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1.
Bioconjug Chem ; 35(2): 254-264, 2024 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-38308817

RESUMO

Preclinical models of neurological diseases and gene therapy are essential for neurobiological research. However, the evaluation of such models lacks reliable reporter systems for use with noninvasive imaging methods. Here, we report the development of a reporter system based on the CLIP-tag enzyme and [18F]pFBC, an 18F-labeled covalent CLIP-tag-ligand synthesized via a DoE-optimized and fully automated process. We demonstrated its specificity using a subcutaneous xenograft model and a model of viral vector-mediated brain gene transfer by engineering HEK293 cells and striatal neurons to express membrane-tethered CLIP-tag protein. After in vitro characterization of the reporter, mice carrying either CLIP-tag expressing or control subcutaneous xenografts underwent dynamic [18F]pFBC PET imaging. The CLIP-tag expressing xenografts showed a significantly higher uptake than control xenografts (tumor-to-muscle ratio 5.0 vs 1.7, p = 0.0379). In vivo, metabolite analysis by radio-HPLC from plasma and brain homogenates showed only one radio-metabolite in plasma and none in the brain. In addition, [18F]pFBC showed fast uptake and rapid clearance from the brain in animals injected with adeno-associated virus (AAV)-CLIP in the right striatum but no right-to-left (R-L) uptake difference in the striata in the acquired PET data. In contrast, autoradiography showed a clear accumulation of radioactivity in the AAV-CLIP-injected right striatum compared to the sham-injected left striatum control. CLIP-tag expression and brain integrity were verified by immunofluorescence and light sheet microscopy. In conclusion, we established a novel reporter gene system for PET imaging of gene expression in the brain and periphery and demonstrated its potential for a wide range of applications, particularly for neurobiological research and gene therapy with viral vectors.


Assuntos
Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Humanos , Camundongos , Animais , Genes Reporter , Células HEK293 , Compostos Radiofarmacêuticos/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo
2.
Curr Biol ; 32(2): 412-427.e8, 2022 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-34883047

RESUMO

Hypoxia, through hypoxia inducible factor (HIF), drives cancer cell invasion and metastatic progression in various cancer types. In epithelial cancer, hypoxia induces the transition to amoeboid cancer cell dissemination, yet the molecular mechanisms, relevance for metastasis, and effective intervention to combat hypoxia-induced amoeboid reprogramming remain unclear. Here, we identify calpain-2 as a key regulator and anti-metastasis target of hypoxia-induced transition from collective to amoeboid dissemination of breast and head and neck (HN) carcinoma cells. Hypoxia-induced amoeboid dissemination occurred through low extracellular matrix (ECM)-adhesive, predominantly bleb-based amoeboid movement, which was maintained by a low-oxidative and -glycolytic energy metabolism ("eco-mode"). Hypoxia induced calpain-2-mediated amoeboid conversion by deactivating ß1 integrins through enzymatic cleavage of the focal adhesion adaptor protein talin-1. Consequently, targeted downregulation or pharmacological inhibition of calpain-2 restored talin-1 integrity and ß1 integrin engagement and reverted amoeboid to elongated phenotypes under hypoxia. Calpain-2 activity was required for hypoxia-induced amoeboid conversion in the orthotopic mouse dermis and upregulated in invasive HN tumor xenografts in vivo, and attenuation of calpain activity prevented hypoxia-induced metastasis to the lungs. This identifies the calpain-2/talin-1/ß1 integrin axis as a druggable mechanosignaling program that conserves energy yet enables metastatic dissemination that can be reverted by interfering with calpain activity.


Assuntos
Calpaína , Neoplasias de Cabeça e Pescoço , Animais , Calpaína/genética , Calpaína/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Humanos , Hipóxia , Integrina beta1/genética , Camundongos , Metástase Neoplásica , Talina/genética , Talina/metabolismo
3.
Front Immunol ; 9: 2199, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30337922

RESUMO

It is increasingly clear that inflammatory diseases and cancers are influenced by cleavage products of the pro-hormone chromogranin A (CgA), such as the 21-amino acids long catestatin (CST). The goal of this review is to provide an overview of the anti-inflammatory effects of CST and its mechanism of action. We discuss evidence proving that CST and its precursor CgA are crucial for maintaining metabolic and immune homeostasis. CST could reduce inflammation in various mouse models for diabetes, colitis and atherosclerosis. In these mouse models, CST treatment resulted in less infiltration of immune cells in affected tissues, although in vitro monocyte migration was increased by CST. Both in vivo and in vitro, CST can shift macrophage differentiation from a pro- to an anti-inflammatory phenotype. Thus, the concept is emerging that CST plays a role in tissue homeostasis by regulating immune cell infiltration and macrophage differentiation. These findings warrant studying the effects of CST in humans and make it an interesting therapeutic target for treatment and/or diagnosis of various metabolic and immune diseases.


Assuntos
Aterosclerose , Cromogranina A/imunologia , Colite , Diabetes Mellitus Experimental , Sistemas de Liberação de Medicamentos/métodos , Macrófagos , Fragmentos de Peptídeos/imunologia , Animais , Aterosclerose/tratamento farmacológico , Aterosclerose/imunologia , Aterosclerose/patologia , Diferenciação Celular/imunologia , Colite/tratamento farmacológico , Colite/imunologia , Colite/patologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/patologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos
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