The genetic source tracking of human urinary exosomes

The genetic origins of nanoscale extracellular vesicles in our body fluids remains unclear. Here, we perform a tracking analysis of urinary exosomes via RNA sequencing, revealing that urine exosomes mostly express tissue-specific genes for the bladder and have close cell-genetic relationships to the endothelial cell, basal cell, monocyte, and dendritic cell. Tracking the differentially expressed genes of cancers and corresponding enrichment analysis show urine exosomes are intensively involved in immune activities, indicating that they may be harnessed as reliable biomarkers of noninvasive liquid biopsy in cancer genomic diagnostics and precision medicine.

Combination of Urine Exosomal mRNAs and lncRNAs as Novel Diagnostic Biomarkers for Bladder Cancer

BackgroundThe recent discovery of miRNAs and lncRNAs in urine exosomes has emerged as promising diagnostic biomarkers for bladder cancer (BCa). However, mRNAs as the direct products of transcription has not been well evaluated in exosomes as biomarkers for BCa diagnosis. The purpose of this study was to identify tumor progression-related mRNAs and lncRNAs in urine exosomes that could be used for detection of BCa.MethodsRNA-sequencing was performed to identify tumor progression-related biomarkers in three matched superficial tumor and deep infiltrating tumor regions of muscle-invasive bladder cancer (MIBC) specimens, differently expressed mRNAs and lncRNAs were validated in TCGA dataset (n = 391) in the discovery stage. Then candidate RNAs were chosen for evaluation in urine exosomes of a training cohort (10 BCa and 10 healthy controls) and a validation cohort (80 BCa and 80 healthy controls) using RT-qPCR. The diagnostic potential of the candidates were evaluated by receiver operating characteristic (ROC) curves.ResultsRNA sequencing revealed 8 mRNAs and 32 lncRNAs that were significantly upregulated in deep infiltrating tumor region. After validation in TCGA database, 10 markedly dysregulated RNAs were selected for further investigation in urine exosomes, of which five (mRNAs: KLHDC7B, CASP14, and PRSS1; lncRNAs: MIR205HG and GAS5) were verified to be significantly dysregulated. The combination of the five RNAs had the highest AUC to disguising the BCa (0.924, 95% CI, 0.875–0.974) or early stage BCa patients (0.910, 95% CI, 0.850 to 0.971) from HCs. The expression levels of these five RNAs were correlated with tumor stage, grade, and hematuria degrees.ConclusionsThese findings highlight the potential of urine exosomal mRNAs and lncRNAs profiling in the early diagnosis and provide new insights into the molecular mechanisms involved in BCa.

Urinary exosomal microRNA profiling in intermediate-risk prostate cancer

MicroRNAs (miRNAs) of urine exosomes have emerged as biomarkers for urological cancers, owing to their high stability. MiRNAs have been linked to factors associated with aggressive prostate cancer such as biochemical recurrence (BCR) and metastasis. In this study, we aimed to identify urinary exosomal miRNAs as prognostic markers associated with BCR in intermediate-risk prostate cancer. We profiled the expression levels of miRNAs via next generation sequencing in urinary exosomes from 21 non-BCR patients and 6 BCR patients of intermediate-risk prostate cancer. A total of 21 urinary exosomal miRNAs were found to be differentially expressed (> twofold) in BCR patients compared to non-BCR patients. For external validation, we validated these results using quantitative reverse transcription PCR in an independent cohort of 28 non-BCR patients and 26 BCR patients. A validation analysis revealed that three miRNAs (miR-26a-5p, miR-532-5p, and miR-99b-3p) were upregulated in exosomes from BCR patients. The univariate and multivariate Cox regression analyses showed that miR-532-5p was an important predictive factor for BCR of intermediate-risk prostate cancer. In conclusion, miR-532-5p in urine exosomes might be a potential biomarker for predicting BCR, which is a poor prognosis in patients with intermediate-risk prostate cancer. Further research is needed on the biological functions and mechanisms of this miRNA.

Biomarker screening for the early detection of prostate cancer using an exosomal-enrichment RNA liquid biopsy test.

e15553 Background: Prostate cancer is one of the most common cancers in men, with approximately 10% of all new cancer cases and ~5% of all cancer deaths in 2019. The standard test for prostate cancer is the Prostate Serum Antigen (PSA) test. The PSA test suffers from low specificity (20-40%) in patients including ‘grey zone’ levels (4-10 ng/mL); moreover, the PSA test fails to identify patients with high-risk cancers. Previously we developed ExoDx Prostate Intelliscore (EPI), a urine-based exosome prostate cancer test optimized to rule out the need for a biopsy (risk stratification for high-grade prostate cancer). This study utilized a next generation exosome-based test that specifically enriches a subtype of prostate cancer exosomes from urine. Early detection of prostate cancer via a non-invasive method is desirable and the identification of patients with high-risk cancer is critical. Here we describe the development of a prostate-specific urinary exosome test for the identification of patients with prostate cancer. Methods: We have developed a prostate-specific enrichment method to isolate exosomes of prostate origin from urine samples. Using an affinity-based method against surface marker proteins found on prostate cells, we were able to selectively enrich for exosomes shed by the prostate gland with demonstrated specificity. Subsequent analysis of exosomal nucleic acids enables a promising panel of gene expression biomarkers capable of distinguishing patients with prostate cancer from healthy individuals. Results: RNA from prostate cancer enriched exosomes was compared to total exosomes from urine. Enrichment of prostate cancer specific exosomes significantly enhanced the RNA signature compared to total urine exosomes. Conclusions: Prostate cancer tests have recently been developed for RNA signatures in urine. Exosomes provide a source of nucleic acids as they are actively shed continuously from living cells as part of their normal life cycle. The urine exosomes can be used for total RNA transcriptome analysis and are therefore a very rich source of biomarkers for prostate cancer that can be tailored to different clinical indications. An affinity-based enrichment for tissue-specific exosomes allow for better resolution of the gene expression profile from the tissue of interest and reduces the RNA targets from non-relevant processes of the bladder and kidneys. The gene signature identified in this ongoing study could potentially provide a non-invasive molecular means for the early diagnosis of prostate cancer.

Phosphoenolpyruvate carboxykinase in urine exosomes reflect impairment in renal gluconeogenesis in early insulin resistance and diabetes

Impaired insulin-induced suppression of renal gluconeogenesis could be a risk for hyperglycemia. Diabetes is associated with elevated renal gluconeogenesis; however, its regulation in early insulin resistance is unclear in humans. A noninvasive marker of renal gluconeogenesis would be helpful. Here, we show that human urine exosomes (uE) contain three gluconeogenic enzymes: phosphoenolpyruvate carboxykinase (PEPCK), fructose 1,6-bisphosphatase, and glucose 6-phosphatase. Their protein levels were positively associated with whole body insulin sensitivity. PEPCK protein in uE exhibited a meal-induced suppression. However, subjects with lower insulin sensitivity had blunted meal-induced suppression. Also, uE from subjects with prediabetes and diabetic rats had higher PEPCK relative to nondiabetic controls. Moreover, uE-PEPCK was higher in drug-naïve subjects with diabetes relative to drug-treated subjects with diabetes. To determine whether increased renal gluconeogenesis is associated with hyperglycemia or PEPCK expression in uE, acidosis was induced in rats by 0.28 M NH4Cl with 0.5% sucrose in drinking water. Control rats were maintained on 0.5% sucrose. At the seventh day posttreatment, gluconeogenic enzyme activity in the kidneys, but not in the liver, was higher in acidotic rats. These rats had elevated PEPCK in their uE and a significant rise in blood glucose relative to controls. The induction of gluconeogenesis in human proximal tubule cells increased PEPCK expression in both human proximal tubules and human proximal tubule-secreted exosomes in the media. Overall, gluconeogenic enzymes are detectable in human uE. Elevated PEPCK and its blunted meal-induced suppression in human urine exosomes are associated with diabetes and early insulin resistance.