ABSTRACT
We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).
Subject(s)
Biomarkers, Tumor/isolation & purification , Carcinoma, Hepatocellular/diagnosis , Early Detection of Cancer/methods , Extracellular Vesicles/genetics , Liver Neoplasms/diagnosis , Aged , Biomarkers, Tumor/genetics , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Case-Control Studies , Click Chemistry/instrumentation , Click Chemistry/methods , Computational Chemistry , Computer Simulation , Diagnosis, Differential , Dimethylpolysiloxanes/chemistry , Disease Progression , Early Detection of Cancer/instrumentation , Female , Hep G2 Cells , Humans , Lab-On-A-Chip Devices , Liquid Biopsy/instrumentation , Liquid Biopsy/methods , Liver Cirrhosis/blood , Liver Cirrhosis/pathology , Liver Neoplasms/blood , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Male , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Middle Aged , Nanostructures/chemistry , Nanowires/chemistry , Neoplasm Staging , RNA, Messenger/genetics , RNA, Messenger/isolation & purification , ROC Curve , Reverse Transcriptase Polymerase Chain Reaction/instrumentation , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
Tumor-derived extracellular vesicles (EVs) present in bodily fluids are emerging liquid biopsy markers for non-invasive cancer diagnosis and treatment monitoring. Because the majority of EVs in circulation are not of tumor origin, it is critical to develop new platforms capable of enriching tumor-derived EVs from the blood. Herein, we introduce a biostructure-inspired NanoVilli Chip, capable of highly efficient and reproducible immunoaffinity capture of tumor-derived EVs from blood plasma samples. Anti-EpCAM-grafted silicon nanowire arrays were engineered to mimic the distinctive structures of intestinal microvilli, dramatically increasing surface area and enhancing tumor-derived EV capture. RNA in the captured EVs can be recovered for downstream molecular analyses by reverse transcription Droplet Digital PCR. We demonstrate that this assay can be applied to monitor the dynamic changes of ROS1 rearrangements and epidermal growth factor receptor T790M mutations that predict treatment responses and disease progression in non-small cell lung cancer patients.
