Extracellular vesicles (EV) are nanometric lipid bilayer coated particles constitutively released from cells. EVs are a heterogeneous population and can be sampled as liquid biopsy. Beyond messengers between cells, EVs act as regulators and mediators of many processes underlying cancer evolution: inflammation, proliferation, invasion, immune modulation, angiogenesis, epithelial mesenchymal transition. The aim of the present research is to describe a new method to obtain high resolution images of individual EVs in patients with advanced cancer.
Plasma from 10 patients with advanced cancer enrolled in the institutional molecular profiling program STING (NCT04932525, sponsor: Gustave Roussy) was diluted in PBS 1:50 and loaded on slides for Single Molecule Localization Microscopy (SMLM) imaging. EVs were stained using a mix of anti-tetraspanin Ab (CD9+CD63+CD81) labelled with AF647 fluorophores to only select tetraspanin+ EVs for imaging and analysis. Single molecule imaging (dSTORM) was performed using Abbelight SMART-kit buffer on a SAFe360 Abbelight super-resolution module mounted on an Olympus Ix83 inverted microscope. Fluorophore labelled EVs were excited with the 640nm laser at 60% of nominal power over a ROI of 80*80 micrometers, by Abbelight Aster technology for homogeneous laser illumination; for each dataset 10000 frames were collected at 40 FPS, with two-three technical replicates per sample (RAW data). Single molecule localization in 3D was performed on the RAW data using Abbelight Neo Software, and localization clusters corresponding to labelled EVs were extracted using DBSCAN and K-Ripley clustering algorithms.
High resolution images of EVs were obtained with SMLM. Number and size distribution of clusters were quantified for each Ab allowing to observe differences between patients samples. Thus, SMLM can be considered as an additional technique to detect and characterize individual EVs in clinical samples.
SMLM imaging with tetraspanin labeling is able to detect demonstrable differences in EV clusters quantity and size distributions in plasma from patients with advanced cancer. Additional investigations are ongoing to further develop the technique and make it applicable in clinical practice.
Gustave Roussy, Abbelight.
Abbelight.
M. Pagliuca: Financial Interests, Personal, Other, Travel expenses: Gilead, Pfizer. M. Triki: Financial Interests, Personal, Full or part-time Employment: Abbelight. C. Schietroma: Financial Interests, Personal, Full or part-time Employment: Abbelight. C. Butler: Financial Interests, Personal, Full or part-time Employment: Abbelight. B. Verret: Financial Interests, Personal, Other, Consultant fees, travel expenses: Lilly, Pfizer; Financial Interests, Personal, Other, Consultant fees: Netcancer, Pierre Fabre, Seagen, Gilead; Financial Interests, Institutional, Other, Consultant fees: Daiichi Sankyo, MSD; Financial Interests, Personal and Institutional, Other, Consultant fees, travel expenses: Novartis; Financial Interests, Personal, Other, Travel expenses: Accord Healthcare, Amgen. A. Italiano: Financial Interests, Personal, Research Grant, Research funding, Consulting fees: AstraZeneca, MSD, Merck; Financial Interests, Personal, Research Grant, Research funding, Consulting fees, Advisory board: Bayer, Roche; Financial Interests, Personal, Other, Research funding, Consulting fees: BMS; Financial Interests, Personal, Advisory Board, Research grants: Chugai; Financial Interests, Personal, Advisory Board: Daiichi Sankyo, Foundation Medicine, GSK, Springworks. D. Planchard: Financial Interests, Personal, Advisory Board: AstraZeneca, BMS, Merck, Novartis, Pfizer, Roche, Samsung, Celgene, AbbVie, Daiichi Sankyo, Janssen; Financial Interests, Personal, Invited Speaker: AstraZeneca, Novartis, Pfizer, priME Oncology, Peer CME, Samsung, AbbVie, Janssen; Non-Financial Interests, Personal, Principal Investigator, Institutional financial interests: AstraZeneca, BMS, Merck, Novartis, Pfizer, Roche, Daiichi Sankyo, Sanofi-Aventis, Pierre Fabre; Non-Financial Interests, Personal, Principal Investigator: AbbVie, Sanofi, Janssen. A. Bayle: Financial Interests, Personal, Advisory Board: Sanofi; Financial Interests, Personal and Institutional, Research Grant: AstraZeneca, BMS, Boehringer Ingelheim, GSK, INCA, Janssen Cilag, Merck, Pfizer, Roche, Sanofi; Non-Financial Interests, Personal, Other, Drug supplied: AstraZeneca, BMS, Boehringer Ingelheim, GSK, MedImmune, Merck, NH TherAGuiX, Pfizer, Roche; Financial Interests, Personal and Institutional, Other, Principal/sub-Investigator of Clinical Trials: AbbVie, Adaptimmune, Adlai Nortye USA Inc, Aduro Biotech, Agios Pharmaceuticals, Amgen, Astex Pharmaceuticals, AstraZeneca Ab, Aveo, Basilea Pharmaceutica International Ltd., Bayer Healthcare Ag, Bbb Technologies Bv, Beigene, BicycleTx Ltd., Blueprint Medi. F. André: Financial Interests, Institutional, Research Grant: AstraZeneca, Lilly, Novartis, Pfizer, Roche, Daiichi Sankyo; Other, Personal, Other, Founder: Pegacsy. S. Delaloge: Financial Interests, Institutional, Advisory Board: AstraZeneca, Novartis, Pierre Fabre, Orion, Sanofi, Rappta, Cellectis, Isis/Servier; Financial Interests, Institutional, Invited Speaker: Exact Sciences, Pfizer, Seagen, Lilly, AstraZeneca, MSD, Roche Genentech, BMS, Puma, AstraZeneca, Orion, Sanofi, Pfizer; Financial Interests, Institutional, Advisory Board, ad board: Besins Healthcare; Financial Interests, Institutional, Invited Speaker, ESMO symposium: Gilead; Financial Interests, Institutional, Advisory Board, scientific board: Elsan; Financial Interests, Institutional, Funding: GE; Financial Interests, Institutional, Invited Speaker, clinical research funding to my institution: Taiho; Non-Financial Interests, Personal, Invited Speaker, Société Française de Sénologie et Pathologie Mammaire: SFSPM; Non-Financial Interests, Personal, Principal Investigator, H2020 funding: European Commission.