Proteins in Tumor-Derived Plasma Extracellular Vesicles Indicate Tumor Origin

Barlin, Meltem; Erdmann-Gilmore, Petra; Mudd, Jacqueline; Zhang, Qiang; Seymour, R. W.; Guo, Zhanfang; Miessner, Julia R.; Goedegebuure, S. Peter; Bi, Ye; Osorio, Omar A.; Alexander‐Brett, Jennifer; Li, Shunqiang; Cynthia, X.; Fields, Ryan C.; Townsend, R. Reid; Held, Jason M.

doi:10.1016/j.mcpro.2022.100476

Cited by 8 publications

(6 citation statements)

References 40 publications

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“…Recently, 14-3-3ε and α-actinin 4 have been identified in cancer-derived EVs in sera or plasma of cancer patients [61,62], while their extracellular oncogenic roles await thorough investigation. A related study reveals that human colorectal cancer cells release vesicles containing 14-3-3ξ along with β-catenin, which can be taken up by neighboring cells and enhance their migration ability via activation of the Wnt signaling [76].…”

Section: Discussionmentioning

confidence: 99%

“…Despite lacking a signal sequence required for conventional protein secretion through the ER-to-Golgi route, 14-3-3ε and α-actinin 4 have been identified in the secretomes of various human cancer cell lines [59,60] and extracellular vesicles (EVs) originating from human cancers [61][62][63][64]. Moreover, 14-3-3ε and α-actinin 4 may be released directly from the cell membranes or liberated from the EVs to serve as ligands for cell-surface proteins.…”

Section: Introductionmentioning

confidence: 99%

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AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells

Yuan,

Wu,

Wang

et al. 2024

Cell Mol Biol Lett

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Background Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis. Methods To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs. Results Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs. Conclusion This study elucidates AGR2’s pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells

Yuan,

Wu,

Wang

et al. 2024

Cell Mol Biol Lett

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“…Although NTA profiles indicate a significant increase in patients with FMA compared to controls, the use of EV quantification as a biomarker requires further research. Firstly, EVs can be increased in the cancer setting 31 , although evidence is conflicting 42 , and EVs in circulation come from a wide variety of origins, not just cancer, making them a heterogenous population of vesicles 43 . It is important to note that increased concentration of EVs may be due to a variety of reasons such as systemic inflammation, haematological diseases, or the presence of viruses, to name a few.…”

Section: Discussionmentioning

confidence: 99%

The protein and miRNA profile of plasma extracellular vesicles (EVs) can distinguish feline mammary adenocarcinoma patients from healthy feline controls

Howard

Browne

Bollard

et al. 2023

Sci Rep

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Feline mammary adenocarcinomas (FMA) are aggressive tumours with metastatic capability and limited treatment options. This study aims to investigate whether miRNAs associated with FMA tumours are secreted in extracellular vesicles (EVs) and whether they can potentially be used as a cancer biomarker in EVs from feline plasma. Tumours and matched tumour free margins from 10 felines with FMA were selected. Following a detailed literature search, RT-qPCR analyses of 90 miRNAs identified 8 miRNAs of interest for further investigation. Tumour tissue, margins and plasma were subsequently collected from a further 10 felines with FMA. EVs were isolated from the plasma. RT-qPCR expression analyses of the 8 miRNAs of interest were carried out in tumour tissue, margins, FMA EVs and control EVs. Additionally, proteomic analysis of both control and FMA plasma derived EVs was undertaken. RT-qPCR revealed significantly increased miR-20a and miR-15b in tumours compared to margins. A significant decrease in miR-15b and miR-20a was detected in EVs from FMAs compared to healthy feline EVs. The proteomic content of EVs distinguished FMAs from controls, with the protein targets of miR-20a and miR-15b also displaying lower levels in the EVs from patients with FMA. This study has demonstrated that miRNAs are readily detectable in both the tissue and plasma derived EVs from patients with FMA. These miRNAs and their protein targets are a detectable panel of markers in circulating plasma EVs that may inform future diagnostic tests for FMA in a non-invasive manner. Moreover, the clinical relevance of miR-20a and miR-15b warrants further investigation.

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“…There is unfortunately no "perfect" method for EV isolation, but many methods are available (Serrano-Pertierra et al, 2019;Janouskova et al, 2022;Shirejini and Inci, 2022;Jalaludin et al, 2023). For our studies, we chose the ExoQuick and ME kits based on previous literature (Ricciardi et al, 2021;Aziz et al, 2022) and collaborator experience (Barlin et al, 2023). There was also research to show that the ME kit was successful for proteomic downstream processing (Ghosh et al, 2014;Knol et al, 2016;Askeland et al, 2020).…”

Section: Discussionmentioning

confidence: 99%

Identification of biomarker candidates for filarial parasite infections by analysis of extracellular vesicles

Yates,

Di Maggio,

Rosa

et al. 2023

Front. Parasitol.

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BackgroundImproved diagnostic tools are needed for detecting active filarial infections in humans. Tests are available that detect adult W. bancrofti circulating filarial antigen, but there are no sensitive and specific biomarker tests for brugian filariasis or loiasis. Here we explored whether extracellular vesicles released by filarial parasites contain diagnostic biomarker candidates.MethodsVesicles were isolated using VN96-affinity purification from supernatants of short-term in vitro cultured B. malayi microfilariae (Mf) and analyzed by mass spectrometry (Bruker timsTOF). Parasite-specific proteins were identified by bioinformatic analysis and a protein was called present if supported by ≥ 2 spectra. After validation with cultures parasites, this approach was then used to analyze vesicles isolated from plasma of animals infected with B. malayi and from humans with heavy Loa loa infections.ResultsVesicles from Mf cultures contained more than 300 B. malayi proteins with high consistency across biological replicates. These included the known Mf excretory antigen BmR1 (AF225296). Over 150 B. malayi proteins were detected in vesicles isolated from plasma samples from two infected animals. Vesicles isolated from plasma from 10 persons with high L. loa Mf densities contained consistently 21 proteins, 9 of them were supported by at least 5 unique peptides and 7 with spectral counts above 10. The protein EN70_10600 (an orthologue of the B. malayi antigen BmR1, GenBank AF225296) was detected in all 10 samples with a total count of 91 spectra and a paralogue (EN70_10598) was detected in 6 samples with a total of 44 spectra.DiscussionExtracellular vesicles released by filarial parasites in vitro and in vivo contain parasite proteins which can be reliably detected by mass spectrometry. This research provides the foundation to develop antigen detection assays to improve diagnosis of active filarial infections in humans.

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Proteins in Tumor-Derived Plasma Extracellular Vesicles Indicate Tumor Origin

Cited by 8 publications

References 40 publications

AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells

AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells

The protein and miRNA profile of plasma extracellular vesicles (EVs) can distinguish feline mammary adenocarcinoma patients from healthy feline controls

Identification of biomarker candidates for filarial parasite infections by analysis of extracellular vesicles

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