RESUMO
Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.
Assuntos
Sistemas CRISPR-Cas , Carcinoma Pulmonar de Células não Pequenas/genética , Técnicas de Inativação de Genes , Neoplasias Pulmonares/genética , Metástase Neoplásica/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Estudo de Associação Genômica Ampla , Humanos , Neoplasias Pulmonares/patologia , Camundongos , RNA Guia de CinetoplastídeosRESUMO
Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.
Assuntos
Copépodes/enzimologia , Vesículas Extracelulares/metabolismo , Luciferases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Linhagem Celular Tumoral , Copépodes/genética , Copépodes/metabolismo , Feminino , Humanos , Luciferases/genética , Proteínas de Membrana/genética , Membranas/metabolismo , Camundongos , Camundongos Nus , Proteínas Recombinantes/genética , Via Secretória/genética , Distribuição TecidualRESUMO
Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type.
Assuntos
Biomarcadores Tumorais/metabolismo , Vesículas Extracelulares/metabolismo , Citometria de Fluxo/métodos , Proteínas de Membrana/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Imunoensaio/métodos , Luciferases/metabolismo , Camundongos , Camundongos Nus , Pessoa de Meia-IdadeRESUMO
Extracellular vesicles (EV) are a family of cell-originating, membrane-enveloped nanoparticles with diverse biological function, diagnostic potential, and therapeutic applications. While EV can be abundant in circulation, their small size (â¼4 order of magnitude smaller than cells) has necessitated bulk analyses, making many more nuanced biological explorations, cell of origin questions, or heterogeneity investigations impossible. Here we describe a single EV analysis (SEA) technique which is simple, sensitive, multiplexable, and practical. We profiled glioblastoma EV and discovered surprising variations in putative pan-EV as well as tumor cell markers on EV. These analyses shed light on the heterogeneous biomarker profiles of EV. The SEA technology has the potential to address fundamental questions in vesicle biology and clinical applications.
Assuntos
Vesículas Extracelulares/patologia , Glioblastoma/patologia , Biomarcadores Tumorais/análise , Linhagem Celular Tumoral , Desenho de Equipamento , Vesículas Extracelulares/química , Glioblastoma/química , Glioblastoma/diagnóstico por imagem , Humanos , Dispositivos Lab-On-A-Chip , Imagem Óptica/instrumentação , Imagem Óptica/métodosRESUMO
Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1-dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.
Assuntos
Antígeno B7-H1/metabolismo , Neoplasias Encefálicas/imunologia , Vesículas Extracelulares/metabolismo , Glioblastoma/imunologia , Evasão da Resposta Imune , Antígenos de Neoplasias/imunologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T , Regulação para CimaRESUMO
Exosomes have recently emerged as highly promising cancer biomarkers because they are abundant in biofluids, carry proteins and RNA reflecting their originating cells and are stable over weeks. Beyond abundance and stability, detailed exosome analyses could be clinically useful for diagnosing and profiling cancers. Despite their clinical potential, simple, reliable and sensitive approaches for rapidly quantifying exosomes and their molecular information has been challenging. Therefore, there is a clear need to develop next-generation sensing technologies for exosome detection and analysis. In this critical review, we will describe three nanotechnology sensing platforms developed for analysis of exosomal proteins and RNAs directly from clinical specimens and discuss future development to facilitate their translation into routine clinical use.
Assuntos
Técnicas Biossensoriais , Exossomos , Nanotecnologia , Biomarcadores Tumorais/análise , Humanos , Técnicas Analíticas Microfluídicas , Proteínas/análise , RNA/análiseRESUMO
Pathogen-activated antibody-secreting cells (ASCs) produce and secrete antigen-specific antibodies. ASCs are detectable in the peripheral blood as early as 3 days after antigen exposure, which makes ASCs a potential biomarker for early disease detection. Here, we present a magnetic capture and detection (MCD) assay for sensitive, on-site detection of ASCs. In this approach, ASCs are enriched through magnetic capture, and secreted antibodies are magnetically detected by a miniaturized nuclear magnetic resonance (µNMR) system. This approach is based entirely on magnetics, which supports high contrast against biological background and simplifies assay procedures. We advanced the MCD system by (i) synthesizing magnetic nanoparticles with high magnetic moments for both cell capture and antibody detection, (ii) developing a miniaturized magnetic device for high-yield cell capture, and (iii) optimizing the µNMR assay for antibody detection. Antibody responses targeting hemolysin E (HlyE) can accurately identify individuals with acute enteric fever. As a proof-of-concept, we applied MCD to detect antibodies produced by HlyE-specific hybridoma cells. The MCD achieved high sensitivity in detecting antibodies secreted from as few as 5 hybridoma cells (50 cells/mL). Importantly, the assay could be performed with whole blood with minimal sample processing.
Assuntos
Células Produtoras de Anticorpos/metabolismo , Infecções Bacterianas/diagnóstico , Nanopartículas de Magnetita/química , Doença Aguda , Anticorpos Antibacterianos/análise , Anticorpos Antibacterianos/imunologia , Células Produtoras de Anticorpos/imunologia , Antígenos de Bactérias/análise , Antígenos de Bactérias/imunologia , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Técnicas Biossensoriais/métodos , Separação Celular/métodos , Proteínas Hemolisinas/análise , Proteínas Hemolisinas/imunologia , Humanos , Hibridomas/citologia , Hibridomas/imunologia , Ferro/química , Dispositivos Lab-On-A-Chip , Óxidos/química , Estudo de Prova de Conceito , Salmonella paratyphi A/imunologia , Salmonella typhi/imunologia , Febre Tifoide/diagnóstico , Febre Tifoide/imunologia , Febre Tifoide/microbiologia , Zinco/químicaRESUMO
Kidney transplant patients require life-long surveillance to detect allograft rejection. Repeated biopsy, albeit the clinical gold standard, is an invasive procedure with the risk of complications and comparatively high cost. Conversely, serum creatinine or urinary proteins are noninvasive alternatives but are late markers with low specificity. We report a urine-based platform to detect kidney transplant rejection. Termed iKEA (integrated kidney exosome analysis), the approach detects extracellular vesicles (EVs) released by immune cells into urine; we reasoned that T cells, attacking kidney allografts, would shed EVs, which in turn can be used as a surrogate marker for inflammation. We optimized iKEA to detect T-cell-derived EVs and implemented a portable sensing system. When applied to clinical urine samples, iKEA revealed high level of CD3-positive EVs in kidney rejection patients and achieved high detection accuracy (91.1%). Fast, noninvasive, and cost-effective, iKEA could offer new opportunities in managing transplant recipients, perhaps even in a home setting.
Assuntos
Técnicas Biossensoriais/métodos , Exossomos/imunologia , Rejeição de Enxerto/urina , Inflamação/urina , Vesículas Extracelulares/imunologia , Vesículas Extracelulares/patologia , Feminino , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/fisiopatologia , Humanos , Inflamação/imunologia , Inflamação/fisiopatologia , Rim/imunologia , Rim/patologia , Transplante de Rim/efeitos adversos , Masculino , Proteômica/métodos , Linfócitos T/imunologiaRESUMO
Health care-associated infections (HAIs) and drug-resistant pathogens have become a major health care issue with millions of reported cases every year. Advanced diagnostics would allow clinicians to more quickly determine the most effective treatment, reduce the nonspecific use of broad-spectrum antimicrobials, and facilitate enrollment in new antibiotic treatments. We present a new integrated system, polarization anisotropy diagnostics (PAD), for rapid detection of HAI pathogens. The PAD uses changes of fluorescence anisotropy when detection probes recognize target bacterial nucleic acids. The technology is inherently robust against environmental noise and economically scalable for parallel measurements. The assay is fast (2 hours) and performed on-site in a single-tube format. When applied to clinical samples obtained from interventional procedures, the PAD determined the overall bacterial burden, differentiated HAI bacterial species, and identified drug resistance and virulence status. The PAD system holds promise as a powerful tool for near-patient, rapid HAI testing.
Assuntos
Bactérias/isolamento & purificação , Infecções Relacionadas a Cateter/diagnóstico , Infecção Hospitalar/diagnóstico , Polarização de Fluorescência/métodos , Bactérias/patogenicidade , Infecções Relacionadas a Cateter/microbiologia , Infecção Hospitalar/microbiologia , Resistência Microbiana a Medicamentos/genética , HumanosRESUMO
Molecular targeting using ligands specific to disease markers has shown great promise for early detection and directed therapy. Bead-based combinatorial libraries have served as powerful tools for the discovery of novel targeting agents. Screening platforms employing magnetic capture have been used to achieve rapid and efficient identification of high-affinity ligands from one-bead-one-compound (OBOC) libraries. Traditional manual methodologies to isolate magnetized "hit" beads are tedious and lack accuracy, and existing instruments to expedite bead sorting tend to be costly and complex. Here, we describe the design and construction of a simple and inexpensive microfluidic magnetic sorting device using standard photolithography and soft lithography approaches to facilitate high-throughput isolation of magnetized positive hit beads from combinatorial libraries. We have demonstrated that the device is able to sort magnetized beads with superior accuracy compared to conventional manual sorting approaches. This chip offers a very convenient yet inexpensive alternative for screening OBOC libraries.
Assuntos
Técnicas de Química Combinatória/métodos , Magnetismo , Microfluídica/métodos , Bibliotecas de Moléculas Pequenas , Biomarcadores , Ensaios de Triagem em Larga Escala/métodos , Humanos , Ligantes , MicroesferasRESUMO
Microvesicles (MVs) are an increasingly important source for biomarker discovery and clinical diagnostics. The small size of MVs and their presence in complex biological environment, however, pose technical challenges in sample preparation, particularly when sample volumes are small. We herein present an acoustic nanofilter system that size-specifically separates MVs in a continuous and contact-free manner. The separation uses ultrasound standing waves to exert differential acoustic force on MVs according to their size and density. By optimizing the design of the ultrasound transducers and underlying electronics, we were able to achieve a high separation yield and resolution. The "filter size-cutoff" can be controlled electronically in situ, which enables versatile MV-size selection. We applied the acoustic nanofilter to isolate nanoscale (<200 nm) vesicles from cell culture media as well as MVs in stored red blood cell products. With the capacity for rapid and contact-free MV isolation, the developed system could become a versatile preparatory tool for MV analyses.
Assuntos
Acústica , Fracionamento Celular/métodos , Micropartículas Derivadas de Células , Fracionamento Celular/instrumentação , Linhagem Celular Tumoral , Eritrócitos/citologia , Exossomos , Humanos , TransdutoresRESUMO
Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O(6)-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Exossomos/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Microfluídica/métodos , Animais , Biomarcadores Tumorais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Separação Imunomagnética , Camundongos Nus , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Temozolomida , Resultado do TratamentoRESUMO
Molecular profiling of central nervous system lymphomas in cerebrospinal fluid (CSF) samples can be challenging due to the paucicellular and limited nature of the samples. Presented herein is a microfluidic platform for complete CSF lymphoid cell analysis, including single cell capture in sub-nanoliter traps, and molecular and chemotherapeutic response profiling via on-chip imaging, all in less than one hour. The system can detect scant lymphoma cells and quantitate their kappa/lambda immunoglobulin light chain restriction patterns. The approach can be further customized for measurement of additional biomarkers, such as those for differential diagnosis of lymphoma subtypes or for prognosis, as well as for imaging exposure to experimental drugs.
Assuntos
Neoplasias do Sistema Nervoso Central/diagnóstico , Líquido Cefalorraquidiano/citologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Linfoma/diagnóstico , Microfluídica/métodos , Neoplasias do Sistema Nervoso Central/patologia , Humanos , Linfoma/patologiaRESUMO
The high incidence of acute and chronic kidney injury due to various environmental factors such as heavy metals or chemicals has been a major problem in developing countries. However, the diagnosis of kidney injury in these areas can be more challenging due to the lack of highly sensitive and specific techniques that can be applied in point-of-care settings. To address this, we have developed a technique called 'micro-urine nanoparticle detection (µUNPD)', that allows the detection of trace amounts of molecular markers in urine. Specifically, this technique utilizes an automated on-chip assay followed by detection with a hand-held device for the read-out. Using the µUNPD technology, the kidney injury markers KIM-1 and Cystatin C were detected down to concentrations of 0.1 ng/ml and 20 ng/ml respectively, which meets the cut-off range required to identify patients with acute or chronic kidney injury. Thus, we show that the µUNPD technology enables point of care and non-invasive detection of kidney injury, and has potential for applications in diagnosing kidney injury with high sensitivity in resource-limited settings.
Assuntos
Injúria Renal Aguda/urina , Biomarcadores/urina , Nanopartículas de Magnetita/análise , Sistemas Automatizados de Assistência Junto ao Leito , Insuficiência Renal Crônica/urina , Injúria Renal Aguda/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Cistatina C/química , Cistatina C/urina , Estudos de Viabilidade , Receptor Celular 1 do Vírus da Hepatite A , Humanos , Espectroscopia de Ressonância Magnética , Nanopartículas de Magnetita/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/urina , Procedimentos Analíticos em Microchip/métodos , Pessoa de Meia-Idade , Receptores Virais/química , Insuficiência Renal Crônica/diagnóstico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto JovemRESUMO
We present a hybrid magnetic/size-sorting (HMSS) chip for isolation and molecular analyses of circulating tumor cells (CTCs). The chip employs both negative and positive cell selection in order to provide high throughput, unbiased CTC enrichment. Specifically, the system utilizes a self-assembled magnet to generate high magnetic forces and a weir-style structure for cell sorting. The resulting device thus can perform multiple functions, including magnetic depletion, size-selective cell capture, and on-chip molecular staining. With such capacities, the HMSS device allowed one-step CTC isolation and single cell detection from whole blood, tested with spiked cancer cells. The system further facilitated the study of individual CTCs for heterogeneity in molecular marker expression.