Extracellular vesicles (EV) have been gaining attention as a promising biomarker for cancer diagnostics and therapeutic monitoring. For example, in recipients of PD1/PD-L1 immune checkpoint inhibitors – a key pillar of cancer immunotherapy, EV-derived PD-L1 molecules are increasingly recognized as a non-invasive key indicator of therapeutic responsiveness. Conventional methods to measure EV-derived PD-L1 molecules requires ultracentrifugation to separate EVs and quantify those that express PD-L1 using nanoscale flow cytometry. These are tedious processes that require trained technologists in centralized facilities, which is a key barrier to the close monitoring of these EV-based indicators of therapeutic responsiveness. However, frequently evaluating the immune responsiveness to the immunotherapies is essential, because prolonged use of a futile therapy can lead to the loss of time in the race with the fatal disease, as well as financial burden on patients, healthcare systems and economy. To bridge the gap, we develop a proof-of-concept microfluidic device that can separate EVs directly from biofluids and quantify PD-L1-positive EVs automatedly. In this device, EVs can be extracted by immunomagnetic beads, and the PD-L1 expression level can be quantified with the electrochemical sensor on-chip < 2 hours. Meanwhile, the device can enable 10X increase in the number of EVs extracted compared with in-tube extraction, with the ability to distinguish 10X differences in EV concentration. This tool can ultimately help to identify futile therapies quickly and prompt personalized alternatives, as well as to collect densely time-longitudinal data points so that we can extrapolate future response trajectories and thus inform strategies to curve the outcome.

Extracellular vesicles (EVs) are membrane-encapsulated nanoparticles shed from all cells. In addition to their critical roles in cellular functions, EVs are emerging as exciting therapeutic delivery vectors and biomarker candidates. Our group has recently developed Single Extracellular VEsicle Nanoscopy (SEVEN), which combines affinity isolation of EVs with super-resolution microscopy to enable a multiparametric characterization of EVs. With ~7 nm precision and molecular sensitivity, this method can robustly provide critical parameters of individual EVs: concentration, size, shape, molecular cargo content, and heterogeneity. We show that SEVEN can specifically detect rare EV populations, including tissue-associated and disease-associated EVs. Because it attains nanoscale precision, SEVEN can also detect small non-vesicular extracellular nanoparticles. Importantly, EV studies may be democratized via a new method that employs super-resolution microscopy and advanced algorithms supported by machine learning-based EV assessment. Our approach – which we termed SEVEN-universal protocol (SEVEN-UP) – can robustly assess EV concentration, size, and molecular content. Altogether, SEVEN and SEVEN-UP may be important tools for significantly propelling our knowledge-base of EVs forward.

Nanoparticle Tracking Analysis (NTA) has emerged as a fast and vital characterization technology for Extracellular Vesicles (EVs), Exosomes and other biological material in the size range from 30 nm to 1 μm. While classic NTA scatter operation feeds back the size and total particle concentration, the user typically cannot discriminate whether the particle is a vesicle, protein aggregate, cellular trash or an inorganic precipitate. The fluorescence detection capabilities of f-NTA however enables the user to gain specific biochemical information for phenotyping of all kinds of vesicles and viruses. Alignment-free switching between excitation wavelengths and measurement modes (scatter and fluorescence) allow quantification of biomarker ratios such as the tetraspanins (CD63, CD81 and CD9) within minutes. Furthermore, specific colocalization studies using c-NTA gives a deeper understanding of the composition of biomarker on single particle.

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