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The profiling of plasma cell-free DNA (cfDNA) is becoming a valuable tool rapidly for tumor diagnosis, monitoring and prognosis. Diverse plasma cfDNA technologies have been in routine or emerging use, including analyses of mutations, copy number alterations, gene fusions and DNA methylation. Recently, new technologies in cfDNA analysis have been developed in laboratories, and potentially reflect the status of epigenetic modification, the immune microenvironment and the microbiome in tumor tissues. In this review, the authors discuss the principles, methods and effects of the current cfDNA assays and provide an overview of studies that may inform clinical applications in the near future.
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OBJECTIVE: Insulinomas and non-functional pancreatic neuroendocrine tumours (NF-PanNETs) have distinctive clinical presentations but share similar pathological features. Their genetic bases have not been comprehensively compared. Herein, we used whole-genome/whole-exome sequencing (WGS/WES) to identify genetic differences between insulinomas and NF-PanNETs. DESIGN: The mutational profiles and copy-number variation (CNV) patterns of 211 PanNETs, including 84 insulinomas and 127 NF-PanNETs, were obtained from WGS/WES data provided by Peking Union Medical College Hospital and the International Cancer Genome Consortium. Insulinoma RNA sequencing and immunohistochemistry data were assayed. RESULTS: PanNETs were categorised based on CNV patterns: amplification, copy neutral and deletion. Insulinomas had CNV amplifications and copy neutral and lacked CNV deletions. CNV-neutral insulinomas exhibited an elevated rate of YY1 mutations. In contrast, NF-PanNETs had all three CNV patterns, and NF-PanNETs with CNV deletions had a high rate of loss-of-function mutations of tumour suppressor genes. NF-PanNETs with CNV alterations (amplification and deletion) had an elevated risk of relapse, and additional DAXX/ATRX mutations could predict an increased relapse risk in the first 2-year period. CONCLUSION: These WGS/WES data allowed a comprehensive assessment of genetic differences between insulinomas and NF-PanNETs, reclassifying these tumours into novel molecular subtypes. We also proposed a novel relapse risk stratification system using CNV patterns and DAXX/ATRX mutations.
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Dosagem de Genes/genética , Insulinoma/genética , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/genética , Sequenciamento Completo do Genoma/métodos , Doenças Assintomáticas/classificação , Biópsia por Agulha , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Insulinoma/classificação , Masculino , Mutação , Tumores Neuroendócrinos/classificação , Proteínas Nucleares/genética , Neoplasias Pancreáticas/classificação , Medição de Risco , Sequenciamento do ExomaRESUMO
BACKGROUND: Successful clinical evaluation of human tumors relies on proper handling of tissue samples to maximally preserve the cellular and metabolic states in vivo. Pancreatic samples are particularly sensitive to sample mishandling due to the abundance of digestive enzymes. We study how the duration of ischemia, in vivo and ex vivo, both of which are unavoidable lagging periods following surgical dissection, significantly impact the utility of pancreatic samples. METHODS: We systematically characterize a wide range of tissue integrity features, including histological patterns, cellular structures, DNA/RNA quality and activity of major signaling pathways in normal pancreases and pancreatic ductal adenocarcinoma (PDAC) tumor tissues from 41 patients with different ischemia. RESULTS: We reveal that tissues experiencing longer periods of ischemia exhibit significant deterioration and could potentially mislead disease diagnosis and preclinical research. Based on these analyses, we propose an optimal procedure that balances better clinical practice and high tissue sample quality. CONCLUSIONS: Our work provides a guideline for pancreatic sample handling and could have wide implications in clinical diagnosis and translational research.
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Isquemia/patologia , Pâncreas/patologia , Manejo de Espécimes/normas , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/patologia , DNA/química , Dissecação , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Pâncreas/irrigação sanguínea , Neoplasias Pancreáticas/patologia , RNA/química , Transdução de SinaisRESUMO
Autophagy is an evolutionarily conserved process by which cytoplasmic proteins and organelles are catabolized. During starvation, the protein TOR (target of rapamycin), a nutrient-responsive kinase, is inhibited, and this induces autophagy. In autophagy, double-membrane autophagosomes envelop and sequester intracellular components and then fuse with lysosomes to form autolysosomes, which degrade their contents to regenerate nutrients. Current models of autophagy terminate with the degradation of the autophagosome cargo in autolysosomes, but the regulation of autophagy in response to nutrients and the subsequent fate of the autolysosome are poorly understood. Here we show that mTOR signalling in rat kidney cells is inhibited during initiation of autophagy, but reactivated by prolonged starvation. Reactivation of mTOR is autophagy-dependent and requires the degradation of autolysosomal products. Increased mTOR activity attenuates autophagy and generates proto-lysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes, thereby restoring the full complement of lysosomes in the cell-a process we identify in multiple animal species. Thus, an evolutionarily conserved cycle in autophagy governs nutrient sensing and lysosome homeostasis during starvation.
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Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/metabolismo , Fenômenos Fisiológicos da Nutrição , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Células HeLa , Homeostase/fisiologia , Humanos , Lisossomos/ultraestrutura , Ratos , Transdução de Sinais , Serina-Treonina Quinases TOR , Células VeroRESUMO
The diagnosis of solid pseudopapillary neoplasm of the pancreas (SPN) can be challenging due to potential confusion with other pancreatic neoplasms, particularly pancreatic neuroendocrine tumors (NETs), using current pathological diagnostic markers. We conducted a comprehensive analysis of bulk RNA sequencing data from SPNs, NETs, and normal pancreas, followed by experimental validation. This analysis revealed an increased accumulation of peroxisomes in SPNs. Moreover, we observed significant upregulation of the peroxisome marker ABCD1 in both primary and metastatic SPN samples compared with normal pancreas and NETs. To further investigate the potential utility of ABCD1 as a diagnostic marker for SPN via immunohistochemistry staining, we conducted verification in a large-scale patient cohort with pancreatic tumors, including 127 SPN (111 primary, 16 metastatic samples), 108 NET (98 nonfunctional pancreatic neuroendocrine tumor, NF-NET, and 10 functional pancreatic neuroendocrine tumor, F-NET), 9 acinar cell carcinoma (ACC), 3 pancreatoblastoma (PB), 54 pancreatic ductal adenocarcinoma (PDAC), 20 pancreatic serous cystadenoma (SCA), 19 pancreatic mucinous cystadenoma (MCA), 12 pancreatic ductal intraepithelial neoplasia (PanIN) and 5 intraductal papillary mucinous neoplasm (IPMN) samples. Our results indicate that ABCD1 holds promise as an easily applicable diagnostic marker with exceptional efficacy (AUC=0.999, sensitivity=99.10%, specificity=100%) for differentiating SPN from NET and other pancreatic neoplasms through immunohistochemical staining.
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Carcinoma Ductal Pancreático , Tumores Neuroendócrinos , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Pâncreas/patologia , Carcinoma Ductal Pancreático/patologia , Tumores Neuroendócrinos/diagnóstico , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Ductos Pancreáticos/química , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análise , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATPRESUMO
The high-throughput sequencing at single-cell resolution requires high-quality samples of inputted 100-10,000 cells with at least 80%-90% viability according to the applied platform. Here, we present a protocol for single-cell isolation from normal and neoplastic human pancreas. We describe steps for sample harvesting, procurement, tissue digestion, and cell purification. Subsequently, isolated cells can be further applied to single-cell profiling, for example, single-cell RNA sequencing and single-cell ATAC-seq using 10× Genomics platform. For complete details on the use and execution of this protocol, please refer to Peng et al. (2019)1 and Li et al. (2021).2.
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Pâncreas , Hormônios Pancreáticos , Humanos , Separação Celular , Genômica , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Interleukin (IL)-17 is a prominent cytokine that promotes pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC) and is associated with the oncogenic pathways in tumor progression. However, the mechanism and therapeutic value of the IL-17 axis remain unclear. In this study, we verified the activation of the IL-17 and Notch pathways in PanIN/PDAC via complementary approaches and validated their pro-tumor effects on tumor progression. Additionally, we found a positive correlation between IL-17 and Notch; the IL-17 axis can upregulate Notch activity via the canonical NF-κB pathway in vitro, thus synergistically promoting PanIN/PDAC. Furthermore, we observed that the co-inhibition of IL-17 and the Notch pathway can enhance the therapeutic effect by restricting tumor growth in vivo. Our study highlights the synergistic effect of the IL-17 axis and Notch pathway in promoting PanIN/PDAC and further suggests that IL-17-Notch co-inhibition is a novel therapeutic strategy with superior potential in treating PDAC.
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Carcinoma Ductal Pancreático/metabolismo , Interleucina-17/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores Notch/metabolismo , Animais , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/terapia , Linhagem Celular Tumoral , Progressão da Doença , Técnicas de Silenciamento de Genes , Células HEK293 , Xenoenxertos , Humanos , Interleucina-17/antagonistas & inibidores , Interleucina-17/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Terapia de Alvo Molecular , NF-kappa B/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Receptores Notch/antagonistas & inibidores , Receptores Notch/genética , Transdução de SinaisRESUMO
Kras-activating mutations display the highest incidence in pancreatic ductal adenocarcinoma. Pancreatic inflammation accelerates mutant Kras-driven tumorigenesis in mice, suggesting high selectivity in the cells that oncogenic Kras transforms, although the mechanisms dictating this specificity are poorly understood. Here we show that pancreatic inflammation is coupled to the emergence of a transient progenitor cell population that is readily transformed in the presence of mutant KrasG12D. These progenitors harbor a proto-oncogenic transcriptional program driven by a transient enhancer network. KrasG12D mutations lock this enhancer network in place, providing a sustained Kras-dependent oncogenic program that drives tumors throughout progression. Enhancer co-option occurs through functional interactions between the Kras-activated transcription factors Junb and Fosl1 and pancreatic lineage transcription factors, potentially accounting for inter-tissue specificity of oncogene transformation. The pancreatic ductal adenocarcinoma cell of origin thus provides an oncogenic transcriptional program that fuels tumor progression beyond initiation, accounting for the intra-tissue selectivity of Kras transformation.
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Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Pancreatite , Adenocarcinoma/patologia , Animais , Carcinogênese , Carcinoma Ductal Pancreático/genética , Inflamação/genética , Metaplasia , Camundongos , Neoplasias Pancreáticas/genética , Pancreatite/induzido quimicamente , Células-Tronco/patologia , Fatores de Transcrição , Neoplasias PancreáticasRESUMO
Background: C-X-C motif chemokine 5 (CXCL5) is an important attractant for immune cell accumulation in tumor tissues. Recent evidence has shown that CXCL5 could promote carcinogenesis and cancer progression in a variety of cancer types. However, the relationships between CXCL5, immune cell infiltration and pancreatic ductal adenocarcinoma (PDAC) remain largely unknown. This study aimed to explore the role and regulative mechanism of CXCL5 in PDAC carcinogenesis. Materials and Methods: The expression of CXCL5 in PDAC was analyzed based on online databases and tissue microarray staining, and Western blotting of CXCL5 in PDAC cell lines and patient samples. The correlation between CXCL5 expression and clinicopathological features, prognosis and immune cell infiltration in tumor tissues was analyzed. Results: High expression of CXCL5 was observed both in PDAC tumor tissue and PDAC cell lines, compared to normal pancreas tissues and normal ductal epithelium cells. High CXCL5 expression in tumor tissues was positively correlated with an advanced T stage (p=0.036), a positive tumor lymph node metastasis (p=0.014), a poor differentiation status (p=0.003) and a poor prognosis (p=0.001). Combination of CA242 and CXCL5 expression (p<0.0001) served as a better prognostic factor than CA242 alone (p=0.006). In addition, PDAC patients with high CXCL5 expression had more intratumoral M2 polarized macrophages (p=0.0248), neutrophils (p=0.0068) and IgG+ plasma cells (p=0.0133) than patients with low CXCL5 expression. Conclusions: The expression of CXCL5 is elevated in pancreatic cancer cells. High CXCL5 expression is positively correlated with poor survival and the increased infiltration of several types of immune suppressive cells. Thus, CXCL5 could be a promising therapeutic target for PDAC immunotherapy.
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Idiopathic pulmonary fibrosis (IPF) and acute lung injury (ALI) are considered two severe public health issues, attributed to malfunctions of neutrophils. They can cause chronic inflammation and have association with subsequent tissue damages. There have been rare drugs applying to the efficient treatment in clinical practice. Existing research revealed that Leukotriene B4 (LTB4) is the critical endogenous molecule to induce neutrophil inflammatory response. LTB4 blocking biosynthesis is the potential strategy treating IPF and ALI. In the present study, 45 hydroxamic acid derivatives were produced, and compound 26 was screened out as a highly selective Lead compound of Leukotriene A4 Hydrolase (LTA4H), i.e., an enzyme critical to the biosynthesis of LTB4. This compound is capable of relieving neutrophilic inflammation in an IPF mouse model at early stage, as well as mitigating LPS-induced acute lung injury via a mechanism of LTB4 blocking biosynthesis in vivo. Whether this compound acts as the potential lead compound for the treatment of IPF and ALI requires further verification.
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Lesão Pulmonar Aguda/tratamento farmacológico , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Fibrose Pulmonar Idiopática/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Animais , Técnicas de Química Sintética , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/uso terapêutico , Leucotrieno B4/biossíntese , CamundongosRESUMO
Tumor-associated macrophages (TAMs) are versatile immune cells that promote a variety of malignant behaviors of pancreatic cancer. CD59 is a GPI-anchored membrane protein that prevents complement activation by inhibiting the formation of the membrane attack complex, which may protect cancer cells from complement-dependent cytotoxicity (CDC). The interactions between CD59, TAMs and pancreatic cancer remain largely unknown. A tissue microarray of pancreatic cancer patients was used to evaluate the interrelationship of CD59 and TAMs and their survival impacts were analyzed. In a coculture system, THP-1 cells were used as a model to study the function of TAMs and the roles of pancreatic cancer-educated macrophages in regulating the expression of CD59 in pancreatic cancer cells were demonstrated by real-time PCR, western blot and immunofluorescence staining. The effects of macrophages on regulating CDC in pancreatic cancer cells were demonstrated by an in vitro study. To explore the potential mechanisms, RNA sequencing of pancreatic cancer cells with or without co-culture of THP-1 macrophages was performed, and the results showed that the IL-6R/STAT3 signaling pathway might participate in the regulation, which was further demonstrated by target-siRNA transfection, antibody neutralization and STAT3 inhibitors. Our data revealed that the infiltration of TAMs and the expression of CD59 of pancreatic cancer were paralleled, and higher infiltration of TAMs and higher expression of CD59 predicted worse survival of pancreatic cancer patients. Pancreatic cancer-educated macrophages could protect cancer cells from CDC by up-regulating CD59 via the IL-6R/STAT3 signaling pathway. These findings uncovered the novel mechanisms between TAMs and CD59, and contribute to providing a new promising target for the immunotherapy of pancreatic cancer.
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Ativação do Complemento/imunologia , Macrófagos/imunologia , Neoplasias Pancreáticas/imunologia , Transdução de Sinais/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD59/imunologia , Feminino , Humanos , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/imunologia , Neoplasias Pancreáticas/patologia , Receptores de Interleucina-6/imunologia , Fator de Transcrição STAT3/imunologia , Células THP-1RESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. To comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, we employed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases, and identified diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, in PDAC. We found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, we found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, our findings provide a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.
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Carcinoma Ductal Pancreático/patologia , Neoplasias Pancreáticas/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Variações do Número de Cópias de DNA , Progressão da Doença , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Inibidores de Proteínas Quinases/farmacologia , RNA-Seq , Análise de Célula Única , TranscriptomaRESUMO
Cells can secrete extracellular vesicles (EVs) to communicate with neighboring or distant cells by EVs which are composed of a lipid bilayer containing transmembrane proteins and enclosing cytosolic proteins, lipids, and nucleic acids. Breast Cancer is the most frequently diagnosed malignancy with more than 1 million new cases each year and ranks the leading cause of cancer mortality in women worldwide. In this review, we will discuss recent progresses of the roles and mechanisms of cancer-derived EVs in metastatic breast cancer, with a special attention on tumor microenvironment construction, progression, and chemo/radiotherapy responses. This review also covers EV roles as biomarker and therapeutic target in clinical application.
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Cancer stem cells (CSCs) have been reported to be involved in tumorigenesis, tumor recurrence, cancer invasion, metastasis, and drug-resistance. Therefore, the development of drug molecules targeting CSCs has become an attractive therapeutic approach. However, the molecules which can selectively ablate CSCs are extremely rare. To explore the leading compounds targeting CSCs, 52 analogues of triterpenoic acids were synthesized in this study, whose biological activities were evaluated. On the basis of the results of tumorsphere assay, two compounds 48 and 51, derived from oleanolic acid, exhibited suppressive effect on elimination of different type of CSCs. Meanwhile, compounds 48 and 51 could significantly inhibit the growth of several tumors both in vitro and in vivo. Furthermore, treatment of cancer cells with both of two compounds would dramatically increase the level of ROS, which might eliminate the CSCs. Collectively, the leading compounds 48 and 51 were promising anti-CSCs agents that merited further validation as a novel class of chemotherapeutics.
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Antineoplásicos/química , Antineoplásicos/farmacologia , Produtos Biológicos/química , Desenho de Fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Triterpenos/química , Triterpenos/farmacologia , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Química Sintética , Ensaios de Seleção de Medicamentos Antitumorais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Células-Tronco Neoplásicas/patologia , Espécies Reativas de Oxigênio/metabolismo , Triterpenos/síntese químicaRESUMO
Cells communicate with each other through secreting and releasing proteins and vesicles. Many cells can migrate. In this study, we report the discovery of migracytosis, a cell migration-dependent mechanism for releasing cellular contents, and migrasomes, the vesicular structures that mediate migracytosis. As migrating cells move, they leave long tubular strands, called retraction fibers, behind them. Large vesicles, which contain numerous smaller vesicles, grow on the tips and intersections of retraction fibers. These fibers, which connect the vesicles with the main cell body, eventually break, and the vesicles are released into the extracellular space or directly taken up by surrounding cells. Since the formation of these vesicles is migration-dependent, we named them "migrasomes". We also found that cytosolic contents can be transported into migrasomes and released from the cell through migrasomes. We named this migration-dependent release mechanism "migracytosis".
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Movimento Celular , Citoplasma/metabolismo , Organelas/metabolismo , Actinas/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Linhagem Celular Tumoral , Citoplasma/ultraestrutura , Humanos , Camundongos , Organelas/ultraestruturaRESUMO
Autophagy is an evolutionarily conserved lysosome-based degradation process. Atg5 plays a very important role in autophagosome formation. Here we show that Atg5 is required for biogenesis of late endosomes and lysosomes in an autophagy-independent manner. In Atg5 (-/-) cells, but not in other essential autophagy genes defecting cells, recycling and retrieval of late endosomal components from hybrid organelles are impaired, causing persistent hybrid organelles and defective formation of late endosomes and lysosomes. Defective retrieval of late endosomal components from hybrid organelles resulting from impaired recruitment of a component of V1-ATPase to acidic organelles blocks the pH-dependent retrieval of late endosomal components from hybrid organelles. Lowering the intracellular pH restores late endosome/lysosome biogenesis in Atg5 (-/-) cells. Our data demonstrate an unexpected role of Atg5 and shed new light on late endosome and lysosome biogenesis.