Multiplex Analysis of CircRNAs from Plasma Extracellular Vesicle-Enriched Samples for the Detection of Early-Stage Non-Small Cell Lung Cancer

Abstract

Background: The analysis of liquid biopsies brings new opportunities in the precision oncology field. Under this context, extracellular vesicle circular RNAs (EV-circRNAs) have gained interest as biomarkers for lung cancer (LC) detection. However, standardized and robust protocols need to be developed to boost their potential in the clinical setting. Although nCounter has been used for the analysis of other liquid biopsy substrates and biomarkers, it has never been employed for EV-circRNA analysis of LC patients. Methods: EVs were isolated from early-stage LC patients (n = 36) and controls (n = 30). Different volumes of plasma, together with different number of pre-amplification cycles, were tested to reach the best nCounter outcome. Differential expression analysis of circRNAs was performed, along with the testing of different machine learning (ML) methods for the development of a prognostic signature for LC. Results: A combination of 500 μL of plasma input with 10 cycles of pre-amplification was selected for the rest of the study. Eight circRNAs were found upregulated in LC. Further ML analysis selected a 10-circRNA signature able to discriminate LC from controls with AUC ROC of 0.86. Conclusions: This study validates the use of the nCounter platform for multiplexed EV-circRNA expression studies in LC patient samples, allowing the development of prognostic signatures.

Selective Internal Radiotherapy Alters the Profiles of Systemic Extracellular Vesicles in Hepatocellular Carcinoma

“…These findings indicate a proinflammatory status induced by SIRT. Immune cell-derived EVs play crucial roles in intercellular communication and regulation of specific mechanisms of both adaptive and innate immune responses [42][43][44]. Similar to our results, previous research by Tung et al demonstrated the influence of EVs on dendritic cell activity and mobility through the study of dendritic cell interaction with exosomes derived from regulatory T cells (Tregs).…”

Metabolic biomarkers in lung cancer screening and early diagnosis (Review)

“…Therefore, it would be of benefit to identify differentiating metabolites in tissues through untargeted metabolomics before testing them in the biofluids through targeted metabolomics. Several studies have recently highlighted the role of extracellular vesicles (EVs) and their cargo (protein and RNAs) in lung cancer diagnosis (82)(83)(84)(85), proposing EVs to be another potential source of cancer biomarkers. Therefore, combined metabolomics approaches for EVs phenotyping would provide vital insights into the characteristics of EVs in cancer and potentially identify novel strategies for the early detection of lung cancer.…”

Extracellular Vesicles in Lung Cancer: Implementation in Diagnosis and Therapeutic Perspectives

“…Recent studies have explored the role of circRNAs derived from the lung and carried by EVs [ 100 ], and most of them are focused on their expression and role in lung cancer [ 101 ] ( Table 2 ).…”

“…A wider analysis of EVs’ composition can support fast stratification and early detection. In this regard, a substantial analysis of EV circRNA signatures can identify lung-cancer-regulated miRNA [ 100 , 102 ]. Furthermore, a proteomic analysis of EV content offers the opportunity to acquire more information about EV biology and identify new biomarkers, contributing to early diagnosis and the design of valid treatments [ 180 ] ( Figure 2 ).…”