| 1. |
- Eldh, Maria, et al.
(författare)
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Proteomic Profiling of Tissue Exosomes Indicates Continuous Release of Malignant Exosomes in Urinary Bladder Cancer Patients, Even with Pathologically Undetectable Tumour
- 2021
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Ingår i: Cancers. - : MDPI. - 2072-6694. ; 13:13
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary Urinary bladder cancer (UBC) has a high recurrence rate, and biomarkers for different treatment strategies are highly needed. This study investigated the release of nanovesicles called exosomes from urinary bladder tissue from tumour-proximal sites as well as tumour-distant sites in transurethrally resected (TUR-B) patients with or without preoperative neoadjuvant chemotherapy prior to ensuing radical cystectomy-all without remaining visible tumour after TUR-B. We show that cancer-promoting exosomes were detected from both sites, suggesting that the previous tumour has altered the whole bladder tissue into a cancer-supporting milieu. The exosomes may originate from remaining pathologically undetectable cancer cells or transformed epithelial cells, and the study supports the notion of exosomes as mediators of metastatic spread and as potential biomarkers. It also supports early and radical removal of the bladder in urinary bladder cancer patients. Invasive urothelial bladder cancer (UBC) has high recurrence rates even after radical cystectomy (RC). Exosomes are membrane-bound nanovesicles, which have been shown to contribute to carcinogenesis and metastasis. We previously showed that urinary exosomes display a malignant profile in UBC patients despite the absence of detectable tumour. Here, we investigated exosomes from sampling sites close to or distant from the former tumour, aiming to understand the effect of the tumour on the local milieu. Ten patients scheduled for cystectomy after transurethral bladder resection (TUR-B), without remaining detectable tumour, were included. Exosomes were isolated from tissue explants of both the previous tumour site and distant bladder tissue. Proteins were quantified by mass spectrometry in seven patients. Exosomes from the previous tumour site were enriched in inflammatory but not cancer-related pathways compared to distant tissue. However, the 69 most abundant proteins in tissue-derived exosomes regardless of site, 20 of which were also found in urinary exosomes from our previous study, were enriched for cancer-related metabolic pathways and associated with poor prognosis in an external mRNA dataset. The enrichment of cancer-related pathways in the most abundant proteins, regardless of sampling site, confirms our hypothesis that despite the absence of detectable tumour, the entire bladder releases exosomes that contribute to metastasis and highlights the need for early RC.
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| 2. |
- Hiltbrunner, Stefanie, et al.
(författare)
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Urinary Exosomes from Bladder Cancer Patients Show a Residual Cancer Phenotype despite Complete Pathological Downstaging
- 2020
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- Invasive urinary bladder cancer shows high recurrence rates after cystectomy even with apparent complete downstaging at cystectomy. Exosomes are nano-sized vesicles important in cell-cell communication, which have been hypothesized to contribute to cancer dissemination and recurrence. The aim of this study was to investigate if pro-carcinogenic exosomes could be detected in urine from histologically downstaged bladder cancer patients. 13 Patients were included in this study. Paired ureter and urine samples from nine patients underwent mass spectrometry, while samples from the remaining patients were used for exosome characterization. At cystectomy, exosomes were isolated from bladder and ureter urine, whereafter quantitative proteome profiling was performed. Urinary exosomes clustered based on whether they came from the bladder, with tumour contact, or the ureters, without tumour contact, even though all came from completely downstaged patients. Proteins overexpressed in exosomes derived from bladder urine contained several oncogenes and were mainly associated with tumour metabolism pathways. Although patients were histologically tumour-free at cystectomy, the bladder urine contained exosomes with a carcinogenic metabolic profile. This suggests a continuous release of exosomes from the bladder, which may promote recurrence at distant sites through metabolic rewiring, even after apparent complete downstaging. These exosomes, coming from either undetected cancer cells or partly transformed cells, are likely to increase the risk of metastasis and encourages cystectomy even in completely downstaged patients.
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| 3. |
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| 4. |
- Veerman, Rosanne E
(författare)
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Analysis of extracellular vesicles and their application in immunotherapy
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Extracellular Vesicles (EVs) are nano-sized particles secreted by most, if not all, cell types. They are important mediators in intercellular communication and carry a broad array of biomolecules such as lipids, nucleic acids and proteins. Because of their ability to deliver functional biomolecules, EVs have been studied and utilized in both physiological and pathological settings. The EV family is composed of multiple types of vesicles, including microvesicles, deriving from the cell membrane, and exosomes which are of endosomal origin. Despite the two different biogenesis pathways of these vesicles, they share many characteristics which makes it difficult to separate them during EV isolation. Hence, the name EVs is preferred when working with vesicles. Many different EV isolation methods exist based on distinct EV characteristics, e.g. size and density, but it still remains largely unknown if different isolation methods also enrich for certain EV subpopulations. Furthermore, it is known that to isolate a pure EV population, a combination of methods needs to be applied. However, depending on the study set up, this is not always possible. Therefore, we isolated EVs with five different methods using two different sample types, i.e. human plasma and conditioned cell culture supernatant, and performed a thorough analysis of the isolated EV fractions (study I). Although each isolation method enriched for EVs, there was a rather large heterogeneity between the samples, both for plasma and cell culture supernatant, regarding particle count, particle size, RNA amount and protein amount. Furthermore, we identified that certain methods enriched for proteins related to cell organelles such as Golgi or endoplasmic reticulum, which originally are not classified as EV-related proteins. We concluded that, depending on the sample type and volume, different isolation methods are preferred. EVs are being applied in a therapeutic setting and especially Dendritic cell (DC) derived EVs have been used, as they can induce antigen-specific T and B cell responses. However, it is not clear whether different EV subtypes can have different immunostimulatory capacities. Therefore, we compared the immunostimulatory capacities of DC derived microvesicles and exosomes, loaded with the antigen Ovalbumin (OVA), in vivo (study II). The results showed that exosomes induce significantly more antigen-specific T cells than microvesicles and higher levels of OVA-specific IgG in the serum. This was likely due to the higher levels of OVA on exosomes, indicating that different EV subsets have different capabilities to load and transfer antigen. To further investigate the immunostimulatory capacities of DC derived EVs, allogeneic exosomes and syngeneic exosomes were compared side by side, as the usage of allogeneic exosomes would enhance the applicability of EVs in the clinic (study III). Both OVA-loaded allogeneic and syngeneic exosomes induced high levels of antigen-specific T cells. Interestingly, allogeneic exosomes induced significantly more T follicular helper cells and increased OVA specific IgG-levels, indicating that allogeneicity might serve as an adjuvant. Since DC derived exosomes are able to induce strong antigen-specific immune responses, we tested if they could sensitize a tumour model that is non-responsive to anti-PD-1 or anti-PD-L1 treatment, into a responsive tumour (study IV). Indeed, we saw that in a prophylactic B16 melanoma tumour model, exosomes and anti-PD-L1 combination improved survival as compared to exosomes or PD-L1 treatment alone. This suggests that exosomes can be used in combination treatment, in which they sensitize the tumour to checkpoint blockade. In conclusion, this thesis provides an improved understanding of EV isolation and specifically on the purity and EV enrichment of different methods. Furthermore, this thesis describes new knowledge on DC-derived EVs, suggesting that exosomes are the preferred EV subtype for EV-based immunotherapies and that allogeneic exosomes can promote the humoral immune responses. Lastly, DC-derived EVs can sensitize non-responsive melanoma to anti-PD-1/PD-L1, showing that DC-derived EVs can be used in cancer combination therapy.
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| 5. |
- Veerman, Rosanne E., et al.
(författare)
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Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin
- 2021
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Ingår i: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 10:9
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 mu l or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.
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