EV-Ident: Identifying Tumor-Specific Extracellular Vesicles by Size Fractionation and Single-Vesicle Analysis.

Tumor-derived extracellular vesicles (EVs) have emerged as a promising source of circulating biomarkers for liquid biopsies. However, understanding the heterogeneous physical and biochemical properties of EVs originating from multiple complex biogenesis pathways remains a major challenge. Here, we introduce EV-Ident for preparation of subpopulations of EVs in three different size fractions: large EVs (EV200 nm; 200-1 000 nm), medium EVs (EV100 nm; 100-200 nm), and small EVs (EV20 nm; 20-100 nm). Furthermore, this technology enables the in situ labeling of fluorescence markers for the protein profiling of individual EVs. As a proof-of-concept, we analyzed the presence of human epidermal growth factor receptor 2 (HER2) and prostate-specific membrane antigen (PSMA) in breast cancer and prostate cancer cell-derived EVs, respectively, using three different size fractions at the single-EV level. By reducing the complexity of EV heterogeneity in each size fraction, we found that HER2-positive breast cancer cells showed the greatest expression of HER2 in EV20 nm, whereas PSMA expression was the highest in EV200 nm derived from PSMA-expressing prostate cancer cells. This increase in HER2 expression in EV20 nm and PSMA expression in EV200 nm was further confirmed in plasma-derived nanoparticles (PNPs) obtained from breast and prostate cancer patients, respectively. Our study demonstrates that single-EV analysis using EV-Ident provides a practical way to understand EV heterogeneity and to successfully identify potent subpopulation of EVs for breast and prostate cancer, which has promising translational implications for cancer theranostics. Furthermore, these findings have the potential to address fundamental questions surrounding the biology and clinical applications of EVs.

The Impact of Androgen Receptor and Histone Deacetylase 1 Expression on the Prognosis of Ductal Carcinoma In Situ.

PURPOSE: Factors associated with invasive recurrence (REC) of ductal carcinoma in situ (DCIS) are less known. This study was aimed at identifying better biomarkers to predict the prognosis of DCIS. METHODS: RNA extracted from formalin-fixed paraffin-embedded blocks of twenty-four pure DCIS cases was subjected to differential gene expression analysis. The DCIS cases were selected by matching age and estrogen receptor status. Sixteen REC-free and 8 invasive-REC cases with disease-free interval of > 5 years were analyzed. Immunohistochemistry (IHC) staining was used to validate sixty-one independent pure DCIS cases, including invasive-REC (n = 16) and REC-free (n = 45) cases. RESULTS: Eight differentially expressed genes (DEGs) were statistically significant (log 2-fold change [FC] < -1 or > 1 and p < 0.001). Less than ½ fold expression of CUL1, androgen receptor (AR), RPS27A, CTNNB1, MAP3K1, PRKACA, GNG12, MGMT genes was observed in the REC group compared to the no evidence of disease group. AR and histone deacetylase 1 (HDAC1) genes were selected for external validation (AR: log 2-FC - 1.35, p < 0.001, and HDAC1: log 2-FC - 0.774, p < 0.001). External validation showed that the absence of AR and high HDAC1 expression were independent risk factors for invasive REC (hazard ratio [HR], 5.04; 95% confidence interval [CI], 1.24-20.4; p = 0.023 and HR, 3.07; 95% CI, 1.04-9.04; p = 0.042). High nuclear grade 3 was also associated with long-term invasive REC. CONCLUSION: Comparative gene expression analysis of pure DCIS revealed 8 DEGs among recurring cases. External validation with IHC suggested that the absence of AR and overexpression of HDAC1 are associated with a greater risk of long-term invasive REC of pure DCIS.