Browsing by Subject "EXOSOMES"

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  • Kyykallio, Heikki; Faria, Alessandra V. S.; Hartmann, Rosabella; Capra, Janne; Rilla, Kirsi; Siljander, Pia R-M (2022)
    Recent advances in cell biology research regarding extracellular vesicles have highlighted an increasing demand to obtain 3D cell culture-derived EVs, because they are considered to more accurately represent EVs obtained in vivo. However, there is still a grave need for efficient and tunable methodologies to isolate EVs from 3D cell cultures. Using nanofibrillar cellulose (NFC) scaffold as a 3D cell culture matrix, we developed a pipeline of two different approaches for EV isolation from cancer spheroids. A batch method was created for delivering high EV yield at the end of the culture period, and a harvesting method was created to enable time-dependent collection of EVs to combine EV profiling with spheroid development. Both these methods were easy to set up, quick to perform, and they provided a high EV yield. When compared to scaffold-free 3D spheroid cultures on ultra-low affinity plates, the NFC method resulted in similar EV production/cell, but the NFC method was scalable and easier to perform resulting in high EV yields. In summary, we introduce here an NFC-based, innovative pipeline for acquiring EVs from 3D cancer spheroids, which can be tailored to support the needs of variable EV research objectives.
  • Rautaniemi, Kaisa; Zini, Jacopo; Löfman, Emilia; Saari, Heikki; Haapalehto, Iida; Laukka, Johanna; Vesamäki, Sami; Efimov, Alexander; Yliperttula, Marjo; Laaksonen, Timo; Vuorimaa-Laukkanen, Elina; Lisitsyna, Ekaterina S. (2022)
    Studies of extracellular vesicles (EVs), their trafficking and characterization often employ fluorescent labelling. Unfortunately, little attention has been paid thus far to a thorough evaluation of the purification of EVs after labelling, although the presence of an unbound dye may severely compromise the results or even lead to wrong conclusions on EV functionality. Here, we systematically studied five dyes for passive EV labelling and meticulously compared five typical purification methods: ultracentrifugation (UC), ultracentrifugation with discontinuous density gradient (UCG), ultrafiltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC). A general methodology for evaluation of EV purification efficiency after the labelling was developed and tested to select the purification methods for the chosen dyes. Firstly, we found that some methods initially lead to high EV losses even in the absence of the dye. Secondly, the suitable purification method needs to be found for each particular dye and depends on the physical and chemical properties of the dye. Thirdly, we demonstrated that the developed parameter E-rp (relative purification efficiency) is a useful tool for the pre-screening of the suitable dye-purification method combinations. Additionally, it was also shown that the labelled EVs properly purified from the unbound dye may show significantly reduced contrast and visibility in the target application, e.g. in the live cell fluorescence lifetime imaging.
  • Multia, Evgen; Liangsupree, Thanaporn; Jussila, Matti; Ruiz-Jimenez, Jose; Kemell, Marianna; Riekkola, Marja-Liisa (2020)
    An automated on-line isolation and fractionation system including controlling software was developed for selected nanosized biomacromolecules from human plasma by on-line coupled immunoaffinity chromatography-asymmetric flow field-flow fractionation (IAC-AsFlFFF). The on-line system was versatile, only different monoclonal antibodies, anti-apolipoprotein B-100, anti-CD9, or anti-CD61, were immobilized on monolithic disk columns for isolation of lipoproteins and extracellular vesicles (EVs). The platelet-derived CD61-positive EVs and CD9-positive EVs, isolated by IAC, were further fractionated by AsFlFFF to their size-based subpopulations (e.g., exomeres and exosomes) for further analysis. Field-emission scanning electron microscopy elucidated the morphology of the subpopulations, and 20 free amino acids and glucose in EV subpopulations were identified and quantified in the ng/mL range using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The study revealed that there were significant differences between EV origin and size-based subpopulations. The on-line coupled IAC-AsFlFFF system was successfully programmed for reliable execution of 10 sequential isolation and fractionation cycles (37–80 min per cycle) with minimal operator involvement, minimal sample losses, and contamination. The relative standard deviations (RSD) between the cycles for human plasma samples were 0.84–6.6%.
  • Al-Samadi, Ahmed; Awad, Shady Adnan; Tuomainen, Katja; Zhao, Yue; Salem, Abdelhakim; Parikka, Mataleena; Salo, Tuula (2017)
    The crosstalk between immune cells, cancer cells, and extracellular vesicles (EVs) secreted by cancer cells remains poorly understood. We created three-dimensional (3D) cell culture models using human leiomyoma discs and Myogel to study the effects of immune cells on highly (HSC-3) and less (SCC-25) invasive oral tongue squamous cell carcinoma (OTSCC) cell lines. Additionally, we studied the effects of EVs isolated from these cell lines on the cytotoxicity of CD8(+) T and NK cells isolated from three healthy donors. Our analysis included the effects of these EVs on innate immunity in zebrafish larvae. Activated immune cells significantly decreased the proliferation of both OTSCC cell lines and associated with a diminished invasion area of HSC-3 cells. In general, EVs from SCC-25 increased the cytotoxic activity of CD8(+) T and NK cells more than those from HSC-3 cells. However, this effect varied depending on the source and the immune and cancer cell subgroups. In zebrafish, the amount of IL-13 mRNA was decreased by SCC-25 EVs. This study describes promising in vitro and in vivo models to investigate interactions between immune cells, cancer cells, and EVs.
  • Lazaro-Ibanez, Elisa; Lunavat, Taral R.; Jang, Su Chul; Escobedo-Lucea, Carmen; Oliver-De La Cruz, Jorge; Siljander, Pia; Lotvall, Jan; Yliperttula, Marjo (2017)
    Background: Multiple types of extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs), are released by all cells constituting part of the cellular EV secretome. The bioactive cargo of EVs can be shuffled between cells and consists of lipids, metabolites, proteins, and nucleic acids, including multiple RNA species from non-coding RNAs to messenger RNAs (mRNAs). In this study, we hypothesized that the mRNA cargo of EVs could differ based on the EV cellular origin and subpopulation analyzed. Methods: We isolated MVs and EXOs from PC-3 and LNCaP prostate cancer cells by differential centrifugation and compared them to EVs derived from the benign PNT2 prostate cells. The relative mRNA levels of 84 prostate cancer-related genes were investigated and validated using quantitative reverse transcription PCR arrays. Results: Based on the mRNA abundance, MVs rather than EXOs were enriched in the analyzed transcripts, providing a snapshot of the tumor transcriptome. LNCaP MVs specifically contained significantly increased mRNA levels of NK3 Homeobox 1 (NKX3-1), transmembrane protease serine 2 (TMPRSS2), and tumor protein 53 (TP53) genes, whereas PC-3 MVs carried increased mRNA levels of several genes including, caveolin-2 (CAV2), glutathione S-transferase pi 1 (GSTP1), pescadillo ribosomal biogenesis factor 1 (PES1), calmodulin regulated spectrin associated protein 1 (CAMSAP1), zinc-finger protein 185 (ZNF185), and others compared to PNT2 MVs. Additionally, ETS variant 1 (ETV1) and fatty acid synthase (FASN) mRNAs identified in LNCaP-and PC-3-derived MVs highly correlated with prostate cancer progression. Conclusions: Our study provides new understandings of the variability of the mRNA cargo of MVs and EXOs from different cell lines despite same cancer origin, which is essential to better understand the the proportion of the cell transcriptome that can be detected within EVs and to evaluate their role in disease diagnosis.
  • Morani, Marco; Duc Mai, Thanh; Krupova, Zuzana; Defrenaix, Pierre; Multia, Evgen; Riekkola, Marja-Liisa; Taverna, Myriam (2020)
    This work reports on the development of the first capillary electrophoresis methodology for the elucidation of extracellular vesicles' (EVs) electrokinetic distributions. The approach is based on capillary electrophoresis coupled with laser-induced fluorescent (LIF) detection for the identification and quantification of EVs after their isolation. Sensitive detection of these nanometric entities was possible thanks to an 'inorganic-species-free' background electrolyte. This electrolyte was made up of weakly charged molecules at very high concentrations to stabilize EVs, and an intra-membrane labelling approach was used to prevent EV morphology modification. The limit of detection for EVs achieved using the developed CE-LIF method reached 8 x 10(9) EV/mL, whereas the calibration curve was acquired from 1.22 x 10(10) to 1.20 x 10(11) EV/mL. The CE-LIF approach was applied to provide the electrokinetic distributions of various EVs of animal and human origins, and visualize different EV subpopulations from our recently developed high-yield EV isolation method. (C) 2020 Elsevier B.V. All rights reserved.
  • Moquin, Alexandre; Ji, Jeff; Neibert, Kevin; Winnik, Francoise M.; Maysinger, Dusica (2018)
    Polymersomes are attractive nanocarriers for hydrophilic and lipophilic drugs; they are more stable than liposomes, tunable, and relatively easy to prepare. The copolymer composition and molar mass are critical features that determine the physicochemical properties of the polymersomes including the rate of drug release. We used the triblockcopolymer, poly(2-methyl-2-oxazoline)-block-poly-(dimethysiloxane)-block-poly(2-methyl-2-oxazoline) (PIVIOXA-PDIVIS-PMOXA), to form amphipathic polymersomes capable of loading proteins and small hydrophobic agents. The selected agents were unstable neurotrophins (nerve growth factor and brain -derived neurotrophic factor), a large protein CD109, and the fluorescent drug curcumin. We prepared, characterized, and tested polymersomes loaded with selected agents in 2D and 3D biological models. Curcumin-loaded and rhodamine-bound PMOXA-PDMS-PMOXA polymersomes were used to visualize them inside cells. NMethyl-D-aspartate receptor (NNIDAR) agonists and antagonists were also covalently attached to the surface of polymersomes for targeting neurons. Labeled and unlabeled polymersomes with or without loaded agents were characterized using dynamic light scattering (DLS), UV-vis fluorescence spectroscopy, and asymmetrical flow field-flow fractionation (AF(4)). Polymersomes were imaged and tested for biological activity in human and murine fibroblasts, murine macrophages, primary murine dorsal root ganglia, and murine hippocampal cultures. Polymersomes were rapidly internalized and there was a clear intracellular co-localization of the fluorescent drug (curcumin) with the fluorescent rhodamine-labeled polymersomes. Polymersomes containing CD109, a glycosylphosphatidylinositol-anchored protein, promoted cell migration in the model of wound healing. Nerve growth factor-loaded polymersomes effectively enhanced neurite outgrowth in dissociated and explanted dorsal root ganglia. Brain -derived neurotrophic factor increased dendritic spine density in serum-deprived hippocampal slice cultures. NMDAR agonist-and antagomst-functionalized polymersomes targeted selectively neurons over filial cells in mixed cultures. Collectively, the study reveals the successful incorporation into polymersomes of biologically active trophic factors and small hydrophilic agents that retain their biological activity in vitro, as demonstrated in selected central and peripheral tissue models.
  • Mannerström, Bettina; Kornilov, Roman; Abu-Shahba, Ahmed G.; Chowdhury, Iftekhar M.; Sinha, Snehadri; Seppänen-Kaijansinkko, Riitta; Kaur, Sippy (2019)
    Extracellular vesicles (EVs) are central to intercellular communication and play an important role in cancer progression and development. Osteosarcoma (OS) is an aggressive bone tumour, characterized by the presence of malignant mesenchymal cells. The specific tumour-driving genetic alterations that are associated with OS development are currently poorly understood. Mesenchymal stem cells (MSCs) of osteogenic lineage have been postulated as likely candidates as the cells of origin for OS, thus indicating that MSCs and OS stroma cells may be related cell types. Therefore, this study set out to examine the EV-mediated intercellular crosstalk of MSCs and OS. MSCs and pre-osteoblasts were treated with OS-EVs at different time points, and the epigenetic signature of OS-EVs was assessed by methylation analysis of LINE-1 (long interspersed element) and tumour suppressor genes. In addition, surface markers and expression of specific genes were also evaluated. Our data indicated that OS-EVs mediated LINE-1 hypomethylation in MSCs, whereas an opposite effect was seen in pre-osteoblasts, indicating that MSCs but not pre-osteoblasts were susceptible to epigenetic transformation. Thus, OS-EVs modulated the fate of MSCs by modulating the epigenetic status, and also influenced the expression of genes related to bone microenvironment remodelling. Overall, this study provided evidence that epigenetic regulation appears to be an early event in the transformation of MSCs during the development of OS. Elucidating the mechanisms of EV-mediated communication may lead to new avenues for therapeutic exploitation.
  • Mannerström, Bettina; Paananen, Riku O; Abu-Shahba, Ahmed; Moilanen, Jukka; Seppänen-Kaijansinkko, Riitta; Kaur, Sippy (2019)
    In the research field of extracellular vesicles (EVs), the use of fetal bovine serum (FBS) depleted of EVs for in vitro studies is advocated to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or purchased commercially. Nevertheless, these preparations do not guarantee an RNA-free FBS for in vitro use. In this study we address the RNA contamination issue, of small non-coding (nc)RNA in vesicular or non-vesicular fractions of FBS, ultracentrifugation EV-depleted FBS, commercial EV-depleted FBS, and in our recently developed filtration based EV-depleted FBS. Commercially available serum- and xeno-free defined media were also screened for small ncRNA contamination. Our small ncRNA sequencing data showed that all EV-depleted media and commercially available defined media contained small ncRNA contaminants. Out of the different FBS preparations studied, our ultrafiltration-based method for EV depletion performed the best in depleting miRNAs. Certain miRNAs such miR-122 and miR-203a proved difficult to remove completely and were found in all media. Compared to miRNAs, other small ncRNA (snRNA,Y RNA, snoRNA, and piRNA) were difficult to eliminate from all the studied media. Additionally, our tested defined media contained miRNAs and other small ncRNAs, albeit at a much lower level than in serum preparations. Our study showed that no media is free of small ncRNA contaminants. Therefore, in order to screen for baseline RNA contamination in culturing media, RNA sequencing data should be carefully controlled by adding a media sample as a control. This should be a mandatory step before performing cell culture experiments in order to eliminate the confounding effects of media.
  • Dourado, Mauricio Rocha; Korvala, Johanna; Åström, Pirjo; De Oliveira, Carine Ervolino; Cervigne, Nilva K.; Mofatto, Luciana Souto; Bastos, Debora Campanella; Pereira Messetti, Ana Camila; Graner, Edgard; Paes Leme, Adriana Franco; Coletta, Ricardo D.; Salo, Tuula (2019)
    As one of the most abundant constituents of the tumour microenvironment (TME), cancer-associated fibroblasts (CAF) display critical roles during tumour progression and metastasis. Multiple classes of molecules including growth factors, cytokines, proteases and extracellular matrix proteins, are produced by CAF to act as mediators of the stroma-tumour interactions. One of the main channels for this communication is associated with extracellular vesicles (EV), which are secreted particles loaded with protein and genetic information. In this study, we evaluated the effects of EV derived from CAF primary human cell lines (n = 5) on proliferation, survival, migration, and invasion of oral squamous cell carcinoma (OSCC) cells. As controls, EV from human primary-established normal oral fibroblasts (NOF, n = 5) were used. Our in vitro assays showed that CAF-EV significantly induces migration and invasion of OSCC cells and promote a disseminated pattern of HSC-3 cell invasion in the 3D organotypic assay. Furthermore, gene expression analysis of EV-treated cancer cells revealed changes in the pathways associated with tumour metabolism and up-regulation of tumour invasion genes. Our findings suggest a significant role of CAF-EV in promoting the migration and invasion of OSCC cells, which are related to the activation of cancer-related pathways.
  • Endzelins, Edgars; Abols, Arturs; Buss, Arturs; Zandberga, Elina; Palviainen, Mari Johanna; Siljander, Pia Riitta-Maria; Line, Aija (2018)
    Background/Aim: Tumor-secreted extracellular vesicles (EVs) play an important role as mediators of intercellular communication. Hypoxia is a common feature of solid tumors frequently associated with an aggressive clinical behavior. This study aimed to gain a deeper understanding into the functions of EVs in intercellular communication between primary and metastatic cancer cells under hypoxic conditions. Materials and Methods: EVs were isolated from two isogenic colorectal cancer (CRC) cell lines SW480 and SW620 cultured under normoxic and hypoxic conditions. Their uptake and effects in SW480 and SW620 cells were studied using EV uptake, proliferation, spheroid-formation, wound healing and invasion assays. Results: Our data showed that hypoxia enhanced the release of EVs from CRC cells in a Hypoxia Induced Factor (HIF)-1-dependent manner. Hypoxic EVs were taken up by CRC cells more efficiently than normoxic EVs. Hypoxic EVs stimulated motility, invasiveness and sternness of primary tumour-derived SW480 cells, whereas they had a little effect on metastasis-derived SW620 cells. Conclusion: Hypoxic colorectal cancer-derived EVs confer aggressiveness and invasiveness to hypoxia-naive cancer cells.
  • Palviainen, Mari; Saraswat, Mayan K.; Varga, Zoltan; Kitka, Diana; Neuvonen, Maarit; Puhka, Maija; Joenväärä, Sakari; Renkonen, Risto; Nieuwland, Rienk; Takatalo, Maarit; Siljander, Pia R. M. (2020)
    Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery.
  • Saari, Heikki; Turunen, Tiia; Lohmus, Andres; Turunen, Mikko; Jalasvuori, Matti; Butcher, Sarah J.; Yla-Herttuala, Seppo; Viitala, Tapani; Cerullo, Vincenzo; Siljander, Pia R. M.; Yliperttula, Marjo (2020)
    Extracellular vesicles (EVs) have been showcased as auspicious candidates for delivering therapeutic cargo, including oncolytic viruses for cancer treatment. Delivery of oncolytic viruses in EVs could provide considerable advantages, hiding the viruses from the immune system and providing alternative entry pathways into cancer cells. Here we describe the formation and viral cargo of EVs secreted by cancer cells infected with an oncolytic adenovirus (IEVs, infected cell-derived EVs) as a function of time after infection. IEVs were secreted already before the lytic release of virions and their structure resembled normally secreted EVs, suggesting that they were not just apoptotic fragments of infected cells. IEVs were able to carry the viral genome and induce infection in other cancer cells. As such, the role of EVs in the life cycle of adenoviruses may be an important part of a successful infection and may also be harnessed for cancer- and gene therapy.
  • Multia, Evgen; Tear, Crystal Jing Ying; Palviainen, Mari; Siljander, Pia R-M; Riekkola, Marja-Liisa (2019)
    A new, fast and selective immunoaffinity chromatographic method including a methacrylate-based convective interaction media (CIM (R)) disk monolithic column, immobilized with anti-human CD61 antibody, was developed for the isolation of CD61-containing platelet-derived extracellular vesicles (EVs) from plasma. The isolated EVs were detected and size characterized by asymmetrical flow field-flow fractionation (AsFlFFF) with multi-angle light-scattering (MALS) and dynamic light-scattering (DLS) detection, and further confirmed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The mean size of platelet-derived EV isolates from the anti-CD61 CIM (R) disk monolithic column were 174 nm (SD 60 nm) based on the NTA results. These results indicated a successful isolation of platelet-derived EVs, which was confirmed by Western blotting the isolates against the EV-specific markers CD9 and TSG101 together with transmission electron microscopy. Additional elucidation of MALS and DLS data provided detailed information of the size distribution of the isolated fractions, confirming the successful isolation of also small platelet-derived EVs ranging from 30 to 130 nm based on the hydrodynamic radii. The isolation procedure took only 19 min and the time can be even further decreased by increasing the flow rate. The same immunoaffinity chromatographic procedure, following AsFlFFF allowed also the isolation and characterization of platelet-derived EVs from plasma in under 60 min. Since it is possible to regenerate the anti-CD61 disk for multiple uses, the methodology developed in this study provides a viable substitution and addition to the conventional EV isolation procedures. (C) 2019 Elsevier B.V. All rights reserved.
  • Lorey, Martina B.; Rossi, Katriina; Eklund, Kari; Nyman, Tuula A.; Matikainen, Sampsa (2017)
    Gram-negative bacteria are associated with a wide spectrum of infectious diseases in humans. Inflammasomes are cytosolic protein complexes that are assembled when the cell encounters pathogens or other harmful agents. The non-canonical caspase-4/5 inflammasome is activated by Gram-negative bacteria-derived lipopolysaccharide (LPS) and by endogenous oxidized phospholipids. Protein secretion is a critical component of the innate immune response. Here, we have used label-free quantitative proteomics to characterize global protein secretion in response to non-canonical inflammasome activation upon intracellular LPS recognition in human primary macrophages. Before proteomics, the total secretome was separated into two fractions, enriched extracellular vesicle (EV) fraction and rest-secretome (RS) fraction using size-exclusion centrifugation. We identified 1048 proteins from the EV fraction and 1223 proteins from the RS fraction. From these, 640 were identified from both fractions suggesting that the non-canonical inflammasome activates multiple, partly overlapping protein secretion pathways. We identified several secreted proteins that have a critical role in host response against severe Gram-negative bacterial infection. The soluble secretome (RS fraction) was highly enriched with inflammation-associated proteins upon intracellular LPS recognition. Several ribosomal proteins were highly abundant in the EV fraction upon infection, and our data strongly suggest that secretion of translational machinery and concomitant inhibition of translation are important parts of host response against Gram-negative bacteria sensing caspase-4/5 inflammasome. Intracellular recognition of LPS resulted in the secretion of two metalloproteinases, a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and MMP14, in the enriched EV fraction. ADAM10 release was associated with the secretion of TNF, a key inflammatory cytokine, and M-CSF, an important growth factor for myeloid cells probably through ADAM10-dependent membrane shedding of these cytokines. Caspase-4/5 inflammasome activation also resulted in secretion of danger-associated molecules S100A8 and prothymosin- in the enriched EV fraction. Both S100A8 and prothymosin- are ligands for toll-like receptor 4 recognizing extracellular LPS, and they may contribute to endotoxic shock during non-canonical inflammasome activation.
  • Arasu, Uma Thanigai; Deen, Ashik Jawahar; Pasonen-Seppänen, Sanna; Heikkinen, Sami; Lalowski, Maciej; Kärnä, Riikka; Härkönen, Kai; Mäkinen, Petri; Lazaro-Ibañez, Elisa; Siljander, Pia R-M; Oikari, Sanna; Levonen, Anna-Liisa; Rilla, Kirsi (2020)
    Intercellular communication is fundamental to the survival and maintenance of all multicellular systems, whereas dysregulation of communication pathways can drive cancer progression. Extracellular vesicles (EVs) are mediators of cell-to-cell communication that regulate a variety of cellular processes involved in tumor progression. Overexpression of a specific plasma membrane enzyme, hyaluronan synthase 3 (HAS3), is one of the factors that can induce EV shedding. HAS3, and particularly its product hyaluronan (HA), are carried by EVs and are known to be associated with the tumorigenic properties of cancer cells. To elucidate the specific effects of cancerous, HAS3-induced EVs on target cells, normal human keratinocytes and melanoma cells were treated with EVs derived from GFP-HAS3 expressing metastatic melanoma cells. We found that the HA receptor CD44 participated in the regulation of EV binding to target cells. Furthermore, GFP-HAS3-positive EVs induced HA secretion, proliferation and invasion of target cells. Our results suggest that HAS3-EVs contains increased quantities of IHH, which activates the target cell hedgehog signaling cascade and leads to the activation of c-Myc and regulation of claspin expression. This signaling of IHH in HAS3-EVs resulted in increased cell proliferation. Claspin immunostaining correlated with HA content in human cutaneous melanocytic lesions, supporting our in vitro findings and suggesting a reciprocal regulation between claspin expression and HA synthesis. This study shows for the first time that EVs originating from HAS3 overexpressing cells carry mitogenic signals that induce proliferation and epithelial-to-mesenchymal transition in target cells. The study also identifies a novel feedback regulation between the hedgehog signaling pathway and HA metabolism in melanoma, mediated by EVs carrying HA and IHH.
  • Sork, Helena; Corso, Giulia; Krjutskov, Kaarel; Johansson, Henrik J.; Nordin, Joel Z.; Wiklander, Oscar P. B.; Lee, Yi Xin Fiona; Westholm, Jakub Orzechowski; Lehtiö, Janne; Wood, Matthew J. A.; Mager, Imre; El Andaloussi, Samir (2018)
    Extracellular vesicles (EVs) mediate cell-to-cell communication by delivering or displaying macromolecules to their recipient cells. While certain broad-spectrum EV effects reflect their protein cargo composition, others have been attributed to individual EV-loaded molecules such as specific miRNAs. In this work, we have investigated the contents of vesicular cargo using small RNA sequencing of cells and EVs from HEK293T, RD4, C2C12, Neuro2a and C17.2. The majority of RNA content in EVs (49-96%) corresponded to rRNA-, coding-and tRNA fragments, corroborating with our proteomic analysis of HEK293T and C2C12 EVs which showed an enrichment of ribosome and translation-related proteins. On the other hand, the overall proportion of vesicular small RNA was relatively low and variable (2-39%) and mostly comprised of miRNAs and sequences mapping to piRNA loci. Importantly, this is one of the few studies, which systematically links vesicular RNA and protein cargo of vesicles. Our data is particularly useful for future work in unravelling the biological mechanisms underlying vesicular RNA and protein sorting and serves as an important guide in developing EVs as carriers for RNA therapeutics.
  • Zini, Jacopo; Saari, Heikki; Ciana, Paolo; Viitala, Tapani; Lohmus, Andres; Saarinen, Jukka; Yliperttula, Marjo (2022)
    Extracellular vesicles (EVs) are a complex and heterogeneous population of nanoparticles involved in cell-to-cell communication. Recently, numerous studies have indicated the potential of EVs as therapeutic agents, drug carriers and diagnostic tools. However, the results of these studies are often difficult to evaluate, since different characterization methods are used to assess the purity, physical and biochemical characteristics of the EV samples. In this study, we compared four methods for the EV sample characterization and purity assessment: i) the particle-to-protein ratio based on particle analyses with nanoparticle tracking and protein concentration by bicinchoninic acid assay, ii) Western Blot analysis for specific EV biomarkers, iii) two spectroscopic lipid-to protein ratios by either the attenuated total reflection Fourier transform infrared (ATR-FTIR) or Raman spectroscopy. The results confirm the value of Raman and ATR-FTIR spectroscopy as robust, fast and operator independent tools that require only a few microliters of EV sample. We propose that the spectroscopic lipid-to protein (Li/Pr) ratios are reliable parameters for the purity assessment of EV preparations. Moreover, apart from determining protein concentrations, we show that ATR-FTIR spectroscopy can also be used for indirect measurements of EV concentrations. Nevertheless, the Li/Pr ratios do not represent full characterization of the EV preparations. For a complete characterization of selected EV preparations, we recommend also additional use of particle size distribution and EV biomarker analysis.
  • Liangsupree, Thanaporn; Multia, Evgen; Forssén, Patrik; Fornstedt, Torgny; Riekkola, Marja-Liisa (2022)
    Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9+, CD63+, and CD81+ EV subpopulations of <50 nm exomeres and 50-80 nm exosomes were isolated and fractionated using our recently developed on-line coupled immunoaffinity chromatography - asymmetric flow field-flow fractionation system. Adaptive Interaction Distribution Algorithm (AIDA), specifically designed for the analysis of complex biological interactions, was used with a four-step procedure for reliable estimation of the degree of heterogeneity in rate constant distributions. Interactions between exomere-sized EVs and anti-tetraspanin antibodies demonstrated two interaction sites with comparable binding kinetics and estimated dissociation constants Kd ranging from nM to fM. Exomeres exhibited slightly higher affinity compared to exosomes. The highest affinity with anti-tetraspanin antibodies was achieved with CD63+ EVs. The interaction of EV subpopulations with ICAM-1 involved in cell internalization of EVs was also investigated. EV - ICAM-1 interaction was also of high affinity (nM to pM range) with overall lower affinity compared to the interactions of anti-tetraspanin antibodies and EVs. Our findings proved that QCM is a valuable label-free tool for kinetic studies with limited sample concentration, and that advanced algorithms, such as AIDA, are crucial for proper determination of kinetic heterogeneity. To the best of our knowledge, this is the first kinetic study on the interaction between plasma-derived EV subpopulations and anti-tetraspanin antibodies and ICAM-1.
  • Koponen, Annika; Kerkelä, Erja; Rojalin, Tatu; Lazaro-Ibanez, Elisa; Suutari, Teemu; Saari, Heikki O.; Siljander, Pia; Yliperttula, Marjo; Laitinen, Saara; Viitala, Tapani (2020)
    Extracellular vesicles (EVs) have the ability to function as molecular vehicles and could therefore be harnessed to deliver drugs to target cells in diseases such as cancer. The composition of EVs determines their function as well as their interactions with cells, which consequently affects the cell uptake efficacy of EVs. In this study, we present two novel label-free approaches for studying EVs; characterization of EV composition by time-gated surface-enhanced Raman spectroscopy (TG-SERS) and monitoring the kinetics and amount of cellular uptake of EVs by surface plasmon resonance (SPR) in real-time. Using these methods, we characterized the most abundant EVs of human blood, red blood cell (RBC)- and platelet (PLT)-derived EVs and studied their interactions with prostate cancer cells. Complementary studies were performed with nanoparticle tracking analysis for concentration and size determinations of EVs, zeta potential measurements for surface charge analysis, and fluorophore-based confocal imaging and flow cytometry to confirm EV uptake. Our results revealed distinct biochemical features between the studied EVs and demonstrated that PLT-derived EVs were more efficiently internalized by PC-3 cells than RBC-derived EVs. The two novel label-free techniques introduced in this study were found to efficiently complement conventional techniques and paves the way for further use of TG-SERS and SPR in EV studies.