DNA Gate-Based CRISPR-Cas Exponential Amplification System for Ultrasensitive Small Extracellular Vesicle Detection to Enhance Breast Cancer Diagnosis.

Tumor-derived small extracellular vesicles (tEVs) as potential biomarkers possess abundant surface proteins closely related to parent cells, which are crucial for noninvasive cancer diagnosis. However, tEVs exhibit phenotype heterogeneity and low abundance, posing a significant challenge for multiplex detection with a high sensitivity. Herein, we developed a DNA gate-based exponential amplification CRISPR-Cas (DGEAC) system for accurate and ultrasensitive detection of tEVs, which can greatly improve the accuracy of breast cancer (BC) diagnosis. Based on the coexpression of CD63 and vascular endothelial growth factor (VEGF) on BC-derived tEVs, we developed a dual-aptamer-based AND gate fluorescent probe by proximity hybridization. By integrating the target recognition and trans-cleavage activity of Cas12a, an autocatalysis-driven exponential amplification circuit was developed for ultrasensitive detection of CD63 and VEGF proteins on tEVs, which could avoid false negative signals from single protein or other interfering proteins. We achieved highly sensitive detection of tEVs over a linear range from 1.75 × 103 to 3.5 × 108 particles/mL with a detection limit as low as 1.02 × 103 particles/mL. Furthermore, the DGEAC system can distinguish tEVs from tEVs derived from different BC cell lines, including MDA-MB-231, MCF-7, SKBR3, and MCF-10A. Compared to linear amplification (AUC 90.0%), the DGEAC system effectively differentiates BC in different stages (AUC 98.3%).

Nucleic acid and nanomaterial-assisted signal-amplified strategies in fluorescent analysis of circulating tumor cells and small extracellular vesicles.

As two main types of liquid biopsy markers, both circulating tumor cells (CTCs) and small extracellular vesicles (sEVs) play important roles in the diagnosis and prognosis of cancers. CTCs are malignant cells that detach from the original tumor tissue and enter the circulation of body fluids. sEVs are nanoscale vesicles secreted by normal cells or pathological cells. However, CTCs and sEVs in body fluids are scarce, leading to great difficulties in the accurate analysis of related diseases. For the sensitive detection of CTCs and sEVs in body fluids, various types of nucleic acid and nanomaterial-assisted signal amplification strategies have been developed. In this review, we summarize the recent advances in fluorescent detection of CTCs and sEVs in liquid biopsy based on nucleic acid and nanomaterial-assisted signal amplification strategies. We also discuss their advantages, challenges, and future prospects.

Nickel-smelting fumes increased the expression of HIF-1α through PI3K/ERK pathway in NIH/3T3 cells.

OBJECTIVE: The purpose of this study was to investigate the effects of Nickel (Ni) -smelting fumes on oncogenic proteins in vivo and in vitro. METHODS: Ni fallout beside a Ni smelting furnace in a factory was sampled to study its toxic effect. The effects of Ni-smelting fumes on the regulation of PI3K and ERK signaling pathways and the important downstream hypoxia inducible factor, HIF-1α, were studied both in NIH/3T3 cells and in the lung tissue of rats. NIH/3T3 cell transformation induced by Ni-smelting fumes was also observed. RESULTS: Ni-smelting fumes activated PI3K, p-AKT, p70S6K1, and ERK proteins and increased HIF-1α expression in a time- and dose-dependent manner. However, activation was suppressed when NIH/3T3 cells were pretreated with PI3K/AKT or ERK inhibitors. Ni-smelting fumes caused malignant transformation of NIH/3T3 cells. CONCLUSIONS: Ni-smelting fumes increased the expression of HIF-1α through the PI3K/ERK pathway in NIH/3T3 cells and induced malignant transformation in these cells indicating that Ni-smelting fumes may be a potential carcinogen in mammalian cells.