RESUMEN

BACKGROUND: Claudin 18.2 (CLDN18.2) is a promising target for targeted therapies in gastric cancer (GC). This study investigated the prevalence of CLDN18.2 expression in patients with stages II-IV GC or gastroesophageal junction (GEJ) adenocarcinoma and its correlation with clinicopathologic features and other crucial GC biomarkers. METHODS: We enrolled 1000 patients diagnosed with stages II-IV GC after surgical treatment. Immunohistochemistry for CLDN18 (43-14A clone), PD-L1 (22C3 pharmDx), HER2, and FGFR2 was performed. CLDN18.2 positivity was defined as moderate-to-strong (2+/3+) membranous staining in ≥75% of tumor cells. CLDN18.2 expression was compared with biomarker expression, Epstein-Barr virus (EBV) association and microsatellite instability status, and clinicopathologic features. RESULT: CLDN18.2 was positive in 34.4% of the patients. CLDN18.2 positivity was significantly higher in the middle and upper thirds than in the lower third gastric location (P < .001), but there was no correlation with age, sex, or stage (P > .05). CLDN18.2 positivity was rare (2.8%) in mucinous adenocarcinoma but frequent (90.9%) in a majority of gastric carcinomas with lymphoid stroma. CLDN18.2 positivity was higher in EBV-associated (P < .001) and PD-L1-positive (PD-L1 CPS ≥ 5) GC (P = .014) but lower in HER2 positive GC (P = .005). CLDN18.2 positivity was not significantly associated with overall survival and disease-free survival. CONCLUSION: This study provides a comprehensive evaluation of CLDN18.2 status and its correlation with the clinicopathologic characteristics of patients with stages II-IV GC in Korea and with crucial biomarkers. It may be valuable for guiding future drug development, expanding treatment options, and ultimately improving patient outcomes in GC.

RESUMEN

Extracellular vesicles (EVs) have emerged as novel biomarkers and therapeutic material. However, the small size (~200 nm) of EVs makes efficient separation challenging. Here, a physical/chemical stress-free separation of EVs based on diffusion through a nanoporous membrane chip is presented. A polycarbonate membrane with 200 nm pores, positioned between two chambers, functions as the size-selective filter. Using the chip, EVs from cell culture media and human serum were separated. The separated EVs were analyzed by nanoparticle tracking analysis (NTA), scanning electron microscopy, and immunoblotting. The experimental results proved the selective separation of EVs in cell culture media and human serum. Moreover, the diffusion-based separation showed a high yield of EVs in human serum compared to ultracentrifuge-based separation. The EV recovery rate analyzed from NTA data was 42% for cell culture media samples. We expect the developed method to be a potential tool for EV separation for diagnosis and therapy because it does not require complicated processes such as immune, chemical reaction, and external force and is scalable by increasing the nanoporous membrane size.

Asunto(s)

Vesículas Extracelulares , Nanoporos , Medios de Cultivo , Humanos , Dispositivos Laboratorio en un Chip , Nanopartículas , Suero

RESUMEN

Extracellular vesicles (EVs) are the cell-secreted nano- and micro-sized particles consisted of lipid bilayer containing nucleic acids and proteins for diagnosis and therapeutic applications. The inherent complexity of EVs is a source of heterogeneity in various potential applications of the biological nanovesicles including analysis. To diminish heterogeneity, EV should be isolated and separated according to their sizes and cargos. However, current technologies do not meet the requirements. We showed noninvasive and precise separation of EVs based on their sizes without any recognizable damages. We separated atto-liter volumes of biological nanoparticles through operation of the present system showing relatively large volume of sample treatment to milliliters within an hour. We observed distinct size and morphological differences of 30 to 100 nm of exosomes and apoptotic bodies through TEM analysis. Indeed, we confirmed the biological moiety variations through immunoblotting with noninvasively separated EVs opening new windows in study and application of the biological nanoparticles.

Asunto(s)

Medios de Cultivo Condicionados/metabolismo , Exosomas/metabolismo , Vesículas Extracelulares/metabolismo , Microfluídica/métodos , Nanopartículas/química , Animales , Exosomas/ultraestructura , Vesículas Extracelulares/ultraestructura , Humanos , Immunoblotting , Membrana Dobles de Lípidos/aislamiento & purificación , Membrana Dobles de Lípidos/metabolismo , Microscopía Electrónica de Transmisión , Nanopartículas/ultraestructura , Neoplasias/metabolismo , Neoplasias/patología , Ácidos Nucleicos/aislamiento & purificación , Ácidos Nucleicos/metabolismo , Tamaño de la Partícula , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Reproducibilidad de los Resultados

RESUMEN

Aminoacyl-tRNA synthetases (ARSs), enzymes that normally control protein synthesis, can be secreted and have different activities in the extracellular space, but the mechanism of their secretion is not understood. This study describes the secretion route of the ARS lysyl-tRNA synthetase (KRS) and how this process is regulated by caspase activity, which has been implicated in the unconventional secretion of other proteins. We show that KRS is secreted from colorectal carcinoma cells within the lumen of exosomes that can trigger an inflammatory response. Caspase-8 cleaved the N-terminal of KRS, thus exposing a PDZ-binding motif located in the C terminus of KRS. Syntenin bound to the exposed PDZ-binding motif of KRS and facilitated the exosomic secretion of KRS dissociated from the multi-tRNA synthetase complex. KRS-containing exosomes released by cancer cells induced macrophage migration, and their secretion of TNF-α and cleaved KRS made a significant contribution to these activities, which suggests a novel mechanism by which caspase-8 may promote inflammation.

Asunto(s)

Caspasa 8/metabolismo , Neoplasias Colorrectales/enzimología , Exosomas/enzimología , Mediadores de Inflamación/metabolismo , Lisina-ARNt Ligasa/metabolismo , Animales , Caspasa 8/genética , Quimiotaxis , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Exosomas/genética , Exosomas/metabolismo , Exosomas/patología , Células HCT116 , Humanos , Lisina-ARNt Ligasa/genética , Macrófagos/metabolismo , Ratones , Complejos Multienzimáticos , Dominios PDZ , Unión Proteica , Células RAW 264.7 , Interferencia de ARN , Transducción de Señal , Sinteninas/metabolismo , Factores de Tiempo , Transfección , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Tumor permeability is a critical determinant of drug delivery and sensitivity, but systematic methods to identify factors that perform permeability barrier functions in the tumor microenvironment are not yet available. Multicellular tumor spheroids have become tractable in vitro models to study the impact of a three-dimensional (3D) environment on cellular behavior. In this study, we characterized the spheroid-forming potential of cancer cells and correlated the resulting spheroid morphologies with genetic information to identify conserved cellular processes associated with spheroid structure. Spheroids generated from 100 different cancer cell lines were classified into four distinct groups based on morphology. In particular, round and compact spheroids exhibited highly hypoxic inner cores and permeability barriers against anticancer drugs. Through systematic and correlative analysis, we reveal JAK-STAT signaling as one of the signature pathways activated in round spheroids. Accordingly, STAT3 inhibition in spheroids generated from the established cancer cells and primary glioblastoma patient-derived cells altered the rounded morphology and increased drug sensitivity. Furthermore, combined administration of the STAT3 inhibitor and 5-fluorouracil to a mouse xenograft model markedly reduced tumor growth compared with monotherapy. Collectively, our findings demonstrate the ability to integrate 3D culture and genetic profiling to determine the factors underlying the integrity of the permeability barrier in the tumor microenvironment, and may help to identify and exploit novel mechanisms of drug resistance.

Asunto(s)

Neoplasias/patología , Factor de Transcripción STAT3/fisiología , Microambiente Tumoral , Animales , Benzoquinonas/farmacología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Resistencia a Antineoplásicos , Fluorouracilo/farmacología , Humanos , Quinasas Janus/fisiología , Lactamas Macrocíclicas/farmacología , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Transducción de Señal , Esferoides Celulares , Tirfostinos/farmacología

RESUMEN

A 7-mer peptide (S-T-L-P-L-P-P) that bound to various divalent cations was selected from a phage display peptide library. Isothermal calorimetric analysis revealed that the peptide bound to Pb²âº, Cd²âº, Hg²âº, and Cu²âº. Through the use of CD studies, no secondary structural changes were observed for the peptide upon binding to divalent cations. Ala scanning mutant peptides bound to Hg²âº with a reduced affinity. However, no single substitution was shown to affect the overall affinity. We suggest that Pro residues chelate divalent cations, while the structure formed by the peptide is also important for the binding process.

Asunto(s)

Cadmio/química , Cobre/química , Plomo/química , Mercurio/química , Péptidos/química , Sitios de Unión , Calorimetría , Cationes Bivalentes/química