Biological reference materials for extracellular vesicle studies.

Extracellular vesicles (EVs) mediate normal physiological homeostasis and pathological processes by facilitating intercellular communication. Research of EVs in basic science and clinical settings requires both methodological standardization and development of reference materials (RM). Here, we show insights and results of biological RM development for EV studies. We used a three-step approach to find and develop a biological RM. First, a literature search was done to find candidates for biological RMs. Second, a questionnaire was sent to EV researchers querying the preferences for RM and their use. Third, a biological RM was selected, developed, characterized, and evaluated. The responses to the survey demonstrated a clear and recognized need for RM optimized for the calibration of EV measurements. Based on the literature, naturally occurring and produced biological RM, such as virus particles and liposomes, were proposed as RM. However, none of these candidate RMs have properties completely matching those of EVs, such as size and refractive index distribution. Therefore, we evaluated the use of nanoerythrosomes (NanoE), vesicles produced from erythrocytes, as a potential biological RM. The strength of NanoE is their resemblance to EVs. Compared to the erythrocyte-derived EVs (eryEVs), NanoE have similar morphology, a similar refractive index (1.37), larger diameter (70% of the NanoE are over 200nm), and increased positive staining for CD235a and lipids (Di-8-ANEPPS) (58% and 67% in NanoE vs. 21% and 45% in eryEVs, respectively). Altogether, our results highlight the general need to develop and validate new RM with similar physical and biochemical properties as EVs to standardize EV measurements between instruments and laboratories.

Determination of the size distribution of blood microparticles directly in plasma using atomic force microscopy and microfluidics.

Microparticles, also known as microvesicles, found in blood plasma, urine, and most other body fluids, may serve as valuable biomarkers of diseases such as cardiovascular diseases, systemic inflammatory disease, thrombosis, and cancer. Unfortunately, the detection and quantification of microparticles are hampered by the microscopic size of these particles and their relatively low abundance in blood plasma. The use of a combination of microfluidics and atomic force microscopy to detect microparticles in blood plasma circumvents both problems. In this study, capture of a specific subset of microparticles directly from blood plasma on antibody-coated mica surface is demonstrated. The described method excludes isolation and washing steps to prepare microparticles, improves the detection sensitivity, and yields the size distribution of the captured particles. The majority of the captured particles have a size ranging from 30 to 90 nm, which is in good agreement with prior results obtained with microparticles immediately isolated from fresh plasma. Furthermore, the qualitative shape of the size distribution of microparticles is shown not to be affected by high-speed centrifugation or the use of the microfluidic circuit, demonstrating the relative stable nature of microparticles ex vivo.

Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles.

BACKGROUND: Microparticles (MPs) are small vesicles released from cells of different origin, bearing surface antigens from parental cells. Elevated numbers of blood MPs have been reported in (cardio)vascular disorders and cancer. Most of these MPs are derived from platelets. OBJECTIVES: To investigate whether atomic force microscopy (AFM) can be used to detect platelet-derived MPs and to define their size distribution. METHODS: Blood MPs isolated from seven blood donors and three cancer patients were immobilized on a modified mica surface coated with an antibody against CD41 prior to AFM imaging. AFM was performed in liquid-tapping mode to detect CD41-positive MPs. In parallel, numbers of CD41-positive MPs were measured using flow cytometry. Mouse IgG1 isotype control was used as a negative control. RESULTS: AFM topography measurements of the number of CD41-positive MPs were reproducible (coefficient of variation=16%). Assuming a spherical shape of unbound MPs, the calculated diameter of CD41-positive MPs (dsph) ranged from 10 to 475 nm (mean: 67.5+/-26.5 nm) and from 5 to 204 nm (mean: 51.4+/-14.9 nm) in blood donors and cancer patients, respectively. Numbers of CD41-positive MPs were 1000-fold higher than those measured by flow cytometry (3-702x10(9) L(-1) plasma vs. 11-626x10(6) L(-1) plasma). After filtration of isolated MPs through a 0.22-microm filter, CD41-positive MPs were still detectable in the filtrate by AFM (mean dsph: 37.2+/-11.6 nm), but not by flow cytometry. CONCLUSIONS: AFM provides a novel method for the sensitive detection of defined subsets of MPs in the nanosize range, far below the lower limit of what can be measured by conventional flow cytometry.

CD13/Aminopeptidase N overexpression by basic fibroblast growth factor mediates enhanced invasiveness of 1F6 human melanoma cells.

CD13/Aminopeptidase N (CD13) is known to play an important role in tumour cell invasion. We examined whether basic fibroblast growth factor (bFGF) is involved in the regulation of CD13 expression in human melanoma cells. 1F6 human melanoma cells were stably transfected with constructs encoding either the 18 kDa (18 kD) or all (ALL) bFGF isoform proteins. We observed highly increased CD13 mRNA and protein expression in the 1F6 clones regardless of the overexpression of either the 18 kD or all isoform proteins. Neutral aminopeptidase activity was increased five-fold and could be inhibited by bestatin and the CD13-neutralising antibody WM15. The enhanced invasion through Matrigel, but not migration in a wound assay, was efficiently abrogated by both bestatin and WM15. Upregulation of CD13 expression was the result of increased epithelial and myeloid promoter activity up to 4.5-fold in 1F6-18 kD and 1F6-ALL clones. Interestingly, in a panel of human melanoma cell lines, a significant correlation (r(2)=0.883, P<0.05) between bFGF and CD13 mRNA and protein expression was detected. High bFGF and CD13 expression were clearly related with an aggressive phenotype. Taken together, our data indicate that high bFGF expression upregulates CD13 expression in human melanoma cells by activating both the myeloid and the epithelial CD13 promoter. In addition, we show that high bFGF and CD13 expression results in enhanced invasive capacity and metastatic behaviour of human melanoma cells.

Recombinant human endostatin administered as a 28-day continuous intravenous infusion, followed by daily subcutaneous injections: a phase I and pharmacokinetic study in patients with advanced cancer.

BACKGROUND: Endostatin is an endogenous collagen XVIII-fragment with anti-angiogenic properties and remarkable antitumor activity in mice. Preclinical data suggest that continuous low dose administration of endostatin is much more potent than intermittent dosing. The feasibility of this approach is tested in a phase I study. PATIENTS AND METHODS: We determined the safety and pharmacokinetic profile of 4-week continuous intravenous infusion of recombinant human (rh)-endostatin, followed after an interval of 1 week by twice daily subcutaneous injections in patients with advanced cancer. Thirty-two patients received rh-endostatin in six dosing cohorts, ranging from 3.75 mg/m(2)/day to 120 mg/m(2)/day. Serum endostatin pharmacokinetics, toxicity and antitumor response were determined. RESULTS: A total of 160 cycles were delivered without significant toxicities. Pharmacokinetic analysis showed a linear increase of steady-state serum endostatin concentrations with dose (i.v. r(2)=0.96; s.c. r(2)=0.99) reaching 300--1,000 ng/ml for the two highest doses, with considerable interpatient variation. The main pharmacokinetic values for both routes of administration were similar. The apparent steady-state concentration and AUC reached at 60--120 mg/m(2)/day were within the range expected to induce anti-angiogenic and antitumor effects based on preclinical tumor models. Although no objective responses were observed, two patients had long-lasting stable disease (defined as a tumor increase<100%). CONCLUSION: rh-endostatin was safely administered both by continuous infusion and by twice daily subcutaneous injections up to 120 mg/m(2)/day. Predictable pK was seen in this dose range and the target endostatin levels were reached from 60 mg/m(2)/day and above.