A specific technique of immunolabelling of urinary small extracellular vesicle biomarkers for the diagnostic of renal cancer

Abstract Small extracellular vesicles (EVs) are characterized by the membrane expression of CD63, CD81 and CD9 tetraspanins. Their size is inferior to 200 nm. They share the same characteristics as the native cells and are found in human fluids. Specific membrane protein biomarkers expressed on small EV are useful for the diagnosis of tumoral pathologies. Clear cells renal cell carcinoma (CCRCC) is diagnosed by imaging examinations and/or tissue biopsy. Carbonic anhydrase IX (CAIX) is a powerful biomarker of CCRCC. The detection of CAIX on small EV from the urine of patients could constitute a liquid biopsy for CCRCC. We have setup a specific protocol for the preparation, immunostaining characterization and the Transmission Electron Microscopy (TEM) observation of small EVs isolated from the urine of CRCC patients. The background labelling was significantly reduced. We successfully detected biomarkers on urinary small EVs from CCRCC patients. This technique could be extended with antibodies directed against other EVs biomarkers for the detection and the monitoring of cancer diseases.