Tumor-derived extracellular vesicle (TEV) protein biomarkers facilitate cancer diagnosis and prognostic evaluations. However, the lack of reliable and convenient quantitative methods for evaluating TEV proteins prevents their clinical application. Methods: Here, based on dual amplification of hybridization chain reaction (HCR) and CRISPR-Cas12a, we developed the apta-HCR-CRISPR assay for direct high-sensitivity detection of TEV proteins. The TEV protein-targeted aptamer was amplified by HCR to produce a long-repeated sequence comprising multiple CRISPR RNA (crRNA) targetable barcodes, and the signals were further amplified by CRISPR-Cas12a collateral cleavage activities, resulting in a fluorescence signal. Results: The established strategy was verified by detecting the TEV protein markers nucleolin and programmed death ligand 1 (PD-L1). Both achieved limit of detection (LOD) values as low as 10 2 particles/µL, which is at least 10 4 -fold more sensitive than aptamer-ELISA and 10 2 -fold more sensitive than apta-HCR-ELISA. We directly applied our assay to a clinical analysis of circulating TEVs from 50 µL of serum, revealing potential applications of nucleolin + TEVs for nasopharyngeal carcinoma cancer (NPC) diagnosis and PD-L1 + TEVs for therapeutic monitoring. Conclusion: The platform was simple and easy to operate, and this approach should be useful for the highly sensitive and versatile quantification of TEV proteins in clinical samples.

Background: Currently, there are no molecular biomarkers for the early detection of non-small-cell lung cancer (NSCLC). This study focused on identifying RNAs found on tumor-educated blood platelets (TEPs) for detecting stage I NSCLC.Methods: Platelet RNAs, isolated from the blood of 9 patients with NSCLC (stages I and II) and 8 healthy controls, were analyzed using RNA-seq. ITGA2B was selected as a candidate marker. Two different Polymerase Chain Reactions (PCR) were used to measure ITGA2B in platelet samples from healthy controls (n = 150), patients with NSCLC (n = 243), and patients with benign pulmonary nodules (n = 141) in two cohorts.Results: Platelet ITGA2B levels were significantly higher (p < 0.001) in patients with NSCLC than in all controls. The diagnostic accuracy of ITGA2B was area under the curve (AUC) of 0.922 [95% confidence interval (CI), 0.892-0.952], sensitivity of 92.8%, and specificity of 78.6% in the test cohort and 0.888, 91.2%, and 56.5% in the validation cohort for NSCLC by quantitative real time PCR (q-PCR). Furthermore, ITGA2B maintained diagnostic accuracy for patients with NSCLC using Droplet Digital PCR (ddPCR) and the other type of internal control, Ribosomal Protein L32 (RPL32) [ddPCR: 0.967 (0.929-1.000) and RPL32: 0.847(0.773-0.920)]. A nomogram incorporating ITGA2B, carcinoembryonic antigen (CEA) and stage could predict the overall survival (C-index = 0.756).Conclusions: TEP ITGA2B is a promising marker to improve identification of patients with stage I NSCLC and differentiate malignant from benign lung nodules.

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To evaluate whether serum Cathepsin S (Cat S) could serve as a biomarker for the diagnosis and prognosis of gastric cancer (GC), Enzyme-linked immuno sorbent assay (ELISA) was used to detect serum Cat S in 496 participants including healthy controls and patients with benign gastric diseases, gastric cancer, esophageal cancer, liver cancer, colorectal cancer, nasopharyngeal cancer and lung cancer. The levels of serum Cat S were significantly increased in cancer patients, especially in GC patients. The qRT-PCR, Western blotting, and immunohistochemical staining revealed the overexpression of Cat S in GC cell lines and tissues. The diagnostic value of serum Cat S for GC patients from controls resulted in an AUC of 0.803 with a sensitivity of 60.7% and a specificity of 90.0%. Moreover, the levels of serum Cat S were associated with GC tumor volume, lymphoid nodal status, metastasis status, and stages. Moreover, the patients with high levels of serum Cat S had a poorer overall survival. Univariate analysis revealed Cat S expression was a prognostic factor. The knockdown of Cat S significantly suppressed the migration and invasion of GC cells. This study suggested serum Cat S may be a potential biomarker for the diagnosis and prognosis of GC.

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