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1.
Int J Mol Sci ; 22(6)2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33806874

RESUMO

Secreted extracellular vesicles (EVs) are heterogeneous cell-derived membranous granules which carry a large diversity of molecules and participate in intercellular communication by transferring these molecules to target cells by endocytosis. In the last decade, EVs' role in several pathological conditions, from etiology to disease progression or therapy evasion, has been consolidated, including in central nervous system (CNS)-related disorders. For this review, we performed a systematic search of original works published, reporting the presence of molecular components expressed in the CNS via EVs, which have been purified from plasma, serum or cerebrospinal fluid. Our aim is to provide a list of molecular EV components that have been identified from both nonpathological conditions and the most common CNS-related disorders. We discuss the methods used to isolate and enrich EVs from specific CNS-cells and the relevance of its components in each disease context.


Assuntos
Biomarcadores , Doenças do Sistema Nervoso Central/diagnóstico , Doenças do Sistema Nervoso Central/metabolismo , Vesículas Extracelulares/metabolismo , Biópsia Líquida , Doenças do Sistema Nervoso Central/etiologia , Fracionamento Químico/métodos , Humanos , Biópsia Líquida/métodos , Técnicas de Diagnóstico Molecular , RNA não Traduzido
2.
Int J Mol Sci ; 22(6)2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33807045

RESUMO

Breast cancer is very heterogenous and the most common gynaecological cancer, with various factors affecting its development. While its impact on human lives and national health budgets is still rising in almost all global areas, many molecular mechanisms affecting its onset and development remain unclear. Conventional treatments still prove inadequate in some aspects, and appropriate molecular therapeutic targets are required for improved outcomes. Recent scientific interest has therefore focused on the non-coding RNAs roles in tumour development and their potential as therapeutic targets. These RNAs comprise the majority of the human transcript and their broad action mechanisms range from gene silencing to chromatin remodelling. Many non-coding RNAs also have altered expression in breast cancer cell lines and tissues, and this is often connected with increased proliferation, a degraded extracellular environment, and higher endothelial to mesenchymal transition. Herein, we summarise the known abnormalities in the function and expression of long non-coding RNAs, Piwi interacting RNAs, small nucleolar RNAs and small nuclear RNAs in breast cancer, and how these abnormalities affect the development of this deadly disease. Finally, the use of RNA interference to suppress breast cancer growth is summarised.


Assuntos
Biomarcadores Tumorais , Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , RNA não Traduzido/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Gerenciamento Clínico , Suscetibilidade a Doenças , Feminino , Humanos , Biópsia Líquida/métodos , Interferência de RNA , RNA não Traduzido/química
3.
Methods Mol Biol ; 2292: 3-15, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33651347

RESUMO

Recent reports suggest that urine is a useful noninvasive tool for the identification of urogenital tumors, including bladder, prostate, kidney, and other nonurological cancers. As a liquid biopsy, urine represents an important source for the improvement of new promising biomarkers, a suitable tool to identify indolent cancer and avoid overtreatment. Urine is enriched with DNAs, RNAs, proteins, circulating tumor cells, exosomes, and other small molecules which can be detected with several diagnostic methodologies.We provide an overview of the ongoing state of urinary biomarkers underlying both their potential utilities to improve cancer prognosis, diagnosis, and therapeutic strategy and their limitations.


Assuntos
Biomarcadores Tumorais/urina , Neoplasias/urina , Animais , Ácidos Nucleicos Livres/urina , Exossomos/patologia , Humanos , Biópsia Líquida/métodos , Neoplasias/diagnóstico , Neoplasias/patologia , Ácidos Nucleicos/urina , Proteínas , Proteinúria/diagnóstico , Proteinúria/patologia , Proteinúria/urina , Urinálise/métodos
4.
Methods Mol Biol ; 2292: 95-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33651354

RESUMO

Application of next generation sequencing techniques in the field of liquid biopsy, in particular urine, requires specific bioinformatics methods in order to deal with its peculiarity. Many aspects of cancer can be explored starting from nucleic acids, especially from cell-free DNA and circulating tumor DNA in order to characterize cancer. It is possible to detect small mutations, as single nucleotide variants, small insertions and deletions, copy-number alterations, and epigenetic profiles. Due to the low fraction of circulating tumor DNA over the whole cell-free DNA, some methods have been exploited. One of them is the application of unique barcodes to each DNA fragment in order to lower the limit of detection of cancer-related variants. Some bioinformatics workflows and tools are the same of a classic analysis of tumor tissue, but there are some steps in which specific algorithms have to be introduced.


Assuntos
Ácidos Nucleicos Livres/urina , Neoplasias/urina , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/urina , Ácidos Nucleicos Livres/genética , Variações do Número de Cópias de DNA , Metilação de DNA , Epigênese Genética , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Biópsia Líquida/métodos , Neoplasias/genética
5.
Methods Mol Biol ; 2292: 115-120, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33651356

RESUMO

The analysis of liquid biopsy as a source of diagnostic, prognostic, and predictive biomarkers is still object of the main research in the prostate cancer field. Many advantages, such as less invasiveness compared to plasma or serum analysis and the rich content, confer to urine a role as an interesting fluid to be analysed especially in urological diseases. Here we report a workflow focused on profile, concentration, and protein surface characterization of EVs from urinary supernatant.


Assuntos
Exossomos/patologia , Neoplasias da Próstata/diagnóstico , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/urina , Humanos , Biópsia Líquida/métodos , Masculino , Neoplasias da Próstata/patologia , Neoplasias da Próstata/urina , Proteínas/análise , Proteinúria/diagnóstico , Proteinúria/patologia , Proteinúria/urina , Coleta de Urina/métodos , Fluxo de Trabalho
6.
Methods Mol Biol ; 2292: 153-172, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33651360

RESUMO

Extracellular vesicles (EVs) are small membrane-bound particles released into extracellular space by almost all cell types, and found in body fluids like blood, urine, and saliva. Mounting evidence has demonstrated the clinical potential of EVs as diagnostic and therapeutic tools to analyse physiological/pathological processes due to their ability to transport biomolecules secreted from diverse tissues of an individual.For example, the urinary EVs (uEVs), released from all regions of the kidney's nephron and from other cells that line the urinary tract, retain proteomic and transcriptomic markers specific to their cell of origin representing a valuable tool for kidney disease diagnosis.Despite the numerous efforts in developing suitable methods to separate EVs from biofluids, providing material of high purity and low variability poses a limit to clinical translation.This chapter focuses on advantages and disadvantages of several EV isolation methodologies, and provides examples of uEV isolation protocols based on time, cost, and equipment considerations, as well as the sample requirements for any downstream analyses.


Assuntos
Vesículas Extracelulares/química , Urinálise/métodos , Animais , Biomarcadores/análise , Cromatografia em Gel/métodos , Humanos , Imunoprecipitação/métodos , Biópsia Líquida/métodos , Ultracentrifugação/métodos
7.
Medicine (Baltimore) ; 100(10): e24010, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33725812

RESUMO

RATIONALE: Renal-occupying lesions positive for urine fluorescence in situ hybridization (FISH) are usually considered urothelial carcinomas. Here, we describe 2 cases of renal metastases with chromosome duplications in urine exfoliated cells. PATIENT SYMPTOMS: Patient 1, a 56-year-old male with a history of esophageal cancer, was admitted to our hospital on May 2017 after presenting with right back pain with microscopic hematuria for 1 month. Magnetic resonance imaging (MRI) showed right renal space-occupying lesions (5.4 cm × 4.6 cm) and multiple enlarged lymph nodes in the right renal hilum and retroperitoneum. The cystoscopy results were negative, and FISH analysis of urine exfoliated cells was positive, indicative of chromosome 3, 7, and 17 amplifications. Patient 2 was a 50-year-old male who was admitted to our hospital on May 2019 with no obvious cause of abdominal pain and abdominal distension (lasting for 7 days), with a serum creatinine level of 844 µmol/L. Patient 2 had no hematuria or fever, and MRI showed left renal inferior and medial space-occupying lesions, and multiple mesenteric nodules at the junction of the left adrenal gland, retroperitoneum, abdomen, and pelvis, which were partially fused. The tumor lesions were approximately 3.1 cm × 2.3 cm in size. The urine FISH results were positive, indicating chromosome 3, 7, and 17 amplifications. DIAGNOSES: Both patients were diagnosed with renal tumors with unknown pathology. INTERVENTIONS: Patient 1 underwent laparoscopic resection of the kidney and ureter, and sleeve cystectomy. The postoperative pathological diagnosis was metastatic keratinized squamous cell carcinoma, with squamous cell carcinoma in the right hilar lymph node. Histological FISH of the primary esophageal cancer and renal metastases were consistent with the urine FISH test results. Patient 2 underwent a biopsy of the left renal inferior and retroperitoneal areas, and was diagnosed with diffuse large B-cell lymphoma. OUTCOMES: Patient 1 survived 6 months after urological surgery. After treating patient 2 with the R-CHOP regimen and kinase inhibitors, his renal function recovered significantly and the mass become undetectable. LESSONS: Our results imply that FISH-positive renal occupying lesions should be considered as potential renal metastases with chromosome aberrations when making a differential diagnosis.


Assuntos
Neoplasias Esofágicas/patologia , Neoplasias Renais/diagnóstico , Linfoma/patologia , Neoplasias Retroperitoneais/patologia , Duplicação Cromossômica , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/urina , Esôfago/diagnóstico por imagem , Humanos , Hibridização in Situ Fluorescente , Rim/diagnóstico por imagem , Rim/patologia , Rim/cirurgia , Neoplasias Renais/genética , Neoplasias Renais/secundário , Neoplasias Renais/urina , Biópsia Líquida/métodos , Linfonodos/diagnóstico por imagem , Linfoma/genética , Linfoma/urina , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Neoplasias Retroperitoneais/genética , Neoplasias Retroperitoneais/urina , Espaço Retroperitoneal/diagnóstico por imagem , Tomografia Computadorizada por Raios X , Urinálise/métodos
8.
Methods Mol Biol ; 2265: 223-233, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704718

RESUMO

The advent of personalized medicines targeting cell signaling pathways has radically improved melanoma patient outcomes. More recently, immune-modulating therapies disrupting the PD-1/PD-L1 axis have become a powerful tool in the treatment of a range of melanoma, showing a profound improvement in the overall survival outcomes. However, immune checkpoint inhibitors (ICIs) are associated with considerable toxicities and appear to only be efficacious in a subset of melanoma patients. Therefore, there is an urgent need to identify biomarkers that can determine if patients will or will not respond to ICI therapy. Here, we describe an optimized method for analyzing PD-L1 expression on circulating melanoma cells following immunomagnetic enrichment from patient blood samples.


Assuntos
Antígeno B7-H1/metabolismo , Separação Imunomagnética/métodos , Melanoma/sangue , Células Neoplásicas Circulantes/imunologia , Anticorpos/imunologia , Antígeno B7-H1/imunologia , Humanos , Leucócitos Mononucleares/citologia , Biópsia Líquida/métodos , Melanoma/diagnóstico
9.
Methods Mol Biol ; 2265: 265-276, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33704721

RESUMO

Liquid biopsy has emerged as the next generation target for diagnostics and therapeutic monitoring of many diseases including cancer. Liquid biopsy offers noninvasive analysis of aberrant biomolecular changes (e.g., aberrant protein expression, DNA mutation) which can provide crucial information on disease stages and therapy responses. As a diagnostically important biomarker for melanoma, the detection of the BRAFV600E aberration at the DNA and protein level in liquid biopsies confers an attractive option. This method describes the preparation and operation of an integrated multimolecular sensor (IMMS) for simultaneous detection of the BRAFV600E aberration in both molecular forms from circulating melanoma cells in liquid biopsy. IMMS integrates specific melanoma cell capture, cell release, cell lysis, and electrochemical BRAFV600E detection on a single device. IMMS is demonstrated for a sample-to-answer workflow of plasma spiked with melanoma cells.


Assuntos
Técnicas Biossensoriais/métodos , Imunoensaio/métodos , Dispositivos Lab-On-A-Chip , Melanoma/metabolismo , Microfluídica/instrumentação , Microfluídica/métodos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Técnicas Biossensoriais/instrumentação , Técnicas de Cultura de Células/métodos , Humanos , Imunoensaio/instrumentação , Biópsia Líquida/métodos , Melanoma/genética , Melanoma/patologia , Mutação , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
10.
J Cancer Res Clin Oncol ; 147(5): 1431-1442, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33760943

RESUMO

BACKGROUND: The recent advancement in massively parallel sequencing technologies has empowered liquid biopsies, in particular circulating tumor DNA (ctDNA) analysis, to be the new paradigm in personalized cancer management. Plasma ctDNA detection overcomes the current limitations in tumor tissue procurement and serves as a convenient and non-invasive method to capture tumor heterogeneity and genetic evolution along patients' cancer journey. In breast cancer, the current clinical application of ctDNA includes real-time monitoring of tumor response, detection of drug-resistant clones, assessing dynamic variations in tumor mutational landscape, identifying actionable mutations, detecting minimal residual disease and screening of early tumor. PURPOSE: This review aims to summarize the current clinical evidence of ctDNA application in the management of breast cancer.


Assuntos
Neoplasias da Mama/sangue , Neoplasias da Mama/genética , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Feminino , Humanos , Biópsia Líquida/métodos , Mutação/genética
11.
RNA Biol ; 18(5): 688-695, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33530819

RESUMO

The COVID-19 emergency pandemic resulting from infection with SARS-CoV-2 represents a major threat to public health worldwide. There is an urgent clinical demand for easily accessible tools to address weaknesses and gaps in the management of COVID-19 patients. In this context, transcriptomic profiling of liquid biopsies, especially microRNAs (miRNAs), has recently emerged as a robust source of potential clinical indicators for medical decision-making. Nevertheless, the analysis of the circulating miRNA signature and its translation to clinical practice requires strict control of a wide array of methodological details. In this review, we indicate the main methodological aspects that should be addressed when evaluating the circulating miRNA profiles in COVID-19 patients, from preanalytical and analytical variables to the experimental design, impact of confounding, analysis of the data and interpretation of the findings, among others. Additionally, we provide practice points to ensure the rigour and reproducibility of miRNA-based biomarker investigations of this condition.Abbreviations: ACE: angiotensin-converting enzyme; ARDS: acute respiratory distress syndrome; COVID-19: coronavirus disease 2019; ERDN: early Detection Research Network; LMWH: low molecular weight heparin; miRNA: microRNA; ncRNA: noncoding RNA; SARS-CoV-2: severe acute respiratory syndrome coronavirus-2; SOP: standard operating procedure.


Assuntos
/sangue , Perfilação da Expressão Gênica/métodos , MicroRNAs/sangue , MicroRNAs/genética , /virologia , Perfilação da Expressão Gênica/normas , Marcadores Genéticos , Humanos , Biópsia Líquida/métodos , Biópsia Líquida/normas , MicroRNAs/isolamento & purificação , Pandemias , Inativação de Vírus
12.
Int J Mol Sci ; 22(3)2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33503982

RESUMO

Studies investigating microRNAs as potential biomarkers for cancer, immune-related diseases, or cardiac pathogenic diseases, among others, have exponentially increased in the last years. In particular, altered expression of specific miRNAs correlates with the occurrence of several diseases, making these molecules potential molecular tools for non-invasive diagnosis, prognosis, and response to therapy. Nonetheless, microRNAs are not in clinical use yet, due to inconsistencies in the literature regarding the specific miRNAs identified as biomarkers for a specific disease, which in turn can be attributed to several reasons, including lack of assay standardization and reproducibility. Technological limitations in circulating microRNAs measurement have been, to date, the biggest challenge for using these molecules in clinical settings. In this review we will discuss pre-analytical, analytical, and post-analytical challenges to address the potential technical biases and patient-related parameters that can have an influence and should be improved to translate miRNA biomarkers to the clinical stage. Moreover, we will describe the currently available methods for circulating miRNA expression profiling and measurement, underlining their advantages and potential pitfalls.


Assuntos
Biomarcadores Tumorais , Testes Genéticos/métodos , MicroRNAs/genética , Neoplasias/diagnóstico , Neoplasias/genética , Ácidos Nucleicos Livres , MicroRNA Circulante , Regulação Neoplásica da Expressão Gênica , Testes Genéticos/normas , Humanos , Biópsia Líquida/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/normas , Prognóstico
13.
J Biomech ; 117: 110235, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33486262

RESUMO

Microfluidic devices can be thought of as comprising interconnected miniaturized compartments performing multiple experimental tasks individually or in parallel in an integrated fashion. Due to its small size, portability, and low cost, attempts have been made to incorporate detection assays into microfluidic platforms for diseases such as cancer and infection. Some of these technologies have served as point-of-care and sample-to-answer devices. The methods for detecting biomarkers in different diseases usually share similar principles and can conveniently be adapted to cope with arising health challenges. The COVID-19 pandemic is one such challenge that is testing the performance of both our conventional and newly-developed disease diagnostic technologies. In this mini-review, we will first look at the progress made in the past few years in applying microfluidics for liquid biopsy and infectious disease detection. Following that, we will use the current pandemic as an example to discuss how such technological advancements can help in the current health challenge and better prepare us for future ones.


Assuntos
/diagnóstico , Biópsia Líquida/métodos , Microfluídica/métodos , Testes Imediatos , Biomarcadores , DNA Tumoral Circulante , Exossomos , Humanos , Dispositivos Lab-On-A-Chip , Aprendizado de Máquina , Neoplasias/diagnóstico , Células Neoplásicas Circulantes
14.
Methods Mol Biol ; 2174: 143-170, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32813249

RESUMO

Extracellular vesicles (EVs) produced by cancer cells function as a unique form of intercellular communication that can promote cell growth and survival, help shape the tumor microenvironment, and increase invasive and metastatic activity. There are two major classes of EVs, microvesicles (MVs) and exosomes, and they differ in how they are formed. MVs are generated by the outward budding and fission of the plasma membrane. On the other hand, exosomes are derived as multivesicular bodies (MVBs) fuse with the plasma membrane and release their contents. What makes EVs especially interesting is how they mediate their effects. Both MVs and exosomes have been shown to contain a wide-variety of bioactive cargo, including cell surface, cytosolic, and nuclear proteins, as well as RNA transcripts, micro-RNAs (miRNAs), and even fragments of DNA. EVs, and their associated cargo, can be transferred to other cancer cells, as well as to normal cell types, causing the recipient cells to undergo phenotypic changes that promote different aspects of cancer progression. These findings, combined with those demonstrating that the amounts and contents of EVs produced by cancer cells can vary depending on their cell of origin, stage of development, or response to therapies, have raised the exciting possibility that EVs can be used for diagnostic purposes. Moreover, the pharmaceutical community is aggressively pursuing the use of EVs as a potential drug delivery platform. Here, in this chapter, we will highlight what is currently known about how EVs are generated, how they impact cancer progression, and the different ways they are being exploited for clinical applications.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Biópsia Líquida/métodos , Neoplasias/patologia , Membrana Celular/metabolismo , Membrana Celular/patologia , Exossomos/metabolismo , Exossomos/patologia , Vesículas Extracelulares/classificação , Humanos , Neoplasias/irrigação sanguínea , Neovascularização Patológica/patologia , Microambiente Tumoral/imunologia
15.
Methods Mol Biol ; 2226: 39-45, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33326092

RESUMO

Liquid biopsies enable noninvasive therapy monitoring in patients with solid tumors. Specific serum markers such as proteins, hormones, or enzymes released from tumor cells or in response to tumor growth can be used for quantification of the tumor burden. However, only a fraction of pediatric tumors has none of these serum markers, but tumor-specific genetic alterations represent reliable alternatives. Here we describe a method for using genomic fusion sequences as liquid biopsy markers in Ewing sarcoma patients.


Assuntos
Neoplasias Ósseas/diagnóstico , Biópsia Líquida , Sarcoma de Ewing/diagnóstico , Biomarcadores Tumorais , DNA Tumoral Circulante/isolamento & purificação , Humanos , Biópsia Líquida/métodos , Reação em Cadeia da Polimerase
16.
Methods Mol Biol ; 2195: 189-223, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32852766

RESUMO

Genomewide association studies (GWAS) have been widely used in recent years to identify common variants that are associated with multiple types of cancer, including testicular germ cell tumors. These studies require no a priori hypotheses and have advantages, including the ability to highlight new pathways relevant to the biology of common diseases. GWAS require collection of germline DNA from individuals with and without the disease of interest. Following DNA extraction and quantification, a variety of array based platforms are available to evaluate common and moderately rare germline variation throughout the genome in an agnostic fashion. Here, we describe DNA extraction methods from samples typically used in the evaluation of germline genetic variation (blood and saliva). We also describe assays used to assess DNA quality and quantity. Finally, we include methods describing array based genotyping using the Illumina platform and validation of relevant variants using the iPLEX Agena Multiplexed Genotyping (formerly Sequenom).


Assuntos
Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Testiculares/genética , Alelos , Biomarcadores Tumorais , Estudo de Associação Genômica Ampla/métodos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida/métodos , Masculino , Neoplasias Embrionárias de Células Germinativas/diagnóstico , Neoplasias Embrionárias de Células Germinativas/epidemiologia , Saliva/metabolismo , Neoplasias Testiculares/diagnóstico , Neoplasias Testiculares/epidemiologia
17.
Int J Mol Sci ; 22(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374539

RESUMO

Extracellular vesicles (EVs) are released by shedding during different physiological processes and are increasingly thought to be new potential biomarkers. However, the impact of pre-analytical processing phases on the final measurement is not predictable and for this reason, the translation of basic research into clinical practice has been precluded. Here we have optimized a simple procedure in combination with polychromatic flow cytometry (PFC), to identify, classify, enumerate, and separate circulating EVs from different cell origins. This protocol takes advantage of a lipophilic cationic dye (LCD) able to probe EVs. Moreover, the application of the newly optimized PFC protocol here described allowed the obtainment of repeatable EVs counts. The translation of this PFC protocol to fluorescence-activated cell sorting allowed us to separate EVs from fresh peripheral blood samples. Sorted EVs preparations resulted particularly suitable for proteomic analyses, which we applied to study their protein cargo. Here we show that LCD staining allowed PFC detection and sorting of EVs from fresh body fluids, avoiding pre-analytical steps of enrichment that could impact final results. Therefore, LCD staining is an essential step towards the assessment of EVs clinical significance.


Assuntos
Biomarcadores , Vesículas Extracelulares/metabolismo , Citometria de Fluxo , Biópsia Líquida , Animais , Citometria de Fluxo/métodos , Humanos , Biópsia Líquida/métodos , Tamanho da Partícula , Plasma , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
18.
Int J Mol Sci ; 21(24)2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33339204

RESUMO

Platelets contribute to several types of cancer through plenty of mechanisms. Upon activation, platelets release many molecules, including growth and angiogenic factors, lipids, and extracellular vesicles, and activate numerous cell types, including vascular and immune cells, fibroblasts, and cancer cells. Hence, platelets are a crucial component of cell-cell communication. In particular, their interaction with cancer cells can enhance their malignancy and facilitate the invasion and colonization of distant organs. These findings suggest the use of antiplatelet agents to restrain cancer development and progression. Another peculiarity of platelets is their capability to uptake proteins and transcripts from the circulation. Thus, cancer-patient platelets show specific proteomic and transcriptomic expression patterns, a phenomenon called tumor-educated platelets (TEP). The transcriptomic/proteomic profile of platelets can provide information for the early detection of cancer and disease monitoring. Platelet ability to interact with tumor cells and transfer their molecular cargo has been exploited to design platelet-mediated drug delivery systems to enhance the efficacy and reduce toxicity often associated with traditional chemotherapy. Platelets are extraordinary cells with many functions whose exploitation will improve cancer diagnosis and treatment.


Assuntos
Plaquetas/metabolismo , Neoplasias/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Carcinogênese/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Biópsia Líquida/métodos , Neoplasias/tratamento farmacológico , Neoplasias/patologia
19.
Nat Commun ; 11(1): 4489, 2020 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-32895384

RESUMO

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).


Assuntos
Biomarcadores Tumorais/isolamento & purificação , Carcinoma Hepatocelular/diagnóstico , Detecção Precoce de Câncer/métodos , Vesículas Extracelulares/genética , Neoplasias Hepáticas/diagnóstico , Idoso , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/sangue , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Química Click/instrumentação , Química Click/métodos , Química Computacional , Simulação por Computador , Diagnóstico Diferencial , Dimetilpolisiloxanos/química , Progressão da Doença , Detecção Precoce de Câncer/instrumentação , Feminino , Células Hep G2 , Humanos , Dispositivos Lab-On-A-Chip , Biópsia Líquida/instrumentação , Biópsia Líquida/métodos , Cirrose Hepática/sangue , Cirrose Hepática/patologia , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Pessoa de Meia-Idade , Nanoestruturas/química , Nanofios/química , Estadiamento de Neoplasias , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
20.
PLoS One ; 15(6): e0234182, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32492056

RESUMO

The development of noninvasive approaches for brain tumor diagnosis and monitoring continues to be a major medical challenge. Although blood-based liquid biopsy has received considerable attention in various cancers, limited progress has been made for brain tumors, at least partly due to the hindrance of tumor biomarker release into the peripheral circulation by the blood-brain barrier. Focused ultrasound (FUS) combined with microbubbles induced BBB disruption has been established as a promising technique for noninvasive and localized brain drug delivery. Building on this established technique, we propose to develop FUS-enabled liquid biopsy technique (FUS-LBx) to enhance the release of brain tumor biomarkers (e.g., DNA, RNA, and proteins) into the circulation. The objective of this study was to demonstrate that FUS-LBx could sufficiently increase plasma levels of brain tumor biomarkers without causing hemorrhage in the brain. Mice with orthotopic implantation of enhanced green fluorescent protein (eGFP)-transfected murine glioma cells were treated using magnetic resonance (MR)-guided FUS system in the presence of systemically injected microbubbles at three peak negative pressure levels (0.59, 1.29, and 1.58 MPa). Plasma eGFP mRNA levels were quantified with the quantitative polymerase chain reaction (qPCR). Contrast-enhanced MR images were acquired before and after the FUS sonication. FUS at 0.59 MPa resulted in an increased plasma eGFP mRNA level, comparable to those at higher acoustic pressures (1.29 MPa and 1.58 MPa). Microhemorrhage density associated with FUS at 0.59 MPa was significantly lower than that at higher acoustic pressures and not significantly different from the control group. MRI analysis revealed that post-sonication intratumoral and peritumoral hyperenhancement had strong correlations with the level of FUS-induced biomarker release and the extent of hemorrhage. This study suggests that FUS-LBx could be a safe and effective brain-tumor biomarker release technique, and MRI could be used to develop image-guided FUS-LBx.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Ultrassonografia de Intervenção/métodos , Animais , Biomarcadores Tumorais/sangue , Barreira Hematoencefálica , Neoplasias Encefálicas/diagnóstico por imagem , Linhagem Celular Tumoral , Meios de Contraste , Feminino , Glioblastoma/diagnóstico por imagem , Proteínas de Fluorescência Verde/sangue , Proteínas de Fluorescência Verde/genética , Hemorragias Intracranianas/etiologia , Hemorragias Intracranianas/patologia , Biópsia Líquida/métodos , Imagem por Ressonância Magnética , Camundongos , Ultrassonografia de Intervenção/efeitos adversos
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