Biology subjects

Linnros, J.

Publications and source records attributed to Linnros, J..

3 recordsLinked to original sources

bioRxiv2021

Multiplexed electrokinetic sensor for detection and therapy monitoring of extracellular vesicles from liquid biopsies of non-small-cell lung cancer patients

Liquid biopsies based on extracellular vesicle (EV) protein profiles represent a promising tool for treatment monitoring of tumors, including non-small-cell lung cancers (NSCLC). In this study, we present the development of an electrokinetic sensor for multiplexed surface protein profiling of EVs and analysis of clinical samples. The method detects the difference in the streaming current obtained as a result of EV binding to the inner surface of a functionalized microcapillary, thereby estimating the expression level of a surface marker. Using multiple microchannels functionalized with different antibodies in a parallel fluidic connection, we first demonstrate the capacity for simultaneous detection of multiple surface markers in small EVs (sEVs) from NSCLC cells. To investigate the prospects of liquid biopsies based on EVs, we then apply the method to profile sEVs isolated from the pleural effusion (PE) fluids of three NSCLC adenocarcinoma patients with different genomic alterations (ALK-fusion, KRAS and EGFR) and applied treatments (chemotherapy, EGFR or ALK tyrosine kinase inhibitors). These vesicles were targeted against CD9 tetraspanin, as well as EGFR and PD-L1, two markers of interest in NSCLC. The electrokinetic signals showed detection of these markers on sEVs yet highlighting distinct interpatient differences, e.g., increased EGFR levels in sEVs from a patient with EGFR mutation as compared to an ALK-mutant one. The sensors also detected differences in PD-L1 expressions, in line with those measured by complementary methods. The analysis of sEVs from a patient prior and post crizotinib treatment also revealed a significant increase in the expression of some markers, e.g. EGFR and PD-L1. The obtained results hold promise for the application of the method for tumor treatment monitoring based on sEVs from liquid biopsies.

bioengineering↗

bioRxiv2021

Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy: Correlation and Heterogeneity in Their Molecular and Biophysical Features

Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from Human Embryonic Kidney 293, Cord Blood Mesenchymal Stromal and Human Acute Monocytic Leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63 and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having on average diameters <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.

bioengineering↗

bioRxiv2021

High throughput imaging of nanoscale extracellular vesicles by scanning electron microscopy for accurate size-based profiling and morphological analysis.

Nanoscale extracellular vesicle (EVs) have been found to play a key role in intercellular communication, offering opportunities for both diagnostics and therapeutics. However, lying below the diffraction limit and also being highly heterogeneous in their size, morphology and abundance, these vesicles pose significant challenges for their physical characterization. Here, we present a direct visual approach for their accurate morphological and size-based profiling by using scanning electron microscopy (SEM). To achieve that, we methodically examined various process steps and developed a protocol to improve the throughput, conformity and image quality while preserving the shape of EVs. The investigation was performed with small EVs (sEVs) isolated from a non-small cell lung cancer (NSCLC) cell line H1975 as well as from a human serum, and the results were compared with those obtained from nanoparticle tracking analysis (NTA). While the comparison of the sEV size distributions showed good agreement between the two methods for large sEVs (diameter >70 nm), the microscopy based approach showed a better capacity for analyses on smaller vesicles, with higher sEV counts compared to NTA. In addition, we demonstrated the possibility of identifying non-EV particles based on size and morphological features. The study also showed process steps that can generate artifacts bearing resemblance with sEVs. The results therefore present a simple way to use a widely available microscopy tool for accurate and high throughput physical characterization of EVs.

bioengineering↗