[Preparation of bacterial outer membrane vesicles with anti-GPC3 single-chain antibody and identification of their targeting effects on hepatocellular carcinoma].

This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti-glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo. The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD600=0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 ℃. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs (P=0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.

Extracellular Membrane Vesicles of Escherichia coli Induce Apoptosis of CT26 Colon Carcinoma Cells.

Escherichia coli (E. coli) is commonly utilized as a vehicle for anti-tumor therapy due to its unique tumor-targeting capabilities and ease of engineering modification. To further explore the role of E. coli in tumor treatment, we consider that E. coli outer membrane vesicles (E. coli-OMVs) play a crucial role in the therapeutic process. Firstly, E. coli-OMVs were isolated and partially purified by filtration and ultracentrifugation, and were characterized using techniques such as nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western Blot (WB). The obtained extracellular nanoparticles, containing OMVs, were found to inhibited the growth of CT26 tumor in mice, while the expression of Bax protein was increased and the expression of Bcl-2 protein decreased. In vitro experiments showed that E. coli-OMVs entered CT26 cells and inhibited cell proliferation, invasion and migration. In addition, in the presence of E. coli-OMVs, we observed an increase in apoptosis rate and a decrease in the ratio of Bcl-2/Bax. These data indicate that E. coli-OMVs inhibits the growth of CT26 colon cancer by inducing apoptosis of CT26 cells. These findings propose E. coli-OMVs as a promising therapeutic drug for colorectal cancer (CRC), providing robust support for further research in related fields.

The prognostic genes model of breast cancer drug resistance based on single-cell sequencing analysis and transcriptome analysis.

Breast cancer (BC) represents a multifaceted malignancy, with escalating incidence and mortality rates annually. Chemotherapy stands as an indispensable approach for treating breast cancer, yet drug resistance poses a formidable challenge. Through transcriptome data analysis, we have identified two sets of genes exhibiting differential expression in this context. Furthermore, we have confirmed the overlap between these genes and those associated with exosomes, which were subsequently validated in cell lines. The investigation screened the identified genes to determine prognostic markers for BC and utilized them to formulate a prognostic model. The disparities in prognosis and immunity between the high- and low-risk groups were validated using the test dataset. We have discerned different BC subtypes based on the expression levels of prognostic genes in BC samples. Variations in prognosis, immunity, and drug sensitivity among distinct subtypes were examined. Leveraging data from single-cell sequencing and prognostic gene expression, the AUCell algorithm was employed to score individual cell clusters and analyze the pathways implicated in high-scoring groups. Prognostic genes (CCT4, CXCL13, MTDH, PSMD2, and RAB27A) were subsewoquently validated using RT-qPCR. Consequently, we have established a model for predicting prognosis in breast cancer that hinges on drug resistance and ERGs. Furthermore, we have evaluated the prognostic value of this model. The genes identified as prognostic markers can now serve as a reference for precise treatment of this condition.

Melanoma extracellular vesicles inhibit tumor growth and metastasis by stimulating CD8 T cells.

Tumor cell-derived extracellular vesicles (EVs) play a crucial role in mediating immune responses by carrying and presenting tumor antigens. Here, we suggested that melanoma EVs triggered cytotoxic CD8 T cell-mediated inhibition of tumor growth and metastasis. Our results indicated that immunization of mice with melanoma EVs inhibited melanoma growth and metastasis while increasing CD8 T cells and serum interferon γ (IFN-γ) in vivo. In vitro experiments showed that melanoma EV stimulates dendritic cells (DCs) maturation, and mature dendritic cells induce T lymphocyte activation. Thus, tumor cell-derived EVs can generate anti-tumor immunity in a prophylactic setting and may be potential candidates for cell-free tumor vaccines.

Bifidobacterium-derived membrane vesicles inhibit triple-negative breast cancer growth by inducing tumor cell apoptosis.

BACKGROUND: Bacterial outer membrane vesicles have gained increasing attention for its antitumor effect and application in drug delivery. However, the bacterial membrane vesicles (MVs) that are secreted by Gram-positive bacteria are rarely mentioned. Bifidobacterium has a certain anti-tumor effect, but there is a certain risk when injected into human body. Here we investigated the potential of Bifidobacterium-derived membrane vesicles (B-MVs) as therapeutic agents to treat triple-negative breast cancer. METHODS AND RESULTS: Firstly, we discovered that Bifidobacterium can produce B-MVs and isolated them. In vivo, we found that B-MVs can inhibit tumor growth in mice and the mice were in good state. H&E staining displayed extensive apoptotic cells in tumor tissues. Western blotting and immunohistochemistry showed that B-MVs increased the expression of Bax, while decreased the expression of Bcl-2. These results suggested that B-MVs may induce apoptosis of tumor cells in vivo. Furthermore, to further confirm this phenomenon, we conducted experiments in vitro. Hoechst 33,258 staining assay, flow cytometry and western blotting also demonstrated B-MVs promoted cell apoptosis in vitro. CONCLUSIONS: We speculate B-MVs may inhibit tumor growth by inducing tumor cell apoptosis in triple-negative breast cancer, which provided a new direction in the treatment of TNBC.

Dauer larva-derived extracellular vesicles extend the life of Caenorhabditis elegans.

There is growing evidence that extracellular vesicles (EVs) play a functional role in tissue repair and anti-aging by transferring the contents of donor cells to recipient cells. We hypothesized that Dauer (C. elegans), known as "ageless" nematodes, can also secrete extracellular vesicles and influence the lifespan of C. elegans. Here, we isolated EVs of dauer larvae (dauer EVs). Dauer EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis (NTA), and Western blot analysis. Wild-type C. elegans were fed in the presence or absence of dauer EVs and tested for a range of phenotypes, including longevity, mobility and reproductive capacity. Results showed that dauer EVs increased the average lifespan of nematodes by 15.74%, improved mobility, slowed age-related pigmentation as well as body length, and reduced the accumulation of reactive oxygen species and lipids, while not impairing nematode reproductive capacity. These findings suggest that dauer EVs can extend the lifespan of C. elegans as well as the healthy lifespan by reducing ROS accumulation, with potential anti-aging capacity.

Comparative proteomics identify HSP90A, STIP1 and TAGLN-2 in serum extracellular vesicles as potential circulating biomarkers for human adenomyosis.

Extracellular vesicles (EVs) carry specific proteins involved in intercellular communication. EVs with different protein contents are released into circulation in different diseases. Recent studies have identified proteins in adenomyosis (AM)-derived EVs (AMEVs) from blood as biomarkers for this disease. AM is an extension of endometrial tissue into the uterine myometrium. Magnetic resonance imaging (MRI) is the most accurate imaging tool for identifying adenomyosis. Therefore, the present study aimed to investigate the role of EVs in diagnosing AM. In the present study, tissue AMEVs (T-AMEVs) were isolated from lesion homogenates of patients with adenomyosis, and blood AMEVs (B-AMEVs) were isolated from peripheral blood of patients with AM via differential centrifugation and density gradient centrifugation. T-AMEVs and B-AMEVs were characterized by electron microscopy, western blotting and mass spectrometry and analysed using FunRich3.1.3 software. T-AMEVs (average diameter, 150.9±102.2 nm) and B-AMEVs (194.1±66.81 nm) expressed the CD9, CD63 and flotillin-2 EV markers. A total of 211 proteins expressed in T-AMEVs and B-AMEVs overlapped with Vesiclepedia database entries, including 2 epithelial-to-mesenchymal transition (EMT)-associated proteins and 6 invasion-associated proteins. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that these 211 proteins were associated with the 'regulation of cell morphogenesis' and 'cytoskeletal organization' terms, as well as the PPAR and HIF-1 signalling pathways, which are related to the proliferation and metastasis of endometrial cells that cannot invade the myometrium under normal circumstances. Among the 211 proteins, HSP90A, STIP1 and TAGLN-2 were expressed in T-AMEVs and B-AMEVs, but not in serum EVs of women without adenomyosis/endometriosis, and these proteins might be the potential biomarkers for adenomyosis. These findings provide insights into the molecular features of adenomyosis and the new candidate biomarkers for diagnosis.

Subgroups of Extracellular Vesicles: Can They Be Defined by "Labels?"

Extracellular vesicles (EVs) are a class of lipid bilayer membranes, containing lipids, nucleic acids (DNA and RNA), proteins, and other substances. They are produced by almost all types of cells and act as signaling intermediaries between cells and/or tissues through different mechanisms involving complex signals. EVs produced by each type of cells are composed of highly heterogeneous and inhomogeneous subgroups with different biological functions. Therefore, in the past few decades, researchers have tried to use different "labels" to define the subgroups of EVs, and explore the differences in them. However, a unified standard for defining the populations of EVs has not yet been established so far. In this study, we review and summarize the use of different "labels" to define subgroups of EVs.

Melanoma-Derived Exosomes Endow Fibroblasts with an Invasive Potential via miR-21 Target Signaling Pathway.

BACKGROUND: Tumor-derived exosomes are messengers that participate in tumor progression. Fibroblasts are associated with the metastasis of cancer depending on their cellular plasticity. We hypothesize that tumor-derived exosomes endow the fibroblasts in tumor microenvironment with invasive phenotype to the benefit of tumor metastasis. MATERIALS AND METHODS: Exosomes derived from B16-F10 cells were identified by nanoparticle tracking analyzer (NTA), dynamic light scattering (DLS), Western blot (WB), and transmission electron microscopy (TEM). Cell invasion and migration assays were performed using the xCELLigence real-time cell analyzer (RTCA). Role of tumor-derived exosomal miR-21 in cell invasion was determined by qPCR. RESULTS: The invasion analysis showed that exosome-treated fibroblast cells had greater invasive capability as compared to untreated fibroblast cells, with the higher expressions of MMP2 and MMP9. miR-21 is at least partially responsible for this effect. After ingestion of melanoma-derived exosomes during incubation, mouse embryonic fibroblasts cells emerged cellular invasiveness with the presentation of a marked increase in miR-21 expression. MiR-21 promoted invasion of fibroblasts by down-regulation of tissue inhibitor of metalloproteinase 3 (TIMP3) expression and increasing of matrix metalloprotein (MMP) expression in fibroblast cells via melanoma-derived exosomes in a time-dependent manner. CONCLUSION: Our results suggest that tumor-derived exosomes may facilitate stromal fibroblasts an aggressive phenotype to equip the tumor progression.

Adenomyosis-derived extracellular vesicles endow endometrial epithelial cells with an invasive phenotype through epithelial-mesenchymal transition.

Extracellular vesicles from highly metastatic tumor cells have been shown to mediate epithelial-mesenchymal transition (EMT)-related events in recipient cells. In endometrial epithelial cells, EMT processes are known to be involved in the development of adenomyosis. We aimed to investigate whether adenomyosis-derived extracellular vesicles (AMEVs) are able to induce an EMT process in endometrial epithelial cells. In this study, AMEVs were isolated from patients with adenomyosis and characterized by transmission electron microscopy, Western blot, and nanoparticle tracking. Primary endometrial epithelial cells (EECs) were derived from normal endometrium tissues from patients with leiomyoma and co-cultured with AMEVs in vitro. AMEV uptake was examined by fluorescence confocal microscopy. The invasion of EECs was confirmed by Transwell assay. Immunohistochemistry, Western blot, and qRT-PCR were performed on EECs to illustrate the expression levels of cytokeratin 19, E-cadherin, vimentin, and zinc finger E-box-binding homeobox 1 (ZEB1). The results indicated that the cellular fluorescence intensity gradually increased after 48 h of co-culture, but decreased after 72 h. After co-culturing with AMEVs for 72 h, EECs expressed significantly lower levels of cytokeratin 19 and E-cadherin, and significantly higher levels of vimentin and ZEB1. Together these results demonstrated that AMEVs induce an EMT process and enhance the invasion of EECs. These changes may contribute to the pathogenesis and progression of adenomyosis.