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1.
World J Gastrointest Oncol ; 16(3): 659-669, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38577461

RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with a 5-year survival rate of less than 10%, owing to its late-stage diagnosis. Early detection of pancreatic cancer (PC) can significantly increase survival rates. AIM: To identify the serum biomarker signatures associated with early-stage PDAC by serum N-glycan analysis. METHODS: An extensive patient cohort was used to determine a biomarker signature, including patients with PDAC that was well-defined at an early stage (stages I and II). The biomarker signature was derived from a case-control study using a case-cohort design consisting of 29 patients with stage I, 22 with stage II, 4 with stage III, 16 with stage IV PDAC, and 88 controls. We used multiparametric analysis to identify early-stage PDAC N-glycan signatures and developed an N-glycan signature-based diagnosis model called the "Glyco-model". RESULTS: The biomarker signature was created to discriminate samples derived from patients with PC from those of controls, with a receiver operating characteristic area under the curve of 0.86. In addition, the biomarker signature combined with cancer antigen 19-9 could discriminate patients with PDAC from controls, with a receiver operating characteristic area under the curve of 0.919. Glyco-model demonstrated favorable diagnostic performance in all stages of PC. The diagnostic sensitivity for stage I PDAC was 89.66%. CONCLUSION: In a prospective validation study, this serum biomarker signature may offer a viable method for detecting early-stage PDAC.

2.
Anal Chem ; 95(48): 17502-17512, 2023 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-38050674

RESUMO

Cell migration is an essential process of cancer metastasis. The spatiotemporal dynamics of signaling molecules influences cellular phenotypic outcomes. It has been increasingly documented that the Abelson (ABL) family kinases play critical roles in solid tumors. However, ABL1's shuttling dynamics in cell migration still remains unexplored. This is mainly because tools permitting the investigation of translocation dynamics of proteins in single living cells are lacking. Herein, to bridge this gap, we developed a unique multifunctional integrated single-cell analysis method that enables long-term observation of cell migration behavior and monitoring of signaling proteins and complexes at the subcellular level. We found that the shuttling of ABL1's to the cytoplasm results in a higher migration speed, while its trafficking back to the nucleus leads to a lower one. Furthermore, our results indicated that fluctuant protein-protein interactions between 14-3-3 and ABL1 modulate ABL1's nucleocytoplasmic fluctuation and eventually affect the cell speed. Importantly, based on these new insights, we demonstrated that disturbing ABL1's nuclear export traffic and 14-3-3-ABL1 complexes formation can effectively suppress cell migration. Thus, our method opens up a new possibility for simultaneous tracking of internal molecular mechanisms and cell behavior, providing a promising tool for the in-depth study of cancer.


Assuntos
Núcleo Celular , Neoplasias , Humanos , Núcleo Celular/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas/metabolismo , Citoplasma/metabolismo , Movimento Celular , Neoplasias/metabolismo
3.
Anal Chem ; 95(14): 6080-6089, 2023 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-36995353

RESUMO

A sound understanding of cell migration behaviors and the internal mechanism is crucial for studying cancer metastasis and invasion. Continuous cell tracking and quantifying cellular and molecular dynamics of cell migration at the single-cell level is essential to elucidate rare, dynamic, and heterogeneous cell responses. Yet, a competent comprehensive analytical platform is lacking. Herein, we present an integrated single living cell analysis platform that enables long-term observation of migration behavioral phenotypes in single cells and simultaneous analysis of the signaling proteins and complexes during cell migration. Considering correlation between pathways and phenotypes, this platform is capable of analyzing multiple phenotypes and signaling protein dynamics at a subcellular resolution, reflecting the molecular mechanism of biological behavior. Using the EGFR-PI3K signaling pathway as a proof-of-principle, we explored how the pathway and related regulators, Rho GTPases, promote different migration phenotypes. Signaling pathway protein complexes p85α-p110α and p85α-PTEN were found to reciprocally modulate each other and subsequently regulate the expression level of the small GTPases by EGFR-related signaling pathways, which governs the cell migratory behavior. Thus, this single-cell analysis platform is a promising tool for rapid molecular mechanism analysis and direct observation of migration phenotypes at the single-cell level, providing insights into the molecular mechanism and phenotypes in cell migration.


Assuntos
Fosfatidilinositol 3-Quinases , Transdução de Sinais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Receptores ErbB/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Sobrevivência Celular
4.
Anal Chem ; 94(37): 12828-12835, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36069705

RESUMO

Queuosine (Q) modification on tRNA plays an essential role in protein synthesis, participating in many tRNA functions such as folding, stability, and decoding. Appropriate analytical tools for the measurement of tRNA Q modifications are essential for the exploration of new roles of Q-modified tRNAs and the rationalization of their exact mechanisms. However, conventional methods for Q modification analysis suffer from apparent disadvantages, such as destructive cells, tedious procedure, and low sensitivity, which much hamper in-depth studies of Q modification-related biological questions. In this study, we developed a new approach called plasmonic affinity sandwich assay that allows for facile and sensitive determination of Q-modified tRNAs in single living cells. This method relies on the combination of plasmon-enhanced Raman scattering detection, base-paring affinity in-cell microextraction, and a set of boronate affinity and molecularly imprinted labeling nanotags for selective recognition of individual Q modifications, including queuosine, galactosyl queuosine (Gal-Q), and mannosyl queuosine (Man-Q). The developed method exhibited high affinity extraction and high specificity recognition. It allowed for the measurement of tRNA Q modifications in not only Q-rich cultured tumor cells but also Q-deficient primary tumor cells. Usefulness of this approach for investigation of the change of the Q modification level in single cells under oxidative stress was demonstrated. Because of its significant advantages over conventional methods, this approach provides a promising analytical tool for the exploration of more roles of Q-modified tRNAs and elucidation of their mechanisms.


Assuntos
Nucleosídeo Q , RNA de Transferência , Humanos , Masculino , Nucleosídeo Q/análise , Nucleosídeo Q/genética , Nucleosídeo Q/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo
5.
Anal Chem ; 93(42): 14204-14213, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34648273

RESUMO

It is of significant importance in cancer biology to identify signaling pathways that play key roles in cell fate determination. Dissecting cellular signaling pathways requires the measurement of a large number of signaling proteins. However, tools for simultaneously monitoring multiple signaling pathway components in single living cells remain limited at present. Herein, we describe an approach, termed multiplexed single-cell plasmonic immunosandwich assay (mxscPISA), for simultaneous detection of multiple signaling proteins in individual living cells. This approach enabled simultaneous non-destructive monitoring of multiple (up to five, currently the highest multiplexing capacity in living cells) cytoplasmic and nucleus signaling proteins in individual cells with ultrahigh detection sensitivity. As a proof of principle, the epidermal growth factor receptor (EGFR) pathway, which plays a central role in cell fate determination, was investigated using this approach in this study. We found that there were differential attenuation rate of pro-survival and accumulation rate of pro-death signaling protein of the EGFR pathway in response to EGFR inactivation. These findings implicate that, after EGFR inactivation, a transient imbalance between survival and apoptotic signaling outputs contributed to the final cell fate of death. The mxscPISA approach can be a promising tool to reveal a signaling dynamic pattern at the single-cell level and to identify key components of signaling pathways that contribute to the final cell fate using only a limited number of cells.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Transdução de Sinais , Núcleo Celular , Citoplasma , Imunoensaio
6.
Nat Protoc ; 16(7): 3522-3546, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34089021

RESUMO

Cellular heterogeneity is pervasive and of paramount importance in biology. Single-cell analysis techniques are indispensable for understanding the heterogeneity and functions of cells. Low-copy-number proteins (fewer than 1,000 molecules per cell) perform multiple crucial functions such as gene expression, cellular metabolism and cell signaling. The expression level of low-copy-number proteins of individual cells provides key information for the in-depth understanding of biological processes and diseases. However, the quantitative analysis of low-copy-number proteins in a single cell still remains challenging. To overcome this, we developed an approach called single-cell plasmonic immunosandwich assay (scPISA) for the quantitative measurement of low-copy-number proteins in single living cells. scPISA combines in vivo microextraction for specific enrichment of target proteins from cells and a state-of-the-art technique called plasmon-enhanced Raman scattering for ultrasensitive detection of low-copy-number proteins. Plasmon-enhanced Raman scattering detection relies on the plasmonic coupling effect (hot-spot) between silver-based plasmonic nanotags and a gold-based extraction microprobe, which dramatically enhances the signal intensity of the surface-enhanced Raman scattering of the nanotags and thereby enables sensitivity at the single-molecule level. scPISA is a straightforward and minimally invasive technique, taking only ~6-15 min (from in vivo extraction to Raman spectrum readout). It is generally applicable to all freely floating intracellular proteins provided that appropriate antibodies or alternatives (for example, molecularly imprinted polymers or aptamers) are available. The entire protocol takes ~4-7 d to complete, including material fabrication, single-cell manipulation, protein labeling, signal acquisition and data analysis.


Assuntos
Dosagem de Genes , Imunoensaio/métodos , Proteínas/metabolismo , Análise de Célula Única , Anticorpos/metabolismo , Calibragem , Linhagem Celular Tumoral , Sobrevivência Celular , Análise de Dados , Ouro/química , Humanos , Proteínas Imobilizadas/metabolismo , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Coloração e Rotulagem
7.
Anal Chem ; 92(18): 12498-12508, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32790289

RESUMO

Single-cell DNA analysis technology has provided unprecedented insights into many physiological and pathological processes. In contrast, technologies that allow protein analysis in single cells have lagged behind. Herein, a method called single-cell Plasmonic ImmunoSandwich Assay (scPISA) that is capable of measuring signaling proteins and protein complexes in single living cells is described. scPISA is straightforward, comprising specific in-cell extraction and ultrasensitive plasmonic detection. It is applied to evaluate the efficacy and kinetics of cytotoxic drugs. It reveals that different drugs exhibit distinct proapoptotic properties at the single-cell level. A set of new parameters is thus proposed for comprehensive and quantitative evaluation of the efficacy of anticancer drugs. It discloses that metformin can dramatically enhance the overall anticancer efficacy when combined with actinomycin D, although it itself is significantly less effective. Furthermore, scPISA reveals that survivin interacts with cytochrome C and caspase-3 in a dynamic fashion in single cells during continuous drug treatment. As compared with conventional assays, scPISA exhibits several significant advantages, such as ultrahigh sensitivity, single-cell resolution, fast speed, and so on. Therefore, this approach may provide a powerful tool for wide, important applications from basic research to clinical applications, particularly precision medicine.


Assuntos
Antineoplásicos/farmacologia , Caspase 3/análise , Citocromos c/análise , Dactinomicina/farmacologia , Imunoensaio , Metformina/farmacologia , Análise de Célula Única , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Dactinomicina/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Cinética , Metformina/química , Tamanho da Partícula , Propriedades de Superfície
8.
Anal Chem ; 91(15): 9993-10000, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31347834

RESUMO

Molecularly imprinted polymers (MIPs), which are synthesized in the presence of a template, have been widely used as antibody mimics for important applications. Through the combination with a highly sensitive detection scheme such as chemiluminescence and surface-enhanced Raman scattering (SERS), MIP-based sandwich assays have emerged as promising analytical tools for the detection of disease biomarkers. However, so far, MIPs have been used only as target-capturing probes, whereas labeling by other means was needed, which limits the application range. Herein, we present a new approach, called a dual MIP-based plasmonic immunosandwich assay (duMIP-PISA), for the specific and sensitive detection of protein biomarkers in complex biological samples. A C-terminal epitope-imprinted self-assembled gold nanoparticle monolayer-coated glass slide was prepared as a plasmonic substrate for the specific extraction of target protein, while N-terminal epitope-imprinted Raman-responsive Ag@SiO2 nanoparticles were prepared as nanotags for the specific labeling of captured protein. The formed MIP-protein-MIP sandwich-like complexes could produce a significantly enhanced SERS signal. The dual MIP-based recognitions ensured high specificity of the assay, while SERS detection provided ultrahigh sensitivity. The duMIP-PISA of neuron-specific enolase (NSE) in human serums was demonstrated, which permitted the differentiation of small cell lung cancer patients from healthy individuals. As compared to regular ELISA, the duMIP-PISA exhibited multiple merits including a simpler procedure, faster speed, lower sample volume requirement, and wider linear range. The approach well demonstrated the great potentials of MIPs and can be easily modified and extended to other protein biomarkers. Therefore, the duMIP-PISA approach holds great promise in many important applications such as disease diagnosis.


Assuntos
Biomarcadores/sangue , Imunoensaio/métodos , Impressão Molecular , Fosfopiruvato Hidratase/sangue , Polímeros/química , Linhagem Celular Tumoral , Humanos , Nanopartículas Metálicas/química , Neoplasias/diagnóstico , Dióxido de Silício/química , Prata/química , Análise Espectral Raman
9.
Anal Chem ; 89(10): 5646-5652, 2017 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-28438017

RESUMO

Recognition of cancer cells is essential for many important areas such as targeted cancer therapy. Multimonosaccharide-based recognition could be a useful strategy to improve the recognition specificity, but such a possibility has not been explored yet. Herein we report pattern recognition of cells via multiplexed imaging with monosaccharide-imprinted quantum dots (QDs). Imprinted with sialic acid, fucose, and mannose as the template, respectively, the QDs exhibited good specificity toward the template monosaccharides. Multiplexed imaging of cells simultaneously stained with these monosaccharide-imprinted QDs revealed the relative expression levels of the monosaccharides on the cells. Pattern recognition constructed using the intensities of multiplexed imaging unveiled the similarities and differences of different cell lines, allowing for the recognition of not only cancer cells from normal cells but also cancer cells of different cell lines. Thus, this study paved a solid ground for the design and preparation of novel cancer-cell targeting reagents and nanoprobes.


Assuntos
Impressão Molecular , Monossacarídeos/química , Pontos Quânticos/química , Ácidos Borônicos/química , Linhagem Celular Tumoral , Análise por Conglomerados , Glicosídeo Hidrolases/metabolismo , Humanos , Microscopia Confocal , Monossacarídeos/metabolismo , Análise de Componente Principal
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