Nanozyme-assisted sensitive profiling of exosomal proteins for rapid cancer diagnosis

Abstract

The proteins expressed on exosomes have emerged as promising liquid-biopsy biomarkers for cancer diagnosis. However, molecular profiling of exosomal proteins remains technically challenging. Herein, we report a nanozyme-assisted immunosorbent assay (NAISA) that enables sensitive and rapid multiplex profiling of exosomal proteins. This NAISA system is based on the installation of peroxidase-like nanozymes onto the phospholipid membranes of exosomes, thus avoiding the need for post-labelling detection antibodies. The exosomal proteins are determined by a sensitive nanozyme-catalyzed colorimetric assay less than 3 h, without the need for multi-step incubation and washing operations. Using NAISA to profile exosomal proteins from different cell lines and clinical samples, we reveal that tumor-associated exosomal proteins can serve as promising biomarkers for accurate cancer diagnosis in a cooperative detection pattern. Methods: Exosomes were engineered with DSPE-PEG-SH through hydrophobic interaction, and then were assembled with gold nanoparticles (2 nm) to produce Exo@Au nanozyme. The proteins on Exo@Au could be selectively captured by their specific antibodies seeded into a 96-well plate. The immobilized Exo@Au shows peroxidase-like activity to perform colorimetric assays by reaction with 3,3′,5,5′-tetramethylbenzidine (TMB) and H 2 O 2 . The protein levels of exosomes were recorded on a microplate reader. Results: The NAISA platform is capable of profiling multiple exosomal proteins from both cancer cell lines and clinical samples. The expression levels of exosomal proteins, such as CD63, CEA, GPC-3, PD-L1 and HER2, were used to classify different cancer cell lines. Moreover, the protein profiles have been applied to differentiate healthy donors, hepatitis B patients, and hepatic cell carcinoma (HCC) patients with high accuracy. Conclusion: The NAISA nanozyme was allowed to rapidly profile multiple exosomal proteins and could have great promise for early HCC diagnosis and identification of other cancer types.

From biomarkers to therapeutic targets: the promise of PD-L1 in thyroid autoimmunity and cancer

“…Altogether, PD-L1 appears to be a helpful diagnostic biomarker for indeterminate nodules, whether at the mRNA or protein level and in tissues or serum 48 , 50 , 52 , 54 , 57 . Nevertheless, no study has been designed specifically for this subgroup of patients.…”

“…Consistent with its prognostic value in other malignancies 56 , Aghajani et al . showed that a high level of soluble PD-L1 in serum, over 0.44 ng/ml, and its detection in matched tumor tissues were associated with a significantly shorter PFS in PTC patients (uni- and multivariate analyses) 57 . As a result, this study places PD-L1 in serum as a marker of bad prognosis for WDTC 57 .…”

“…It is worth mentioning that the above PD-L1 IHC analyses of surgery biopsies remain invasive. Thus, exploiting the release by cancer cells into the blood of high amounts of PD-L1 (multiple myeloma, and hepatocellular carcinoma, among others 56 , 57 , Aghajani et al recently detected a soluble form of PD-L1 (sPD-L1) in the serum (but not in plasma) from PTC patients but not in healthy controls 58 .…”

“…Of clinical relevance, the overexpression of PD-L1 appears to predict worse prognosis, identifying patients at-risk of TC progression, representing real hope for the early management of advanced WDTC (18 clinical studies totaling 3107 patients, Table 3 ) 34 - 37 , 48 - 50 , 57 , 61 - 70 . Indeed, the expression of PD-L1 independently correlated with all clinicopathological markers of poor TC prognosis 5 : such as male gender, an age > 45 years 49 , 50 , 69 , tumor size (including extra-thyroid or multifocal lesions) 36 , 49 , 57 , 63 , 66 , 69 , lymphovascular invasion and lymph node metastasis 34 , 36 , 49 , 62 , 64 , 65 , 69 , 70 , or TNM stage 48 , 50 , 63 . Not surprisingly given its role in tumor immune escape, PD-L1 overexpression (protein and mRNA) is associated with reduced progression-free survival (PFS) in PTC patients 48 , 49 , 57 , 64 , 69 .…”

“…Indeed, the expression of PD-L1 independently correlated with all clinicopathological markers of poor TC prognosis 5 : such as male gender, an age > 45 years 49 , 50 , 69 , tumor size (including extra-thyroid or multifocal lesions) 36 , 49 , 57 , 63 , 66 , 69 , lymphovascular invasion and lymph node metastasis 34 , 36 , 49 , 62 , 64 , 65 , 69 , 70 , or TNM stage 48 , 50 , 63 . Not surprisingly given its role in tumor immune escape, PD-L1 overexpression (protein and mRNA) is associated with reduced progression-free survival (PFS) in PTC patients 48 , 49 , 57 , 64 , 69 . Along this line, its expression tends to persist in up to 50% of the corresponding metastatic lymph nodes, being associated with a higher density of immunosuppressive Tregs, especially in cases of extranodal extension or cancer relapse 50 , 64 , 65 , 71 .…”