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1.
J Vis Exp ; (198)2023 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-37607103

RESUMEN

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility adjustments. Conventionally, their contractility is gauged by observing morphological shifts and nearby capillary diameter changes under specific circumstances. Yet, post-tissue fixation, evaluating vitality and ensuing pericyte contractility of imaged brain pericytes becomes compromised. Similarly, genetically labeling brain pericytes falls short in distinguishing between viable and non-viable pericytes, particularly in neurologic conditions like subarachnoid hemorrhage (SAH), where our preliminary investigation validates brain pericyte demise. A reliable protocol has been devised to surmount these constraints, enabling simultaneous fluorescent tagging of both functional and non-functional brain pericytes in brain sections. This labeling method allows high-resolution confocal microscope visualization, concurrently marking the brain slice microvasculature. This innovative protocol offers a means to appraise brain pericyte contractility, its impact on capillary diameter, and pericyte structure. Investigating brain pericyte contractility within the SAH context yields insightful comprehension of its effects on cerebral microcirculation.


Asunto(s)
Hemorragia Subaracnoidea , Humanos , Pericitos , Encéfalo , Diagnóstico por Imagen , Circulación Cerebrovascular
2.
J Exp Med ; 220(4)2023 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-36749798

RESUMEN

Metastatic castration-resistant prostate cancer (PC) is the final stage of PC that acquires resistance to androgen deprivation therapies (ADT). Despite progresses in understanding of disease mechanisms, the specific contribution of the metastatic microenvironment to ADT resistance remains largely unknown. The current study identified that the macrophage is the major microenvironmental component of bone-metastatic PC in patients. Using a novel in vivo model, we demonstrated that macrophages were critical for enzalutamide resistance through induction of a wound-healing-like response of ECM-receptor gene expression. Mechanistically, macrophages drove resistance through cytokine activin A that induced fibronectin (FN1)-integrin alpha 5 (ITGA5)-tyrosine kinase Src (SRC) signaling cascade in PC cells. This novel mechanism was strongly supported by bioinformatics analysis of patient transcriptomics datasets. Furthermore, macrophage depletion or SRC inhibition using a novel specific inhibitor significantly inhibited resistant growth. Together, our findings elucidated a novel mechanism of macrophage-induced anti-androgen resistance of metastatic PC and a promising therapeutic approach to treat this deadly disease.


Asunto(s)
Neoplasias Óseas , Neoplasias de la Próstata Resistentes a la Castración , Masculino , Humanos , Antagonistas de Andrógenos/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Línea Celular Tumoral , Macrófagos/metabolismo , Receptores Androgénicos/genética , Nitrilos/uso terapéutico , Microambiente Tumoral
3.
Nat Med ; 29(6): 1424-1436, 2023 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-37280275

RESUMEN

Gemcitabine plus cisplatin (GP) chemotherapy is the standard of care for nasopharyngeal carcinoma (NPC). However, the mechanisms underpinning its clinical activity are unclear. Here, using single-cell RNA sequencing and T cell and B cell receptor sequencing of matched, treatment-naive and post-GP chemotherapy NPC samples (n = 15 pairs), we show that GP chemotherapy activated an innate-like B cell (ILB)-dominant antitumor immune response. DNA fragments induced by chemotherapy activated the STING type-I-interferon-dependent pathway to increase major histocompatibility complex class I expression in cancer cells, and simultaneously induced ILB via Toll-like receptor 9 signaling. ILB further expanded follicular helper and helper type 1 T cells via the ICOSL-ICOS axis and subsequently enhanced cytotoxic T cells in tertiary lymphoid organ-like structures after chemotherapy that were deficient for germinal centers. ILB frequency was positively associated with overall and disease-free survival in a phase 3 trial of patients with NPC receiving GP chemotherapy ( NCT01872962 , n = 139). It also served as a predictor for favorable outcomes in patients with NPC treated with GP and immunotherapy combined treatment (n = 380). Collectively, our study provides a high-resolution map of the tumor immune microenvironment after GP chemotherapy and uncovers a role for B cell-centered antitumor immunity. We also identify and validate ILB as a potential biomarker for GP-based treatment in NPC, which could improve patient management.


Asunto(s)
Cisplatino , Neoplasias Nasofaríngeas , Humanos , Carcinoma Nasofaríngeo/tratamiento farmacológico , Carcinoma Nasofaríngeo/patología , Cisplatino/uso terapéutico , Gemcitabina , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/etiología , Neoplasias Nasofaríngeas/patología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Desoxicitidina/uso terapéutico , Microambiente Tumoral
4.
Commun Biol ; 5(1): 510, 2022 05 30.
Artículo en Inglés | MEDLINE | ID: mdl-35637301

RESUMEN

High-throughput single-cell RNA sequencing (scRNA-seq) is a popular method, but it is accompanied by doublet rate problems that disturb the downstream analysis. Several computational approaches have been developed to detect doublets. However, most of these methods may yield satisfactory performance in some datasets but lack stability in others; thus, it is difficult to regard a single method as the gold standard which can be applied to all types of scenarios. It is a difficult and time-consuming task for researchers to choose the most appropriate software. We here propose Chord which implements a machine learning algorithm that integrates multiple doublet detection methods to address these issues. Chord had higher accuracy and stability than the individual approaches on different datasets containing real and synthetic data. Moreover, Chord was designed with a modular architecture port, which has high flexibility and adaptability to the incorporation of any new tools. Chord is a general solution to the doublet detection problem.


Asunto(s)
Aprendizaje Automático , Análisis de la Célula Individual , Algoritmos , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Programas Informáticos
5.
Nat Commun ; 12(1): 4091, 2021 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-34215748

RESUMEN

Little is known about the transcriptomic plasticity and adaptive mechanisms of circulating tumor cells (CTCs) during hematogeneous dissemination. Here we interrogate the transcriptome of 113 single CTCs from 4 different vascular sites, including hepatic vein (HV), peripheral artery (PA), peripheral vein (PV) and portal vein (PoV) using single-cell full-length RNA sequencing in hepatocellular carcinoma (HCC) patients. We reveal that the transcriptional dynamics of CTCs were associated with stress response, cell cycle and immune-evasion signaling during hematogeneous transportation. Besides, we identify chemokine CCL5 as an important mediator for CTC immune evasion. Mechanistically, overexpression of CCL5 in CTCs is transcriptionally regulated by p38-MAX signaling, which recruites regulatory T cells (Tregs) to facilitate immune escape and metastatic seeding of CTCs. Collectively, our results reveal a previously unappreciated spatial heterogeneity and an immune-escape mechanism of CTC, which may aid in designing new anti-metastasis therapeutic strategies in HCC.


Asunto(s)
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/inmunología , Heterogeneidad Genética , Evasión Inmune , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Células Neoplásicas Circulantes/inmunología , Anciano , Animales , Biomarcadores de Tumor/metabolismo , Ciclo Celular , Línea Celular Tumoral , Quimiocina CCL5/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Metástasis de la Neoplasia , Células Neoplásicas Circulantes/metabolismo , Pronóstico , RNA-Seq , Transcriptoma , Microambiente Tumoral
6.
Cell Res ; 30(11): 1024-1042, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32686767

RESUMEN

Nasopharyngeal carcinoma (NPC) is an aggressive malignancy with extremely skewed ethnic and geographic distributions. Increasing evidence indicates that targeting the tumor microenvironment (TME) represents a promising therapeutic approach in NPC, highlighting an urgent need to deepen the understanding of the complex NPC TME. Here, we generated single-cell transcriptome profiles for 7581 malignant cells and 40,285 immune cells from fifteen primary NPC tumors and one normal sample. We revealed malignant signatures capturing intratumoral transcriptional heterogeneity and predicting aggressiveness of malignant cells. Diverse immune cell subtypes were identified, including novel subtypes such as CLEC9A+ dendritic cells (DCs). We further revealed transcriptional regulators underlying immune cell diversity, and cell-cell interaction analyses highlighted promising immunotherapeutic targets in NPC. Moreover, we established the immune subtype-specific signatures, and demonstrated that the signatures of macrophages, plasmacytoid dendritic cells (pDCs), CLEC9A+ DCs, natural killer (NK) cells, and plasma cells were significantly associated with improved survival outcomes in NPC. Taken together, our findings represent a unique resource providing in-depth insights into the cellular heterogeneity of NPC TME and highlight potential biomarkers for anticancer treatment and risk stratification, laying a new foundation for precision therapies in NPC.


Asunto(s)
Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/inmunología , Análisis de la Célula Individual , Transcriptoma/genética , Linfocitos B/inmunología , Comunicación Celular , Diferenciación Celular , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunidad , Células Asesinas Naturales/inmunología , Macrófagos/metabolismo , Monocitos/metabolismo , Células Mieloides/metabolismo , Carcinoma Nasofaríngeo/patología , Fenotipo , Pronóstico , Procesos Estocásticos , Análisis de Supervivencia , Linfocitos T/inmunología , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología
7.
J Exp Clin Cancer Res ; 38(1): 261, 2019 Jun 14.
Artículo en Inglés | MEDLINE | ID: mdl-31200735

RESUMEN

BACKGROUND & AIMS: Although the prognosis of patients with occult hepatitis B virus (HBV) infection (OBI) is usually benign, a small portion may undergo cirrhosis and subsequently hepatocellular carcinoma (HCC). We studied the mechanism of life-long Integration of virus DNA into OBI host's genome, of which may induce hepatocyte transformation. METHODS: We applied HBV capture sequencing on single cells from an OBI patient who, developed multiple HCC tumors and underwent liver resection in May 2013 at Tongji Hospital in China. Despite with the undetectable virus DNA in serum, we determined the pattern of viral integration in tumor cells and adjacent non-tumor cells and obtained the details of the viral arrangement in host genome, and furthermore the HBV integrated region in cancer genome. RESULTS: HBV captured sequencing of tissues and individual cells revealed that samples from multiple tumors shared two viral integration sites that could affect three host genes, including CSMD2 on chr1 and MED30/EXT1 on chr8. Whole genome sequencing further indicated one hybrid chromosome formed by HBV integrations between chr1 and chr8 that was shared by multiple tumors. Additional 50 poorly differentiated liver tumors and the paired adjacent non-tumors were evaluated and functional studies suggested up-regulated EXT1 expression promoted HCC growth. We further observed that the most somatic mutations within the tumor cell genome were common among the multiple tumors, suggesting that HBV associated, multifocal HCC is monoclonal in origin. CONCLUSION: Through analyzing the HBV integration sites in multifocal HCC, our data suggested that the tumor cells were monoclonal in origin and formed in the absence of active viral replication, whereas the affected host genes may subsequently contribute to carcinogenesis.


Asunto(s)
Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/patología , Virus de la Hepatitis B , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/virología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/patología , Integración Viral , Biopsia , Carcinoma Hepatocelular/diagnóstico por imagen , Línea Celular Tumoral , Mapeo Cromosómico , ADN Viral/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Haplotipos , Virus de la Hepatitis B/genética , Humanos , Neoplasias Hepáticas/diagnóstico por imagen , Imagen por Resonancia Magnética , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , N-Acetilglucosaminiltransferasas/genética , Carga Tumoral
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