| 1. |
- Bojmar, Linda, 1983-, et al.
(författare)
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Extracellular vesicle and particle isolation from human and murine cell lines, tissues, and bodily fluids
- 2021
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Ingår i: STAR protocols. - : Cell Press. - 2666-1667. ; 2:1
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Tidskriftsartikel (refereegranskat)abstract
- We developed a modified protocol, based on differential ultracentrifugation (dUC), to isolate extracellular vesicles and particles (specifically exomeres) (EVPs) from various human and murine sources, including cell lines, surgically resected tumors and adjacent tissues, and bodily fluids, such as blood, lymphatic fluid, and bile. The diversity of these samples requires robust and highly reproducible protocols and refined isolation technology, such as asymmetric-flow field-flow fractionation (AF4). Our isolation protocol allows for preparation of EVPs for various downstream applications, including proteomic profiling. For complete details on the use and execution of this protocol, please refer to Hoshino et al. (2020).
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| 2. |
- Bojmar, Linda, et al.
(författare)
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Protocol for cross-platform characterization of human and murine extracellular vesicles and particles
- 2024
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Ingår i: STAR PROTOCOLS. - : ELSEVIER. - 2666-1667. ; 5:1
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Tidskriftsartikel (refereegranskat)abstract
- Characterization of isolated extracellular vesicles and particles (EVPs) is crucial for determining functions and biomarker potential. Here, we present a protocol to analyze size, number, morphology, and EVP protein cargo and to validate EVP proteins in both humans and mice. We describe steps for nanoparticle tracking analysis, transmission electron microscopy, single-EVP immunodetection, EVP proteomic mass spectrometry and bioinformatic analysis, and EVP protein validation by ExoELISA and western blot analysis. This allows for EVP cross -validation across different platforms.For complete details on the use and execution of this protocol, please refer to Hoshino et al.1
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| 3. |
- Hoshino, Ayuko, et al.
(författare)
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Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers
- 2020
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Ingår i: Cell. - : CELL PRESS. - 0092-8674 .- 1097-4172. ; 182:4, s. 1044-
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Tidskriftsartikel (refereegranskat)abstract
- There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n =151) and plasma-derived (n =120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.
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| 4. |
- Hoshino, Ayuko, et al.
(författare)
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Tumour exosome integrins determine organotropic metastasis
- 2015
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Ingår i: Nature. - : NATURE PUBLISHING GROUP. - 0028-0836 .- 1476-4687. ; 527:7578, s. 329-
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Tidskriftsartikel (refereegranskat)abstract
- Ever since Stephen Pagets 1889 hypothesis, metastatic organotropism has remained one of cancers greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver-and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins alpha(6)beta(4) and alpha(6)beta(1) were associated with lung metastasis, while exosomal integrin alpha(v)beta(5) was linked to liver metastasis. Targeting the integrins alpha(6)beta(4) and alpha(v)beta(5) decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.
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| 5. |
- Prakash, Varsha, et al.
(författare)
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Ribosome biogenesis during cell cycle arrest fuels EMT in development and disease
- 2019
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Ribosome biogenesis is a canonical hallmark of cell growth and proliferation. Here we show that execution of Epithelial-to-Mesenchymal Transition (EMT), a migratory cellular program associated with development and tumor metastasis, is fueled by upregulation of ribosome biogenesis during G1/S arrest. This unexpected EMT feature is independent of species and initiating signal, and is accompanied by release of the repressive nucleolar chromatin remodeling complex (NoRC) from rDNA, together with recruitment of the EMT-driving transcription factor Snai1 (Snail1), RNA Polymerase I (Pol I) and the Upstream Binding Factor (UBF). EMT-associated ribosome biogenesis is also coincident with increased nucleolar recruitment of Rictor, an essential component of the EMT-promoting mammalian target of rapamycin complex 2 (mTORC2). Inhibition of rRNA synthesis in vivo differentiates primary tumors to a benign, Estrogen Receptor-alpha (ER alpha) positive, Rictor-negative phenotype and reduces metastasis. These findings implicate the EMT-associated ribosome biogenesis program with cellular plasticity, de-differentiation, cancer progression and metastatic disease.
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