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Microglia are a specialized population of innate immune cells located in the central nervous system. In response to physiological and pathological changes in their microenvironment, microglia can polarize into pro-inflammatory or anti-inflammatory phenotypes. A dysregulation in the pro-/anti-inflammatory balance is associated with many pathophysiological changes in the brain and nervous system. Therefore, the balance between microglia pro-/anti-inflammatory polarization can be a potential biomarker for the various brain pathologies. A non-invasive method of detecting microglia polarization in patients would have promising clinical applications. Here, we perform proteomic analysis of small extracellular vesicles (sEVs) derived from microglia cells to identify sEVs biomarkers indicative of pro-inflammatory and anti-inflammatory phenotypic changes. sEVs were isolated from microglia cell lines under different inflammatory conditions and analyzed by proteomics by liquid chromatography with mass spectrometry. Our findings provide the potential roles of sEVs that could be related to the pathogenesis of various brain diseases.
Assuntos
Vesículas Extracelulares , Microglia , Proteômica , Microglia/metabolismo , Humanos , Vesículas Extracelulares/metabolismo , Proteômica/métodos , Linhagem Celular , Proteoma/análise , Proteoma/metabolismo , Biomarcadores/metabolismo , Biomarcadores/análise , Inflamação/metabolismoRESUMO
The implementation of accurate and sensitive molecular detection for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is paramount to effectively control the ongoing coronavirus disease 2019 (COVID-19) pandemic. In this regard, we herein propose the specific and highly sensitive SARS-CoV-2 detection based on nanoyeast single-chain-variable fragment (scFv) and ultrasensitive plasmonic nanobox-integrated nanomixing microassay. Importantly, this designed platform showcases the utility of nanoyeast-scFvs as specific capture reagents targeting the receptor-binding domain (RBD) of the virus and as monoclonal antibody alternatives suitable for cost-effective mass production and frequent testing. By capitalizing on single-particle active nanoboxes as plasmonic nanostructures for surface-enhanced Raman scattering (SERS), the microassay utilizes highly sensitive Raman signals to indicate virus infection. The developed microassay further integrated nanomixing for accelerating molecular collisions. Through the synergistic working of nanoyeast-scFv, plasmonic nanoboxes, and nanomixing, the highly specific and sensitive SARS-CoV-2 detection is achieved as low as 17 virus/µL without any molecular amplification. We successfully demonstrate SARS-CoV-2 detection in saliva samples of simulated patients at clinically relevant viral loads, suggesting the possibility of this platform for accurate and noninvasive patient screening.
Assuntos
COVID-19 , Anticorpos de Cadeia Única , Humanos , SARS-CoV-2 , Saliva , Análise Espectral RamanRESUMO
A manner in which cells can communicate with each other is via secreted nanoparticles termed exosomes. These vesicles contain lipids, nucleic acids, and proteins, and are said to reflect the cell-of-origin. However, for the exosomal protein content, there is limited evidence in the literature to verify this statement. Here, proteomic assessment combined with pathway-enrichment analysis is used to demonstrate that the protein cargo of exosomes reflects the epithelial/mesenchymal phenotype of secreting breast cancer cells. Given that epithelial-mesenchymal plasticity is known to implicate various stages of cancer progression, the results suggest that breast cancer subtypes with distinct epithelial and mesenchymal phenotypes may be distinguished by directly assessing the protein content of exosomes. Additionally, the work is a substantial step toward verifying the statement that cell-derived exosomes reflect the phenotype of the cells-of-origin.
Assuntos
Neoplasias da Mama/patologia , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/ultraestrutura , Linhagem Celular Tumoral , Cromatografia Líquida , Transição Epitelial-Mesenquimal/fisiologia , Exossomos/metabolismo , Exossomos/patologia , Exossomos/ultraestrutura , Feminino , Humanos , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de TransmissãoRESUMO
While tumour cells are classically known to communicate via direct cell-to-cell contact and the secretion of soluble protein-based factors such as cytokines and growth factors, alternative novel mechanisms that promote tumour progression have recently emerged. Now, new critical components of the secretome thought to be involved in tumour progression are exosomes, small vesicles of endocytic origin that carry a variety of bioactive molecules, including proteins, lipids, RNA, as well as DNA molecules. Cancer cell-derived exosomes have been shown to participate in crucial steps of metastatic spread of a primary tumour, ranging from oncogenic reprogramming of malignant cells to formation of pre-metastatic niches. These effects are achieved through the mediation of intercellular cross-talk and subsequent modification of both local and distant microenvironments in an autocrine and paracrine fashion. Here, we summarise the recent findings that implicate this non-canonical signalling within the tumour as a critical driver of metastatic disease progression, and discuss how understanding the molecular mechanisms involved in exosome-mediated metastasis is of great value for the development of new therapeutic strategies to prevent cancer progression.
Assuntos
Exossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias/metabolismo , Microambiente Tumoral/genética , Transporte Biológico , Comunicação Celular , Citocinas/genética , Citocinas/metabolismo , Progressão da Doença , Exossomos/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Metabolismo dos Lipídeos/genética , Metástase Linfática , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , Ácidos Nucleicos/genética , Ácidos Nucleicos/metabolismoRESUMO
Selenium, a trace element with anticancer properties, can reduce harmful toxicities of chemotherapy and radiotherapy without compromising efficacy. However, the dose-response relationship in normal versus malignant human cells is unclear. We evaluated how methylseleninic acid (MSA) modulates the toxicity and efficacy of chemotherapy and radiation on malignant and non-malignant human mononuclear blood cells in vitro. We specifically investigated its effects on endoplasmic reticulum stress induction, intracellular glutathione concentration, DNA damage and viability of peripheral blood mononuclear cells and THP1 monocytic leukaemia cells in response to radiation, cytosine arabinoside or doxorubicin chemotherapy. MSA, at lower concentrations, induced protective responses in normal cells but cytotoxic effects in malignant cells, alone and in conjunction with chemotherapy or radiation. However, in normal cells higher concentrations of MSA were directly toxic and increased the cytotoxicity of radiation but not chemotherapy. In malignant cells higher MSA concentrations were generally more effective in combination with cancer treatments. Thus, optimal MSA concentrations differed between normal and malignant cells and treatments. This work supports clinical reports that selenium can significantly reduce dose-limiting toxicities of anticancer therapies and potentially improve efficacy of anticancer treatments. The optimal selenium compound and dose is not yet determined.
Assuntos
Antineoplásicos/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/efeitos da radiação , Radiação , Selênio/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Interações Medicamentosas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos da radiação , Glutationa/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Compostos Organosselênicos/farmacologiaRESUMO
Lung cancer is responsible for the highest rate of cancer mortality worldwide. Lung cancer patients are often ineligible for tumor biopsies due to comorbidities. As a result, patients may not have the most effective treatment regimens administered. Patients with mutations in the epidermal growth factor receptor (EGFR) have improved survival in response to EGFR tyrosine kinase inhibitors. A noninvasive method of determining EGFR mutations in patients would have promising clinical applications. Exosomes have the potential to be noninvasive novel diagnostic markers in cancer. Using MS analysis, we identify differentially abundant cell and exosome proteins induced by mutations in p53 and EGFR in lung cells. Importantly, mutations in p53 and EGFR alter cell and exosome protein content compared to an isogenic normal lung epithelial cell. For some proteins, mutation had similar effects in the cell of origin and exosomes. Differences between the cells of origin and exosomes were also apparent, which may reflect specific packaging of proteins into exosomes. These findings that mutations alter protein abundance in exosomes suggest that analysis of exosomes may be beneficial in the diagnosis of oncogenic mutations.
Assuntos
Transformação Celular Neoplásica/metabolismo , Receptores ErbB/genética , Exossomos/metabolismo , Neoplasias Pulmonares/metabolismo , Mutação , Proteína Supressora de Tumor p53/genética , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Non-small cell lung cancer (NSCLC) is the most common lung cancer type and the most common cause of mortality in lung cancer patients. NSCLC is often associated with resistance to chemotherapeutics and together with rapid metastatic spread, results in limited treatment options and poor patient survival. NSCLCs are heterogeneous, and consist of epithelial and mesenchymal NSCLC cells. Mesenchymal NSCLC cells are thought to be responsible for the chemoresistance phenotype, but if and how this phenotype can be transferred to other NSCLC cells is currently not known. We hypothesised that small extracellular vesicles, exosomes, secreted by mesenchymal NSCLC cells could potentially transfer the chemoresistance phenotype to surrounding epithelial NSCLC cells. To explore this possibility, we used a unique human bronchial epithelial cell (HBEC) model in which the parental cells were transformed from an epithelial to mesenchymal phenotype by introducing oncogenic alterations common in NSCLC. We found that exosomes derived from the oncogenically transformed, mesenchymal HBECs could transfer chemoresistance to the parental, epithelial HBECs and increase ZEB1 mRNA, a master EMT transcription factor, in the recipient cells. Additionally, we demonstrate that exosomes from mesenchymal, but not epithelial HBECs contain the ZEB1 mRNA, thereby providing a potential mechanism for the induction of a mesenchymal phenotype in recipient cells. Together, this work demonstrates for the first time that exosomes derived from mesenchymal, oncogenically transformed lung cells can transfer chemoresistance and mesenchymal phenotypes to recipient cells, likely via the transfer of ZEB1 mRNA in exosomes.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Exossomos/patologia , Neoplasias Pulmonares/patologia , Mesoderma/patologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Proliferação de Células/efeitos dos fármacos , Exossomos/efeitos dos fármacos , Exossomos/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Células Tumorais CultivadasRESUMO
Glycosylation is the most common post-translational modification of proteins and regulates a myriad of fundamental biological processes under normal, and pathological conditions. Altered protein glycosylation is linked to malignant transformation, showing distinct glycopatterns that are associated with cancer initiation and progression by regulating tumor proliferation, invasion, metastasis, and therapeutic resistance. The glycopatterns of small extracellular vesicles (sEVs) released by cancer cells are promising candidates for cancer monitoring since they exhibit glycopatterns similar to their cell-of-origin. However, the clinical application of sEV glycans is challenging due to the limitations of current analytical technologies in tracking the trace amounts of sEVs specifically derived from tumors in circulation. Herein, a sEV GLYcan PHenotype (EV-GLYPH) assay that utilizes a microfluidic platform integrated with surface-enhanced Raman scattering for multiplex profiling of sEV glycans in non-small cell lung cancer is clinically validated. For the first time, the EV-GLYPH assay effectively identifies distinct sEV glycan signatures between non-transformed and malignantly transformed lung cells. In a clinical study evaluated on 40 patients, the EV-GLYPH assay successfully differentiates patients with early-stage malignant lung nodules from benign lung nodules. These results reveal the potential to profile sEV glycans for noninvasive diagnostics and prognostics, opening up promising avenues for clinical applications and understanding the role of sEV glycosylation in lung cancer.
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Immune checkpoint proteins (ICPs) play a major role in a patient's immune response against cancer. Tumour cells usually express those proteins to communicate with immune cells as a process of escaping the anti-cancer immune response. Detecting the major functional immune checkpoint proteins present on cancer cells (such as circulating tumor cells or CTCs) and examining the heterogeneity in their expression at the single-cell level could play a crucial role in both cancer diagnosis and the monitoring of therapy. In this study, we develop a mesoporous gold biosensor to precisely assess ICP heterogeneity in individual cancer cells within a lung cancer model. The platform utilizes a nanostructured mesoporous gold surface to capture CTCs and a Surface Enhanced Raman Scattering (SERS) readout to identify and monitor the expression of key ICP proteins (PD-L1, B7H4, CD276, CD80) in lung cancer cells. The homogeneous and abundant pores in mesoporous 3D gold nanostructures enable increased antibody loading on-chip and an enhanced SERS signal, which are key to our single cell capture, and accurate analysis of ICPs in cancer cells with high sensitivity. Our lung cancer cell line model data showed that our method can detect single cells and analyse the expression of four lung cancer associated ICPs on individual cell surfaces during treatment. To show the potential of our mesoporous gold biosensor in analysing clinical samples, we tested 9 longitudinal Peripheral Blood Mononuclear Cells (PBMC) samples from lung cancer patient before and after therapy. Our mesoporous biosensor successfully captured single CTCs and found that the expression of ICPs in CTCs is highly heterogeneous in both pre-treatment and treated PBMC samples isolated from lung cancer patient blood. We suggest that our findings will help clinicians in selecting the most appropriate therapy for patients.
Assuntos
Técnicas Biossensoriais , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Humanos , Proteínas de Checkpoint Imunológico , Leucócitos Mononucleares , Ouro , Células Neoplásicas Circulantes/patologia , Antígenos B7RESUMO
Accurate and early detection of biomarkers provides the molecular evidence for disease management, allowing prompt actions and timely treatments to save lives. Multivalent biomolecular interactions between the probe and biomarker as well as controlled probe orientation on material surfaces are keys for highly sensitive detection. Here we report the bioengineering of programmable and multifunctional nanoprobes, which can provide rapid, specific and highly sensitive detection of emerging diseases in a range of widely used diagnostic systems. These nanoprobes composed of nanosized cell wall fragments, termed as synthetic bionanofragments (SynBioNFs), are generated by the fragmentation of genetically programmed yeast cells. SynBioNFs display multiple copies of biomolecules for high-affinity target binding and molecular handles for the precisely orientated attachment on surfaces used in diagnostic platforms. SynBioNFs are demonstrated for the capture and detection of SARS-CoV-2 virions using multiple diagnostic platforms, including surface-enhanced Raman scattering, fluorescence, electrochemical and colorimetric-based lateral flow systems with sensitivity comparable with the gold-standard reverse-transcription quantitative polymerase chain reaction.
Assuntos
SARS-CoV-2 , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Indicadores e Reagentes , SARS-CoV-2/genéticaRESUMO
Despite significant therapeutic advances, lung cancer remains the leading cause of cancer-related death worldwide1. Non-small cell lung cancer (NSCLC) patients have a very poor overall five-year survival rate of only 10-20%. Currently, TNM staging is the gold standard for predicting overall survival and selecting optimal initial treatment options for NSCLC patients, including those with curable stages of disease. However, many patients with locoregionally-confined NSCLC relapse and die despite curative-intent interventions, indicating a need for intensified, individualised therapies. Epithelial-to-mesenchymal transition (EMT), the phenotypic depolarisation of epithelial cells to elongated, mesenchymal cells, is associated with metastatic and treatment-refractive cancer. We demonstrate here that EMT-induced protein changes in small extracellular vesicles are detectable in NSCLC patients and have prognostic significance. Overall, this work describes a novel prognostic biomarker signature that identifies potentially-curable NSCLC patients at risk of developing metastatic NSCLC, thereby enabling implementation of personalised treatment decisions.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Vesículas Extracelulares , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Prognóstico , Recidiva Local de Neoplasia , Vesículas Extracelulares/metabolismo , Transição Epitelial-Mesenquimal/genéticaRESUMO
Accurate identification of malignant lung lesions is a prerequisite for rational clinical management to reduce morbidity and mortality of lung cancer. However, classification of lung nodules into malignant and benign cases is difficult as they show similar features in computer tomography and sometimes positron emission tomography imaging, making invasive tissue biopsies necessary. To address the challenges in evaluating indeterminate nodules, the authors investigate the molecular profiles of small extracellular vesicles (sEVs) in differentiating malignant and benign lung nodules via a liquid biopsy-based approach. Aiming to characterize phenotypes between malignant and benign groups, they develop a single-molecule-resolution-digital-sEV-counting-detection (DECODE) chip that interrogates three lung-cancer-associated sEV biomarkers and a generic sEV biomarker to create sEV molecular profiles. DECODE capturessEVs on a nanostructured pillar chip, confines individual sEVs, and profiles sEV biomarker expression through surface-enhanced Raman scattering barcodes. The author utilize DECODE to generate a digitally acquired sEV molecular profiles in a cohort of 33 people, including patients with malignant and benign lung nodules, and healthy individuals. Significantly, DECODE reveals sEV-specific molecular profiles that allow the separation of malignant from benign (area under the curve, AUC = 0.85), which is promising for non-invasive characterisation of lung nodules found in lung cancer screening and warrants further clinincal validaiton with larger cohorts.
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Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.
Assuntos
Vesículas Extracelulares , Animais , Cromatografia em Gel , Vesículas Extracelulares/química , Filtração/métodos , Humanos , Camundongos , Reprodutibilidade dos Testes , Ultracentrifugação/métodosRESUMO
With five-year survival rates as low as 3%, lung cancer is the most common cause of cancer-related mortality worldwide. The severity of the disease at presentation is accredited to the lack of early detection capacities, resulting in the reliance on low-throughput diagnostic measures, such as tissue biopsy and imaging. Interest in the development and use of liquid biopsies has risen, due to non-invasive sample collection, and the depth of information it can provide on a disease. Small extracellular vesicles (sEVs) as viable liquid biopsies are of particular interest due to their potential as cancer biomarkers. To validate the use of sEVs as cancer biomarkers, we characterised cancer sEVs using miRNA sequencing analysis. We found that miRNA-3182 was highly enriched in sEVs derived from the blood of patients with invasive breast carcinoma and NSCLC. The enrichment of sEV miR-3182 was confirmed in oncogenic, transformed lung cells in comparison to isogenic, untransformed lung cells. Most importantly, miR-3182 can successfully distinguish early-stage NSCLC patients from those with benign lung conditions. Therefore, miR-3182 provides potential to be used for the detection of NSCLC in blood samples, which could result in earlier therapy and thus improved outcomes and survival for patients.
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Bioengineered yeast bio-nanomaterials termed nanoyeasts displaying antibody single-chain variable fragments (scFvs) against diagnostic targets are a promising alternative to monoclonal antibodies (mAbs). A potential limitation for translating nanoyeasts into diagnostic tools is batch-to-batch variability. Herein, we demonstrate a systematic approach for cost-efficient production of highly specific nanoyeasts that enabled accurate dengue virus (DENV) detection by immunoassay (2.5% CV). Yeasts bioengineered to surface express DENV-specific scFvs (up to 66% of the total cell population) were fragmented into nanoyeast fractions trialing sonication, bead beating, and high-pressure disruption methods. Nanoyeast fractions from sonication had optimal target binding, uniform particle size (±89 nm), were stable, and retained diagnostic activity for 7 days at 37 °C compared to traditional mAbs that lost activity after 1 day at 37 °C. We engineered a panel of nanoyeast scFvs targeting DENV nonstructural protein 1 (NS1): (i) specific for serotyping DENV 1-4 and (ii) cross-reactive anti-DENV scFvs that are suitable for "yes/no" diagnostic applications. We demonstrate highly specific nanoyeast scFvs for serotyping DENV. We show that nanoyeast scFvs specifically detect NS1 in simulated patient plasma with a limit of detection of 250 ng/mL, the concentration found in infected patients.
Assuntos
Vírus da Dengue , Dengue , Anticorpos de Cadeia Única , Anticorpos Antivirais , Materiais Biocompatíveis , Dengue/diagnóstico , Vírus da Dengue/genética , Humanos , Anticorpos de Cadeia Única/genética , Proteínas não Estruturais ViraisRESUMO
Metastatic spread of a cancer to secondary sites is a coordinated, non-random process. Cancer cell-secreted vesicles, especially exosomes, have recently been implicated in the guidance of metastatic dissemination, with specific surface composition determining some aspects of organ-specific localization. Nevertheless, whether the tumor microenvironment influences exosome biodistribution has yet to be investigated. Here, we show that microenvironmental cytokines, particularly CCL2, decorate cancer exosomes via binding to surface glycosaminoglycan side chains of proteoglycans, causing exosome accumulation in specific cell subsets and organs. Exosome retention results in changes in the immune landscape within these organs, coupled with a higher metastatic burden. Strikingly, CCL2-decorated exosomes are directed to a subset of cells that express the CCL2 receptor CCR2, demonstrating that exosome-bound cytokines are a crucial determinant of exosome-cell interactions. In addition to the finding that cytokine-conjugated exosomes are detected in the blood of cancer patients, we discovered that healthy subjects derived exosomes are also associated with cytokines. Although displaying a different profile from exosomes isolated from cancer patients, it further indicates that specific combinations of cytokines bound to exosomes could likewise affect other physiological and disease settings.
Assuntos
Neoplasias da Mama/sangue , Quimiocina CCL2/metabolismo , Exossomos/metabolismo , Receptores CCR2/metabolismo , Microambiente Tumoral , Animais , Neoplasias da Mama/patologia , Citocinas/metabolismo , Exossomos/imunologia , Exossomos/patologia , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Proteoglicanas/metabolismo , Receptores de Citocinas/metabolismo , Baço/imunologia , Baço/metabolismo , Baço/patologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologiaRESUMO
Identifying small extracellular vesicle (sEV) subpopulations based on their different molecular signatures could potentially reveal the functional roles in physiology and pathology. However, it is a challenge to achieve this aim due to the nano-sized dimensions of sEVs, low quantities of biological cargo each sEV carries, and our incomplete knowledge of identifying features capable of separating heterogeneous sEV subpopulations. Here, a sensitive, multiplexed, and nano-mixing-enhanced sEV subpopulation characterization platform (ESCP) is proposed to precisely determine the sEV phenotypic heterogeneity and understand the role of sEV heterogeneity in cancer progression and metastasis. The ESCP utilizes spatially patterned anti-tetraspanin-functionalized micro-arrays for sEV subpopulation sorting and nanobarcode-based surface-enhanced Raman spectroscopy for multiplexed read-outs. An ESCP has been used for investigating sEV phenotypic heterogeneity in terms of canonical sEV tetraspanin molecules and cancer-associated protein biomarkers in both cancer cell line models and cancer patient samples. Our data explicitly demonstrate the selective enrichment of tetraspanins and cancer-associated protein biomarkers, in particular sEV subpopulations. Therefore, it is believed that the ESCP could enable the evaluation and broader application of sEV subpopulations as potential diagnostic disease biomarkers.
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Vesículas Extracelulares , Neoplasias , Humanos , Neoplasias/diagnósticoRESUMO
Cancer-derived extracellular vesicles (EVs) are regarded as having promising potential to be used as therapeutics and disease biomarkers. Mechanistically, EVs have been shown to function in most, if not all, steps of cancer progression. Cancer EVs, including small EVs (sEVs), contain unique biomolecular cargo, consisting of protein, nucleic acid and lipids. Through progress in the identification of this specific cargo, cancer biomarkers have been identified and developed, opening up novel and interesting opportunities for cancer diagnosis and prognosis. Intriguingly, we still lack a comprehensive understanding of the cancer-specific pathways that govern EV biogenesis in cancer cells. Filling this knowledge gap will rapidly improve cancer EV biomarkers, as it will also allow discrimination of the procancer and anticancer actions of those EVs. Even more promising is uncovering therapeutically targetable, tumour-specific EV pathways and content, which will generate novel classes of cancer therapies. This Review highlights the progress the cancer sEV field has made in the areas of biomarker discovery and validation as well as sEV-based therapeutics, highlights the challenges we are facing and identifies gaps in our knowledge, which currently prevent us from developing the full potential of sEVs in cancer diagnostic and therapy.
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Vesículas Extracelulares/fisiologia , Neoplasias/etiologia , Biomarcadores Tumorais , Progressão da Doença , Humanos , Neoplasias/diagnóstico , Neoplasias/imunologia , Neoplasias/terapia , Microambiente TumoralRESUMO
The progression of a solid cancer from a localised disease to metastatic stages is a key reason for mortality in patients. Amongst the drivers of cancer progression, Epithelial-to-Mesenchymal Transition (EMT) has been shown to be of crucial importance. EMT results in the phenotypic shift of an immotile, treatment-sensitive epithelial cell into an elongated, metastatic and treatment-resistant mesenchymal cell. Depending on the cellular and molecular setting, a myriad of studies have demonstrated that EMT causes increased cancer cell motility, invasiveness, resistance to therapies, dormancy and cancer-stem cell phenotypes, all of which are prerequisites for metastasis. The alteration of non-canonical intercellular signalling events in cancer EMT is a phenomenon that is not completely understood. Recently, extracellular vesicles, especially small vesicles called exosomes, have shown to be involved in cancer cell EMT. Most intriguingly, across different cancer types, cancer-derived exosomes have demonstrated to be capable of transferring a mesenchymal phenotype upon recipient epithelial cells, including epithelial cancer cells. The uptake of EMT-inducing exosomes results in molecular changes, altering miRNA, mRNA, and protein levels, either through direct transfer of these components, or by altering gene expression networks involved in EMT. In this review, we are presenting the current state of research of exosomes in cancer EMT, highlight gaps in our current knowledge and propose strategies for future experiments in this area.
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Transição Epitelial-Mesenquimal/genética , Exossomos/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Células-Tronco Neoplásicas/metabolismo , Animais , Exossomos/metabolismo , Humanos , MicroRNAs/genética , Neoplasias/metabolismo , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , RNA Mensageiro/genética , Transdução de Sinais/genéticaRESUMO
Anthracyclines are amongst the most effective chemotherapeutics ever developed, but they produce grueling side-effects, serious adverse events and resistance often develops over time. We found that these compounds can be sequestered by secreted cellular Prion protein (PrPC), blocking their cytotoxic activity. This effect was dose-dependent using either cell line-conditioned medium or human serum as a source of PrPC. Genetic depletion of PrPC or inhibition of binding via chelation of ionic copper prevented the interaction and restored cytotoxic activity. This was more pronounced for doxorubicin than its epimer, epirubicin. Investigating the relevance to breast cancer management, we found that the levels of PRNP transcript in pre-treatment tumor biopsies stratified relapse-free survival after neoadjuvant treatment with anthracyclines, particularly amongst doxorubicin-treated patients with residual disease at surgery (p=2.8E-08). These data suggest that local sequestration could mediate treatment resistance. Consistent with this, tumor cell expression of PrPC protein correlated with poorer response to doxorubicin but not epirubicin in an independent cohort analyzed by immunohistochemistry, particularly soluble isoforms released into the extracellular environment by shedding (p=0.015). These findings have important potential clinical implications for frontline regimen decision-making. We suggest there is warranted utility for prognostic PrPC/PRNP assays to guide chemo-sensitization strategies that exploit an understanding of PrPC-anthracycline-copper ion complexes.