Abstract
The current lack of reliable methods for quantifying extracellular vesicles (EVs) isolated from complex biofluids significantly hinders translational applications in EV research. The recently developed fluorescence nanoparticle tracking analysis (FL-NTA) allows for the detection of EV-associated proteins, enabling EV content determination. In this study, we present the first comprehensive phenotyping of bronchopulmonary lavage fluid (BALF)-derived EVs from non-small cell lung cancer (NSCLC) patients using classical EV-characterization methods as well as the FL-NTA method. We found that EV immunolabeling for the specific EV marker combined with the use of the fluorescent mode NTA analysis can provide the concentration, size, distribution, and surface phenotype of EVs in a heterogeneous solution. However, by performing FL-NTA analysis of BALF-derived EVs in comparison to plasma-derived EVs, we reveal the limitations of this method, which is suitable only for relatively pure EV isolates. For more complex fluids such as plasma, this method appears to not be sensitive enough and the measurements can be compromised. Our parallel presentation of NTA-based phenotyping of plasma and BALF EVs emphasizes the great impact of sample composition and purity on FL-NTA analysis that has to be taken into account in the further development of FL-NTA toward the detection of EV-associated cancer biomarkers.
Highlights
In recent years, a lot of attention has been given to studies of extracellular vesicles (EVs) as a potential diagnostic and prognostic biomarker for many diseases including cancer
We developed an experimental setup based on fluorescence nanoparticle tracking analysis (FL-NTA) that can reveal the amount of bona fide EVs in isolates from heterogeneous particle solutions such as biological fluids
We have shown that EV immunolabeling for specific EV markers combined with the differential use of the scatter mode and fluorescent mode NTA analysis can provide the concentration, size, distribution, and surface phenotype of bona fide EVs in a heterogeneous solution
Summary
A lot of attention has been given to studies of extracellular vesicles (EVs) as a potential diagnostic and prognostic biomarker for many diseases including cancer. In the case of non-small cell lung cancer (NSCLC), the bronchopulmonary lavage fluid (BALF) seems to be a good source of EVs from the tumor microenvironment [2]. BALF is currently extensively studied as a source of lung cancer-specific genetic or protein biomarkers [3]. Some reports suggest that BALF-derived biomarkers might be superior to serum biomarkers because they appear earlier during the cancer progression and at the higher concentrations [4]. The same can be true for BALF-derived EVs. Because of tumor proximity, EVs released by tumor cells may appear in BALF in the earlier disease stage and the higher concentration than in peripheral blood and reflect the tumor microenvironment more accurately. A thorough study on the composition and function of BALF-EVs, representing EVs from the tumor microenvironment in NSCLC patients, can contribute to the development of biomarkers for patient therapy
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