Methods for Systematic Identification of Membrane Proteins for Specific Capture of Cancer-Derived Extracellular Vesicles

Zaborowski, Mikołaj; Lee, Kyungheon; Na, Young Jeong; Sammarco, Alessandro; Zhang, Xuan; Iwanicki, Marcin P.; Cheah, Pike See; Lin, Hua‐Kuo; Zinter, Max; Chou, Che-Chung; Fulci, Giulia; Tannous, Bakhos A.; Lai, Charles P.; Birrer, Michael J.; Weissleder, Ralph; Lee, Hakho; Breakefield, Xandra O.

doi:10.1016/j.celrep.2019.03.003

Cited by 39 publications

(34 citation statements)

References 52 publications

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“…EVs are often classified into: (1) exosomes which are secreted by exocytosis of intra-luminal vesicles via membrane fusion of multivesicular bodies with plasma membrane and their size range between 50 to 200 nm, (2) ectosomes aka microvesicles or microparticles formed by cytoplasmic membrane budding, and fission; their size typically ranges from 200 nm to 1000 nm in diameter [5][6][7]. These populations of EVs are often distinguished based on their biogenesis, size, content, and function [1,8,9]. In addition to these EV types, other vesicles have been reported including; oncosomes, large oncosomes (1-10 µm) [10,11], matrix vesicles [12][13][14], migrasomes (50 nm to 3 µm) [15,16], exopheres (~4 µm), exomeres (~35 nm), and bacterial outer membrane vesicles (OMV) [4,[17][18][19].…”

Section: Introductionmentioning

confidence: 99%

“…Several fluorescence-based methods have been developed to monitor EV biogenesis, transmission (or uptake), distribution, and subcellular localization. Such materials and methods for EV analysis include lipid-based fluorescence labeling [34,46], transmembrane proteins (i.e., CD63) fused with fluorescent proteins [8,47], and a membrane lipid-binding palmitoylation (palm) signal fused with fluorescent proteins such as tandem dimer Tomato (tdTomato) or enhanced green fluorescence protein (EGFP) [48]. In the present study, we utilize the palm system and abbreviate as palmGFP (palmG) and palmtdTomato (palmT).…”

Section: Introductionmentioning

confidence: 99%

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Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

Taha

Sogawa

Okusha

et al. 2020

Cancers

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The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200–1000 nm) and small EVs (50–200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

Taha

Sogawa

Okusha

et al. 2020

Cancers

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“…It is reported that KCNG1 encodes extracellular antigenic epitopes with the highest difference between ovarian tumors and normal tissues. [90] PPCS is an enzyme that catalyzes the chemical reaction which constitutes the second of five steps involved in the conversion of pantothenate to Coenzyme A. The molecular function of PPCS consists of regulating phosphopantothenate--cysteine ligase activity and ligase activity.…”

Section: Survival Analysismentioning

confidence: 99%

Identification of Potential Key Genes Associated with the Pathogenesis and Prognosis of Glioblastoma Based on Integrated Bioinformatics Analysis

Tu¹,

Zhou²,

Zhang³

et al. 2020

Preprint

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Background: Despite striking advances in multimodality management, the low survival rate of Glioblastoma (GBM) patients has not been significantly improved and identifying novel diagnostic and prognostic biomarkers is urgently demanded. The present study aimed to identify potential key genes associated with the pathogenesis and prognosis of GBM.Methods: Differentially expressed genes between GBM and normal brain tissue samples were screened by an integrated analysis of multiple gene expression profile datasets. Key genes related to the pathogenesis and prognosis of GBM were identified by employing protein–protein interaction network and Cox proportional hazards model analyses.Results: We identified nine hub genes (TP53, FN1, EGFR, MYC, RRM2, EZH2, FOXM1, CD44 and MMP2) which might be closely associated with the pathogenesis of GBM. A prognostic gene signature consisted of RAB33B, KIAA1199, TEK, EVC, SOD2, CXCR4, hCG_40738, CHD9, GCSH, SUHW1, RPS6KA5, PDCD4, ZG16, KCNG1, DECR1, PPCS, SERPINF1, TMSB10, NAT10, HIC2, PIR and OR2W1 was constructed with a good performance in predicting overall survivals (OS).Conclusions: The findings of present study would provide certain reference for further predicting the diagnostic and prognostic biomarkers to facilitate the molecular targeting therapy of GBM.

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“…To date, most available protocols to recover EVs from patients' blood resulted in the recovery of a heterogeneous population of vesicles of uncertain origin, but the implementation of more robust technologies have facilitated the isolation of specific subpopulations of as few as ten EVs per microliter of plasma with direct impact on the performance of revealing predictive biomarkers [111]. Novel approaches have for the first time enabled the identification of cancer-derived EVs through a specific immunocapture method that targets membrane proteins of the tumor tissue of origin, separating cancer EVs from the bulk of background blood EVs [122,123]. Selective isolation of tumor-associated EVs can also be coupled with direct PCR-based quantification of oncogenic mRNAs following nanodroplet encapsulation of plasma EVs, and dramatically improve the sensitivity potential of liquid biopsy of BRAF and KRAS mutations [123].…”

Section: Detection Of Extracellular Vesicles and Their Application Asmentioning

confidence: 99%

Extracellular Vesicles in Non-Small-Cell Lung Cancer: Functional Role and Involvement in Resistance to Targeted Treatment and Immunotherapy

Pasini

Ulivi

2019

Cancers

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Targeted and immunological therapies have become the gold standard for a large portion of non-small cell lung cancer (NSCLC) patients by improving significantly clinical prognosis. However, resistance mechanisms inevitably develop after a first response, and almost all patients undergo progression. The knowledge of such a resistance mechanism is crucial to improving the efficacy of therapies. So far, monitoring therapy responses through liquid biopsy has been carried out mainly in terms of circulating tumor (ctDNA) analysis. However, other particles of tumor origin, such as extracellular vehicles (EVs) represent an emerging tool for the studying and monitoring of resistance mechanisms. EVs are now considered to be ubiquitous mediators of cell-to-cell communication, allowing cells to exchange biologically active cargoes that vary in response to the microenvironment and include proteins, metabolites, RNA species, and nucleic acids. Novel findings on the biogenesis and fate of these vesicles reveal their fundamental role in cancer progression, with foreseeable and not-far-to-come clinical applications in NSCLC.

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Methods for Systematic Identification of Membrane Proteins for Specific Capture of Cancer-Derived Extracellular Vesicles

Cited by 39 publications

References 52 publications

Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

Identification of Potential Key Genes Associated with the Pathogenesis and Prognosis of Glioblastoma Based on Integrated Bioinformatics Analysis

Extracellular Vesicles in Non-Small-Cell Lung Cancer: Functional Role and Involvement in Resistance to Targeted Treatment and Immunotherapy

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