Extracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still... Show moreExtracellular vesicles (EVs) in blood plasma are recognized as potential biomarkers for disease. Although blood plasma is easily obtainable, analysis of EVs at the single particle level is still challenging due to the biological complexity of this body fluid. Besides EVs, plasma contains different types of lipoproteins particles (LPPs), that outnumber EVs by orders of magnitude and which partially overlap in biophysical properties such as size, density and molecular makeup. Consequently, during EV isolation LPPs are often co-isolated. Furthermore, physical EV-LPP complexes have been observed in purified EV preparations. Since co-isolation or association of LPPs can impact EV-based analysis and biomarker profiling, we investigated the presence and formation of EV-LPP complexes in biological samples by using label-free atomic force microscopy, cryo-electron tomography and synchronous Rayleigh and Raman scattering analysis of optically trapped particles and fluorescence-based high sensitivity single particle flow cytometry. Furthermore, we evaluated the impact on flow cytometric analysis in the presence of LPPs using in vitro spike-in experiments of purified tumour cell line-derived EVs in different classes of purified human LPPs. Based on orthogonal single-particle analysis techniques we demonstrate that EV-LPP complexes can form under physiological conditions. Furthermore, we show that in fluorescence-based flow cytometric EV analysis staining of LPPs, as well as EV-LPP associations, can influence quantitative and qualitative EV analysis. Lastly, we demonstrate that the colloidal matrix of the biofluid in which EVs reside impacts their buoyant density, size and/or refractive index (RI), which may have consequences for down-stream EV analysis and EV biomarker profiling. Show less
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts within the kidney due to mutations in PKD1 or PKD2. Although the disease remains... Show moreAutosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid-filled cysts within the kidney due to mutations in PKD1 or PKD2. Although the disease remains incompletely understood, one of the factors associated with ADPKD progression is the release of nucleotides (including ATP), which can initiate autocrine or paracrine purinergic signaling by binding to their receptors. Recently, we and others have shown that increased extracellular vesicle (EVs) release from PKD1 knockout cells can stimulate cyst growth through effects on recipient cells. Given that EVs are an important communicator between different nephron segments, we hypothesize that EVs released from PKD1 knockout distal convoluted tubule (DCT) cells can stimulate cyst growth in the downstream collecting duct (CD). Here, we show that administration of EVs derived from Pkd1(-/-) mouse distal convoluted tubule (mDCT15) cells result in a significant increase in extracellular ATP release from Pkd1(-/-) mouse inner medullary collecting duct (iMCD3) cells. In addition, exposure of Pkd1(-/-) iMCD3 cells to EVs derived from Pkd1(-/-) mDCT15 cells led to an increase in the phosphorylation of the serine/threonine-specific protein Akt, suggesting activation of proliferative pathways. Finally, the exposure of iMCD3 Pkd1(-/-) cells to mDCT15 Pkd1(-/-) EVs increased cyst size in Matrigel. These findings indicate that EVs could be involved in intersegmental communication between the distal convoluted tubule and the collecting duct and potentially stimulate cyst growth. Show less
Uveal melanoma (UM) is the second most frequent type of melanoma. Therapeutic options for UM favor minimally invasive techniques such as irradiation for vision preservation. As a consequence, no... Show moreUveal melanoma (UM) is the second most frequent type of melanoma. Therapeutic options for UM favor minimally invasive techniques such as irradiation for vision preservation. As a consequence, no tumor material is obtained. Without available tissue, molecular analyses for gene expression, mutation or copy number analysis cannot be performed. Thus, proper patient stratification is impossible and patients' uncertainty about their prognosis rises. Minimally invasive techniques have been studied for prognostication in UM. Blood-based biomarker analysis has become more common in recent years; however, no clinically standardized protocol exists. This review summarizes insights in biomarker analysis, addressing new insights in circulating tumor cells, circulating tumor DNA, extracellular vesicles, proteomics, and metabolomics. Additionally, medical imaging can play a significant role in staging, surveillance, and prognostication of UM and is addressed in this review. We propose that combining multiple minimally invasive modalities using tumor biomarkers should be the way forward and warrant more attention in the coming years. Show less
Pharmacogenomics (PGx) entails the study of heritability of drug response. This may include both variability in genes related to pharmacokinetics (drug absorption, distribution, metabolism and... Show morePharmacogenomics (PGx) entails the study of heritability of drug response. This may include both variability in genes related to pharmacokinetics (drug absorption, distribution, metabolism and excretion) and pharmacodynamics (e.g., drug receptors or signaling pathways). Individualizing drug therapy taking into account the genetic profile of the patient has the potential to make drug therapy safer and more effective. Currently, this approach relies on the determination of genetic variants in pharmacogenes by genotyping. However, it is widely acknowledged that large variability in gene expression is attributed to non-structural genetic variants. Therefore, at least from a theoretical viewpoint individualizing drug therapy based upon expression of pharmacogenes rather than on genotype may be advantageous but has been difficult to implement in the clinical setting. Extracellular vesicles (EVs) are lipid encapsulated structures that contain cargo such as lipids, nucleic acids and proteins. Since their cargo is tissue- and cell-specific they can be used to determine the expression of pharmacogenes in the liver. In this review, we describe methods of EV isolation and the potential of EVs isolated from liquid biopsies as a tool to determine the expression of pharmacogenes for use in personalized medicine. Show less
Li, C.; Teixeira, A.F.; Zhu, H.J.; Dijke, P. ten 2021
To identify novel cancer therapies, the tumor microenvironment (TME) has received a lot of attention in recent years in particular with the advent of clinical successes achieved by targeting immune... Show moreTo identify novel cancer therapies, the tumor microenvironment (TME) has received a lot of attention in recent years in particular with the advent of clinical successes achieved by targeting immune checkpoint inhibitors (ICIs). The TME consists of multiple cell types that are embedded in the extracellular matrix (ECM), including immune cells, endothelial cells and cancer associated fibroblasts (CAFs), which communicate with cancer cells and each other during tumor progression. CAFs are a dominant and heterogeneous cell type within the TME with a pivotal role in controlling cancer cell invasion and metastasis, immune evasion, angiogenesis and chemotherapy resistance. CAFs mediate their effects in part by remodeling the ECM and by secreting soluble factors and extracellular vesicles. Exosomes are a subtype of extracellular vesicles (EVs), which contain various biomolecules such as nucleic acids, lipids, and proteins. The biomolecules in exosomes can be transmitted from one to another cell, and thereby affect the behavior of the receiving cell. As exosomes are also present in circulation, their contents can also be explored as biomarkers for the diagnosis and prognosis of cancer patients. In this review, we concentrate on the role of CAFs-derived exosomes in the communication between CAFs and cancer cells and other cells of the TME. First, we introduce the multiple roles of CAFs in tumorigenesis. Thereafter, we discuss the ways CAFs communicate with cancer cells and interplay with other cells of the TME, and focus in particular on the role of exosomes. Then, we elaborate on the mechanisms by which CAFs-derived exosomes contribute to cancer progression, as well as and the clinical impact of exosomes. We conclude by discussing aspects of exosomes that deserve further investigation, including emerging insights into making treatment with immune checkpoint inhibitor blockade more efficient. Show less
Photodynamic therapy (PDT) is a promising and clinically approved method for the treatment of cancer. However, the efficacy of PDT is often limited by the poor selectivity and distribution of the... Show morePhotodynamic therapy (PDT) is a promising and clinically approved method for the treatment of cancer. However, the efficacy of PDT is often limited by the poor selectivity and distribution of the photosensitizers (PS) toward the malignant tumors, resulting in prolonged periods of skin photosensitivity. In this work, we present a simple and straightforward strategy to increase the tumor distribution, selectivity, and efficacy of lipophilic PS zinc phthalocyanine (ZnPc) in colon cancer by their stabilization in purified, naturally secreted extracellular vesicles (EVs). The PS ZnPc was incorporated in EVs (EV-ZnPc) by a direct incubation strategy that did not affect size distribution or surface charge. By using co-culture models simulating a tumor microenvironment, we determined the preferential uptake of EV-ZnPc toward colon cancer cells when compared with macrophages and dendritic cells. We observed that PDT promoted total tumor cell death in normal and immune cells, but showed selectivity against cancer cells in co-culture models. In vivo assays showed that after a single intravenous or intratumoral injection, EV-ZnPc were able to target the tumor cells and strongly reduce tumor growth over 15 days. These data expose opportunities to enhance the potential and efficacy of PDT using simple non-synthetic strategies that might facilitate translation into clinical practice. Show less
Solinge, T.S. van; Abels, E.R.; Haar, L.L. van de; Hanlon, K.S.; Maas, S.L.N.; Schnoor, R.; ... ; Broekman, M.L.D. 2020
Introduction: Glioma cells exert influence over the tumor-microenvironment in part through the release of extracellular vesicles (EVs), membrane-enclosed structures containing proteins, lipids, and... Show moreIntroduction: Glioma cells exert influence over the tumor-microenvironment in part through the release of extracellular vesicles (EVs), membrane-enclosed structures containing proteins, lipids, and RNAs. In this study, we evaluated the function of Ras-associated protein 27a (Rab27a) in glioma and evaluated the feasibility of assessing its role in EV release in glioma cells in vitro and in vivo.Methods: Rab27a was knocked down via a short hairpin RNA (shRNA) stably expressed in mouse glioma cell line GL261, with a scrambled shRNA as control. EVs were isolated by ultracentrifugation and quantified with Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS). CellTiter-Glo viability assays and cytokine arrays were used to evaluate the impact of Rab27a knockdown. GL261.shRab27a cells and GL261.shControl were implanted into the left striatum of eight mice to assess tumor growth and changes in the tumor microenvironment.Results: Knockdown of Rab27a in GL261 glioma cells decreased the release of small EVs isolated at 100,000 x g in vitro (p = 0.005), but not the release of larger EVs, isolated at 10,000 x g. GL261.shRab27a cells were less viable compared to the scramble control in vitro (p < 0.005). A significant increase in CCL2 expression in shRab27a GL261 cells was also observed (p < 0.001). However, in vivo there was no difference in tumor growth or overall survival between the two groups, while shRab27a tumors showed lower proliferation at the tumor borders. Decreased infiltration of IBA1 positive macrophages and microglia, but not FoxP3 positive regulatory T cells was observed.Conclusion: Rab27a plays an important role in the release of small EVs from glioma cells, and also in their viability and expression of CCL2 in vitro. As interference in Rab27a expression influences glioma cell viability and expression profiles, future studies should be cautious in using the knockdown of Rab27a as a means of studying the role of small EVs in glioma growth. Show less
Extracellular vesicles (EVs) are important mediators of intercellular communication that participate in many physiological/pathological processes. As such, EVs have unique properties related to... Show moreExtracellular vesicles (EVs) are important mediators of intercellular communication that participate in many physiological/pathological processes. As such, EVs have unique properties related to their origin, which can be exploited for drug delivery applications in cell regeneration, immunosuppression, inflammation, cancer treatment or cardioprotection. Moreover, their cell-like membrane organization facilitates uptake and accumulation in specific tissues and organs, which can be exploited to improve selectivity of cargo delivery. The combination of these properties with the inclusion of drugs or imaging agents can significantly improve therapeutic efficacy and selectivity, reduce the undesirable side effects of drugs or permit earlier diagnosis of diseases. In this review, we will describe the natural properties of EVs isolated from different cell sources and discuss strategies that can be applied to increase the efficacy of targeting drugs or other contents to specific locations. The potential risks associated with the use of EVs will also be addressed. Show less
