Detection of Tumor-Associated Membrane Receptors on Extracellular Vesicles from Non-Small Cell Lung Cancer Patients via Immuno-PCR

Stiller, Christiane; Viktorsson, Kristina; Gomero, Elizabeth Paz; Hååg, Petra; Arapi, Vasiliki; Kaminskyy, Vitaliy O.; Kamali, Caroline; Petris, Luigi De; Ekman, Simon; Lewensohn, Rolf; Karlström, Amelie Eriksson

doi:10.3390/cancers13040922

Cited by 20 publications

(34 citation statements)

References 48 publications

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“…The expressions of CD9 and EGFR in these sEVs are previously reported. 30 (b) Surface expression levels of CD9, EGFR, and PD-L1 for sEVs from cell culture media of H1975 cells prior and post treatments with 1 μM erlotinib or 0.1 μM osimertinib for 48 h. The cell morphology and cell viability of H1975 upon tyrosine kinase inhibitor (TKI) treatment are shown in Figure S3a,b and CD9 expression from Western blot analyses is presented in Figure S3c . (c) sEV samples isolated from PE-fluid of two NSCLC adenocarcinoma patients PE002 and PE011 using the same capturing and affinity reagents as in (b).…”

Section: Resultsmentioning

confidence: 99%

Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor

Sahu

Cavallaro

Hååg

et al. 2021

ACS Appl. Mater. Interfaces

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We present an approach to improve the detection sensitivity of a streaming current-based biosensor for membrane protein profiling of small extracellular vesicles (sEVs). The experimental approach, supported by theoretical investigation, exploits electrostatic charge contrast between the sensor surface and target analytes to enhance the detection sensitivity. We first demonstrate the feasibility of the approach using different chemical functionalization schemes to modulate the zeta potential of the sensor surface in a range −16.0 to −32.8 mV. Thereafter, we examine the sensitivity of the sensor surface across this range of zeta potential to determine the optimal functionalization scheme. The limit of detection (LOD) varied by 2 orders of magnitude across this range, reaching a value of 4.9 × 10 6 particles/mL for the best performing surface for CD9. We then used the optimized surface to profile CD9, EGFR, and PD-L1 surface proteins of sEVs derived from non-small cell lung cancer (NSCLC) cell-line H1975, before and after treatment with EGFR tyrosine kinase inhibitors, as well as sEVs derived from pleural effusion fluid of NSCLC adenocarcinoma patients. Our results show the feasibility to monitor CD9, EGFR, and PD-L1 expression on the sEV surface, illustrating a good prospect of the method for clinical application.

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Section: Resultsmentioning

confidence: 99%

Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor

Sahu

Cavallaro

Hååg

et al. 2021

ACS Appl. Mater. Interfaces

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“…In particular, the isolation of the sEVs from the cell culture media of NSCLC cells followed the protocol reported in our earlier studies (Cavallaro et al, 2019(Cavallaro et al, , 2021. For the PE fluid sEVs, we followed the procedure described in another earlier work (Stiller et al, 2021).…”

Section: Sev Collection and Isolationmentioning

confidence: 99%

“…Following validation, we applied the multiplexed platform for detection and profiling of EVs isolated from malignant pleural effusion (PE) fluids of three NSCLC patients with different genomic mutations and during different stages of their treatment courses. PE refers to the accumulation of fluid in the space between the lungs and the chest walls in both malignant and non-malignant conditions (Baburaj et al, 2020;Stiller et al, 2021). With respect to advanced LC patients, PE fluid accumulation is seen in about 10-15% of all cases with a predomination of adenocarcinomas due to their growth pattern in the lung (Baburaj et al, 2020).…”

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confidence: 99%

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

Cavallaro

aringg

Sahu

et al. 2021

Preprint

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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, 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.

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“…The study of cellular membranes has a long history, and the study and discovery of cell structures “keep biologists glued to their microscopes” [ 14 ]. Cellular membrane receptors are important in conditions such as Alzheimer’s disease [ 15 ], cancer [ 16 ] and Helicobacter pylori infection [ 17 ]. The geometry of the membranes is also important, for instance the shape [ 18 , 19 ], curvature [ 20 , 21 ] and protuberances [ 22 ] have been studied.…”

Section: Introductionmentioning

confidence: 99%

Volumetric Semantic Instance Segmentation of the Plasma Membrane of HeLa Cells

2021

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In this work, an unsupervised volumetric semantic instance segmentation of the plasma membrane of HeLa cells as observed with serial block face scanning electron microscopy is described. The resin background of the images was segmented at different slices of a 3D stack of 518 slices with 8192 × 8192 pixels each. The background was used to create a distance map, which helped identify and rank the cells by their size at each slice. The centroids of the cells detected at different slices were linked to identify them as a single cell that spanned a number of slices. A subset of these cells, i.e., the largest ones and those not close to the edges were selected for further processing. The selected cells were then automatically cropped to smaller regions of interest of 2000 × 2000 × 300 voxels that were treated as cell instances. Then, for each of these volumes, the nucleus was segmented, and the cell was separated from any neighbouring cells through a series of traditional image processing steps that followed the plasma membrane. The segmentation process was repeated for all the regions of interest previously selected. For one cell for which the ground truth was available, the algorithm provided excellent results in Accuracy (AC) and the Jaccard similarity Index (JI): nucleus: JI =0.9665, AC =0.9975, cell including nucleus JI =0.8711, AC =0.9655, cell excluding nucleus JI =0.8094, AC =0.9629. A limitation of the algorithm for the plasma membrane segmentation was the presence of background. In samples with tightly packed cells, this may not be available. When tested for these conditions, the segmentation of the nuclear envelope was still possible. All the code and data were released openly through GitHub, Zenodo and EMPIAR.

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Detection of Tumor-Associated Membrane Receptors on Extracellular Vesicles from Non-Small Cell Lung Cancer Patients via Immuno-PCR

Cited by 20 publications

References 48 publications

Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor

Exploiting Electrostatic Interaction for Highly Sensitive Detection of Tumor-Derived Extracellular Vesicles by an Electrokinetic Sensor

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

Volumetric Semantic Instance Segmentation of the Plasma Membrane of HeLa Cells

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