Cancer-ID: Toward Identification of Cancer by Tumor-Derived Extracellular Vesicles in Blood.

Extracellular vesicles (EVs) have great potential as biomarkers since their composition and concentration in biofluids are disease state dependent and their cargo can contain disease-related information. Large tumor-derived EVs (tdEVs, >1 µm) in blood from cancer patients are associated with poor outcome, and changes in their number can be used to monitor therapy effectiveness. Whereas, small tumor-derived EVs (<1 µm) are likely to outnumber their larger counterparts, thereby offering better statistical significance, identification and quantification of small tdEVs are more challenging. In the blood of cancer patients, a subpopulation of EVs originate from tumor cells, but these EVs are outnumbered by non-EV particles and EVs from other origin. In the Dutch NWO Perspectief Cancer-ID program, we developed and evaluated detection and characterization techniques to distinguish EVs from non-EV particles and other EVs. Despite low signal amplitudes, we identified characteristics of these small tdEVs that may enable the enumeration of small tdEVs and extract relevant information. The insights obtained from Cancer-ID can help to explore the full potential of tdEVs in the clinic.

Refractive index to evaluate staining specificity of extracellular vesicles by flow cytometry.

Extracellular vesicles (EVs) in plasma are commonly identified by staining with antibodies and generic dyes, but the specificity of antibodies and dyes to stain EVs is often unknown. Previously, we showed that platelet-depleted platelet concentrate contains two populations of particles >200 nm, one population with a refractive index (RI) < 1.42 that included the majority of EVs, and a second population with an RI > 1.42, which was thought to include lipoproteins. In this study, we investigated whether EVs can be distinguished from lipoproteins by the RI and whether the RI can be used to determine the specificity of antibodies and generic dyes used to stain plasma EVs. EVs and lipoproteins present in platelet-depleted platelet concentrate were separated by density gradient centrifugation. The density fractions were analyzed by Western blot and transmission electron microscopy, the RI of particles was determined by Flow-SR. The RI was used to evaluate the staining specificity of an antibody against platelet glycoprotein IIIa (CD61) and the commonly used generic dyes calcein AM, calcein violet, di-8-ANEPPS, and lactadherin in plasma. After density gradient centrifugation, EV-enriched fractions (1.12 to 1.07 g/mL) contained the highest concentration of particles with an RI < 1.42, and the lipoprotein-enriched fractions (1.04 to 1.03 g/mL) contained the highest concentration of particles with an RI > 1.42. Application of the RI showed that CD61-APC had the highest staining specificity for EVs, followed by lactadherin and calcein violet. Di-8-ANEPPS stained mainly lipoproteins and calcein AM stained neither lipoproteins nor EVs. Taken together, the RI can be used to distinguish EVs and lipoproteins, and thus allows evaluation of the specificity of antibodies and generic dyes to stain EVs.

Quality of extracellular vesicle images by transmission electron microscopy is operator and protocol dependent.

Transmission electron microscopy (TEM) has nanometre resolution and can be used to distinguish single extracellular vesicles (EVs) from non-EV particles. TEM images of EVs are a result of operator image selection. To which extent operator image selection reflects the overall sample quality, and to which extent the images are comparable and reproducible, is unclear. In a first attempt to improve the comparability and reproducibility of TEM to visualise EVs, we compared operator image selection to images taken at predefined locations from the same grids, using four EV TEM preparation protocols, a single EV-containing sample and a single TEM instrument. Operator image selection leads to high-quality images that are more similar between the protocols. In contrast, images taken at predefined locations reveal differences between the protocols, for example in number of EVs per image and background quality. From the evaluated protocols, for only one protocol the operator image selection is comparable to the TEM images taken at predefined locations. Taken together, operator image selection can be used to demonstrate the presence of EVs in a sample, but seem less suitable to demonstrate the quality of a sample. Because images taken at predefined locations reflect the overall quality of the EV-containing sample rather than the presence of EVs alone, this is a first step to improve the comparability and reproducibility of TEM for monitoring the quality of EV-containing samples.

Extracellular vesicles from human saliva promote hemostasis by delivering coagulant tissue factor to activated platelets.

Essentials Human salivary extracellular vesicles (EVs) expose coagulant tissue factor (TF). Salivary EVs expose CD24, a ligand of P-selectin. CD24 and coagulant TF co-localize on salivary EVs. TF+ /CD24+ salivary EVs bind to activated platelets and trigger coagulation. SUMMARY: Background Extracellular vesicles (EVs) from human saliva expose coagulant tissue factor (TF). Whether such TF-exposing EVs contribute to hemostasis, however, is unknown. Recently, in a mice model, tumor cell-derived EVs were shown to deliver coagulant TF to activated platelets at a site of vascular injury via interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin. Objectives We hypothesized that salivary EVs may deliver coagulant TF to activated platelets via interaction with P-selectin. Methods We investigated the presence of two ligands of P-selectin on salivary EVs, PSGL-1 and CD24. Results Salivary EVs expose CD24 but PSGL-1 was not detected. Immune depletion of CD24-exposing EVs completely abolished the TF-dependent coagulant activity of cell-free saliva, showing that coagulant TF and CD24 co-localize on salivary EVs. In a whole blood perfusion model, salivary EVs accumulated at the surface of activated platelets and promoted fibrin generation, which was abolished by an inhibitory antibody against human CD24. Conclusions A subset of EVs in human saliva expose coagulant TF and CD24, a ligand of P-selectin, suggesting that such EVs may facilitate hemostasis at a site of skin injury where the wound is licked in a reflex action.

Wound scabs protect regenerating tissue against harmful ultraviolet radiation.

Benefits attributed to wound scabs include prevention of blood loss and protection against infection. However, when formation of a wound scab is prevented, the risk of infection is reduced. Moreover, in the absence of a wound scab, wounds heal faster and scar formation is reduced. The question arises why we develop a wound scab. Here we show that wound scabs inhibit transmission of ultraviolet radiation (UVR). We compared the UVR transmittance of human wound scabs to sunscreen by measuring the sun protection factor (SPF) with diffuse transmittance spectroscopy. Three wound scabs showed SPFs of 70, 84, and 300, which is more effective than the most protective commercially available sun block. Because our results demonstrate that a wound scab offers natural protection against UVR, and because no beneficial trait is attributed to wound scabs, we hypothesize that the main function of wound scabs is to limit DNA damage in underlying cells during regeneration of wound tissue exposed to sunlight, thereby reducing the risk of developing skin cancer.

Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing.

BACKGROUND: Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25-fold in size, 300,000-fold in concentration, 20,000-fold in volume, and 10,000,000-fold in scattered light. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 10(4) to 10(12) vesicles mL(-1) . OBJECTIVES: To investigate whether the large variation in the detected concentration of vesicles is caused by the minimum detectable vesicle size of five widely used techniques. METHODS: The size and concentration of vesicles and reference beads were measured with transmission electron microscopy (TEM), a conventional flow cytometer, a flow cytometer dedicated to detecting submicrometer particles, nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS). RESULTS: Each technique gave a different size distribution and a different concentration for the same vesicle sample. CONCLUSION: Differences between the detected vesicle concentrations are primarily caused by differences between the minimum detectable vesicle sizes. The minimum detectable vesicle sizes were 70-90 nm for NTA, 70-100 nm for RPS, 150-190 nm for dedicated flow cytometry, and 270-600 nm for conventional flow cytometry. TEM could detect the smallest vesicles present, albeit after adhesion on a surface. Dedicated flow cytometry was most accurate in determining the size of reference beads, but is expected to be less accurate on vesicles, owing to heterogeneity of the refractive index of vesicles. Nevertheless, dedicated flow cytometry is relatively fast and allows multiplex fluorescence detection, making it most applicable to clinical research.

Circulating melanoma cells and survival in metastatic melanoma.

A validated assay for the enumeration of circulating melanoma cells (CMCs) may facilitate the development of more effective therapies for metastatic melanoma patients. In this study CD146+ cells were immunomagnetically enriched from 7.5 ml of blood. Isolated cells were fluorescently stained with DAPI, anti-molecular weight melanoma-associated antigen (HMW-MAA), anti-CD45 and CD34 and Ki67. CMCs were identified as CD146+, HMW-MAA+, CD45-, CD34-, Ki67-/+ cells. Eighty-eight percent of spiked SK-MEL28 cells in 7.5 ml blood were recovered. In all 55 healthy donors ≤1 CMCs were detected in 7.5 ml of blood. A retrospective analysis was conducted comparing CMC counts and overall survival in 79 blood samples from 44 melanoma patients. CMCs ranged from 0 to 8,042 per 7.5 ml. Two or more CMCs were detected in 18 (23%) of the patients and 30-100% (mean 84%) of the CMCs expressed the proliferation marker Ki67. Patients with ≥2 CMCs per 7.5 ml of whole blood, as compared with the group with <2 CMCs, had a shorter overall survival (2.0 months vs. 12.1 months, P=0.001).

All circulating EpCAM+CK+CD45- objects predict overall survival in castration-resistant prostate cancer.

BACKGROUND: Presence of five or more circulating tumor cells (CTC) in patients with metastatic carcinomas is associated with poor survival. Although many objects positive for epithelial cell adhesion molecules and cytokeratin (EpCAM+CK+) are not counted as CTC, they may be an important predictor for survival. We evaluated the association between these objects and survival in patients with prostate cancer. PATIENTS AND METHODS: Included in this follow-up study were 179 patients with castration-resistant prostate cancer. CellSearch was used to isolate EpCAM+ objects and to stain DNA, cytokeratin and CD45. All EpCAM+CK+ objects were subdivided into seven classes on the basis of predefined morphological appearance in 63 independent samples. Association of each class with survival was studied using Kaplan-Meier and Cox regression analyses. RESULTS: Each EpCAM+CK+CD45- class showed a strong association with overall survival (P < 0.001). This included small tumor microparticles (S-TMP), which did not require a nucleus and thus are unable to metastasize. A higher number of objects in any class was associated with decreased survival. A good prediction model included large tumor cell fragments (L-TCF), age, hemoglobin and lactate dehydrogenase. Models with S-TMP or CTC instead of L-TCF performed similarly. CONCLUSION: EpCAM+CK+CD45- that do not meet strict definitions for CTC are strong prognostic markers for survival.