamina/fisiologia , Malonatos/farmacologia , Proteínas de Membrana Transportadoras , Neurônios/metabolismo , Neuropeptídeos , 2-etil-1,3,4,6,7,11b-hexaidro-3-isobutil-9,10-dimetoxi-2H-benzo(a)quinolizin-2-ol/farmacologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Inibidores da Captação Adrenérgica/farmacologia , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Radioisótopos de Carbono , Células Cultivadas , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopamina/farmacocinética , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Radicais Livres/metabolismo , Ácido Láctico/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Microdiálise , Degeneração Neural/induzido quimicamente , Degeneração Neural/metabolismo , Neurônios/efeitos dos fármacos , Doença de Parkinson/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Tetrabenazina/farmacologia , Trítio , Proteínas Vesiculares de Transporte de Aminas Biogênicas , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacocinéticaRESUMO
The neuroprotective effects of lowering body temperature have been well documented in various models of neuronal injury. The present study investigated the effects a lower ambient or core body temperature would have on damage to striatal dopamine (DA) neurons produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Mice received systemic MPTP treatment at two different temperatures, 4 degrees C and 22 degrees C. MPTP-treated mice maintained at 4 degrees C demonstrated (1) a greater hypothermic response, (2) a significant reduction in striatal DA content and tyrosine hydroxylase (TH) activity, and (3) significantly greater striatal 1-methyl-4-phenylpyridinium (MPP+) levels, as compared to mice dosed with MPTP at room temperature. Parallel studies with methamphetamine (METH) were conducted since temperature appears to play a pivotal role in the mediation of damage to DA neurons by this CNS stimulant in rodents. As previously reported, METH-induced hyperthermia and the subsequent loss of striatal DA content were attenuated in animals dosed at 4 degrees C. We also evaluated the effects a hypothermic state induced by pharmacological agents would have on striatal neurochemistry and MPP+ levels following MPTP treatment. Concurrent administration of MK-801 or 8-OHDPAT increased the striatal MPP+ levels following MPTP treatment. However, only 8-OHDPAT potentiated the MPTP-induced decrements of striatal DA content and TH activity; MK-801 did not affect MPTP decreases in these striatal markers of dopaminergic damage. Altogether, these findings indicate that temperature has a profound effect on striatal MPP+ levels and MPTP-induced damage to DA neurons in mice.
Assuntos
1-Metil-4-fenilpiridínio/toxicidade , Temperatura Corporal/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopaminérgicos/toxicidade , Intoxicação por MPTP , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacocinética , 1-Metil-4-fenilpiridínio/farmacocinética , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Corpo Estriado/citologia , Maleato de Dizocilpina/farmacologia , Dopamina/metabolismo , Dopaminérgicos/farmacocinética , Antagonistas de Aminoácidos Excitatórios/farmacologia , Metanfetamina/farmacologia , Camundongos , Degeneração Neural/induzido quimicamente , Degeneração Neural/metabolismo , Neurônios/química , Neurônios/enzimologia , Neurotoxinas/farmacocinética , Agonistas do Receptor de Serotonina/farmacologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Major advances have recently been made in understanding the nucleotide excision repair pathway in mammalian cells. Although the signaling events responsible for initiating this process are not known, they probably involve proteins, i.e., damage recognition proteins (DRPs), which detect specific types of DNA damage. In this report, we describe a technique for labeling DNA damage recognition proteins. The procedure utilizes iodogen to radio-iodinate proteins bound to DNA modified with the cancer chemotherapy drug, cisplatin. Following iodination, bound proteins are eluted and analyzed on SDS-polyacrylamide gels. We have optimized this procedure such that the labeling reactions are rapid and employ small amounts of 125I. Using this procedure, we demonstrate that proteins of 28 and 40 kDa in MCF7 human breast epithelial cells bind to CDDP-DNA. This technique is sensitive and potentially will facilitate the identification of DRPs in samples containing limited amounts of protein, such as small tissue biopsy specimens obtained from patients undergoing diagnostic and/or therapeutic treatment.
Assuntos
Dano ao DNA , Proteínas de Ligação a DNA/isolamento & purificação , Animais , Mama/metabolismo , Bovinos , Linhagem Celular , Cisplatino/metabolismo , Adutos de DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Feminino , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/isolamento & purificação , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Técnicas In Vitro , Radioisótopos do Iodo , Peso MolecularRESUMO
Epidemiological studies suggest that human populations consuming diets rich in protease inhibitors have a reduced incidence of cancer at several sites including the breast. Protease inhibitors, such as the Bowman-Birk inhibitor (BBI) have been shown to be highly effective at suppressing carcinogenesis in a variety of experimental model systems. In this study, we have identified a protease activity in human breast epithelial cells which is inhibited by BBI. This enzyme has a molecular mass of 43 kDa, cleaves gelatin and is primarily localized in the cytosol. Protease activity is maximal at pH 8 and is inhibited by DFP, but unaffected by EDTA, indicating that this enzyme is a serine protease. The protease identified in MCF7 cells has characteristics which are similar to a protease present in human fibroblasts. Hence, our results suggest that BBI targets a common enzyme in human epithelial cells and fibroblasts.