Improved recovery of urinary small extracellular vesicles by differential ultracentrifugation.

Extracellular vesicles (EVs) are lipid-membrane enclosed structures that are associated with several diseases, including those of genitourinary tract. Urine contains EVs derived from urinary tract cells. Owing to its non-invasive collection, urine represents a promising source of biomarkers for genitourinary disorders, including cancer. The most used method for urinary EVs separation is differential ultracentrifugation (UC), but current protocols lead to a significant loss of EVs hampering its efficiency. Moreover, UC protocols are labor-intensive, further limiting clinical application. Herein, we sought to optimize an UC protocol, reducing the time spent and improving small EVs (SEVs) yield. By testing different ultracentrifugation times at 200,000g to pellet SEVs, we found that 48 min and 60 min enabled increased SEVs recovery compared to 25 min. A step for pelleting large EVs (LEVs) was also evaluated and compared with filtering of the urine supernatant. We found that urine supernatant filtering resulted in a 1.7-fold increase on SEVs recovery, whereas washing steps resulted in a 0.5 fold-decrease on SEVs yield. Globally, the optimized UC protocol was shown to be more time efficient, recovering higher numbers of SEVs than Exoquick-TC (EXO). Furthermore, the optimized UC protocol preserved RNA quality and quantity, while reducing SEVs separation time.

Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches.

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Validation of potential RNA biomarkers for prostate cancer diagnosis and monitoring in plasma and urinary extracellular vesicles.

Introduction: Prostate cancer (PCa), one of the most prevalent malignancies affecting men worldwide, presents significant challenges in terms of early detection, risk stratification, and active surveillance. In recent years, liquid biopsies have emerged as a promising non-invasive approach to complement or even replace traditional tissue biopsies. Extracellular vesicles (EVs), nanosized membranous structures released by various cells into body fluids, have gained substantial attention as a source of cancer biomarkers due to their ability to encapsulate and transport a wide range of biological molecules, including RNA. In this study, we aimed to validate 15 potential RNA biomarkers, identified in a previous EV RNA sequencing study, using droplet digital PCR. Methods: The candidate biomarkers were tested in plasma and urinary EVs collected before and after radical prostatectomy from 30 PCa patients and their diagnostic potential was evaluated in a test cohort consisting of 20 benign prostate hyperplasia (BPH) and 20 PCa patients' plasma and urinary EVs. Next, the results were validated in an independent cohort of plasma EVs from 31 PCa and 31 BPH patients. Results: We found that the levels of NKX3-1 (p = 0.0008) in plasma EVs, and tRF-Phe-GAA-3b (p < 0.0001) tRF-Lys-CTT-5c (p < 0.0327), piR-28004 (p = 0.0081) and miR-375-3p (p < 0.0001) in urinary EVs significantly decreased after radical prostatectomy suggesting that the main tissue source of these RNAs is prostate and/or PCa. Two mRNA biomarkers-GLO1 and NKX3-1 showed promising diagnostic potential in distinguishing between PCa and BPH with AUC of 0.68 and 0.82, respectively, in the test cohort and AUC of 0.73 and 0.65, respectively, in the validation cohort, when tested in plasma EVs. Combining these markers in a biomarker model yielded AUC of 0.85 and 0.71 in the test and validation cohorts, respectively. Although the PSA levels in the blood could not distinguish PCa from BPH in our cohort, adding PSA to the mRNA biomarker model increased AUC from 0.71 to 0.76. Conclusion: This study identified two novel EV-enclosed RNA biomarkers-NKX3-1 and GLO1-for the detection of PCa, and highlights the complementary nature of GLO1, NKX3-1 and PSA as combined biomarkers in liquid biopsies of PCa.

Darolutamide added to docetaxel augments antitumor effect in models of prostate cancer through cell cycle arrest at the G1-S transition.

Resistance to taxane chemotherapy is frequently observed in metastatic prostate cancer. The androgen receptor (AR) is a major driver of prostate cancer and a key regulator of the G1-S cell cycle checkpoint, promoting cancer cell proliferation by irreversible passage to the S-phase. We hypothesized that AR signaling inhibitor (ARSi) darolutamide in combination with docetaxel could augment antitumor effect by impeding the proliferation of taxane-resistant cancer cells. We monitored cell viability in organoids, tumor volume and PSA secretion in patient-derived xenografts (PDXs) and analyzed cell cycle and signaling pathway alterations. Combination treatment increased anti-tumor effect in androgen-sensitive, AR-positive prostate cancer organoids and PDXs. Equally beneficial effects of darolutamide added to docetaxel were observed in a castration-resistant model, progressive on docetaxel, enzalutamide and cabazitaxel. In vitro studies showed that docetaxel treatment with simultaneous darolutamide resulted in a reduction of cells entering the S-phase in contrast to only docetaxel. Molecular analysis in the prostate cancer cell line LNCaP revealed an upregulation of Cyclin Dependent Kinase inhibitor p21, supporting blockade of S-phase entry and cell proliferation. Our results provide a preclinical support for combining taxanes and darolutamide as a multimodal treatment strategy in metastatic prostate cancer patients progressive on ARSi and taxane chemotherapy.

Site-specific analysis of ribosomal 2'O-methylation by quantitative reverse transcription PCR under low deoxynucleotide triphosphate concentrations.

Ribose 2'O-methylation (Nm, ribomethylation) is the most abundant RNA modification present in rRNA. It has been shown that alterations in ribosomal 2'O-methylation at individual Nm sites likely reflect regulated cellular processes. Although several analytical approaches for Nm detection and profiling have been developed, a simple and affordable method for the screening and measurement of individual Nm sites in large numbers of tissue samples is required to examine their potential for clinical translation. Here, we describe a new quantitative reverse transcription PCR-based method that can sensitively assess ribomethylation levels at specific rRNA sites at single-nucleotide resolution in low input amounts of total RNA.

CRISPRs in the human genome are differentially expressed between malignant and normal adjacent to tumor tissue.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) have been identified in bacteria, archaea and mitochondria of plants, but not in eukaryotes. Here, we report the discovery of 12,572 putative CRISPRs randomly distributed across the human chromosomes, which we termed hCRISPRs. By using available transcriptome datasets, we demonstrate that hCRISPRs are distinctively expressed as small non-coding RNAs (sncRNAs) in cell lines and human tissues. Moreover, expression patterns thereof enabled us to distinguish normal from malignant tissues. In prostate cancer, we confirmed the differential hCRISPR expression between normal adjacent and malignant primary prostate tissue by RT-qPCR and demonstrate that the SHERLOCK and DETECTR dipstick tools are suitable to detect these sncRNAs. We anticipate that the discovery of CRISPRs in the human genome can be further exploited for diagnostic purposes in cancer and other medical conditions, which certainly will lead to the development of point-of-care tests based on the differential expression of the hCRISPRs.

Extracellular vesicles as a source of prostate cancer biomarkers in liquid biopsies: a decade of research.

Prostate cancer is a global cancer burden and considerable effort has been made through the years to identify biomarkers for the disease. Approximately a decade ago, the potential of analysing extracellular vesicles in liquid biopsies started to be envisaged. This was the beginning of a new exciting area of research investigating the rich molecular treasure found in extracellular vesicles to identify biomarkers for a variety of diseases. Vesicles released from prostate cancer cells and cells of the tumour microenvironment carry molecular information about the disease that can be analysed in several biological fluids. Numerous studies document the interest of researchers in this field of research. However, methodological issues such as the isolation of vesicles have been challenging. Remarkably, novel technologies, including those based on nanotechnology, show promise for the further development and clinical use of extracellular vesicles as liquid biomarkers. Development of biomarkers is a long and complicated process, and there are still not many biomarkers based on extracellular vesicles in clinical use. However, the knowledge acquired during the last decade constitutes a solid basis for the future development of liquid biopsy tests for prostate cancer. These are urgently needed to bring prostate cancer treatment to the next level in precision medicine.

The role of OncoSnoRNAs and Ribosomal RNA 2'-O-methylation in Cancer.

Ribosomes are essential nanomachines responsible for all protein production in cells. Ribosome biogenesis and function are energy costly processes, they are tightly regulated to match cellular needs. In cancer, major pathways that control ribosome biogenesis and function are often deregulated to ensure cell survival and to accommodate the continuous proliferation of tumour cells. Ribosomal RNAs (rRNAs) are abundantly modified with 2'-O-methylation (Nm, ribomethylation) being one of the most common modifications. In eukaryotic ribosomes, ribomethylation is performed by the methyltransferase Fibrillarin guided by box C/D small nucleolar RNAs (snoRNAs). Accumulating evidences indicate that snoRNA expression and ribosome methylation profiles are altered in cancer. Here we review our current knowledge on differential snoRNA expression and rRNA 2'-O methylation in the context of human malignancies, and discuss the consequences and opportunities for cancer diagnostics, prognostics, and therapeutics.

Androgens alter the heterogeneity of small extracellular vesicles and the small RNA cargo in prostate cancer.

Proliferation and survival of prostate cancer cells are driven by the androgen receptor (AR) upon binding to androgen steroid hormones. Manipulating the AR signalling axis is the focus for prostate cancer therapy; thus, it is crucial to understand the role of androgens and AR on extracellular vesicle (EV) secretion and cargo. In this study, we report that plasma-derived circulating vesicles consisting of CD9 and double-positive for CD9 and Prostate Specific Membrane Antigen (PSMA) are increased in patients with advanced metastatic prostate cancer, whereas double positives for CD9 and CD63 small extracellular vesicles (S-EVs) are significantly higher in patients with localised prostate cancer. Androgen manipulation by dihydrotestosterone (DHT) and the clinical antagonist enzalutamide (ENZ) altered the heterogeneity and size of CD9 positive S-EVs in AR expressing prostate cancer cells, while assessment of the total number and protein cargo of total S-EVs was unaltered across different treatment groups. Furthermore, hormone stimulation caused strong and specific effects on the small RNA cargo of S-EVs. A total of 543 small RNAs were found to be regulated by androgens including miR-19-3p and miR-361-5p. Analysis of S-EVs heterogeneity and small RNA cargo may provide clinical utility for prostate cancer and be informative to understand further the mechanism of resistance to androgen targeted therapy in castration-resistant prostate cancer.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

VIRMA-Dependent N6-Methyladenosine Modifications Regulate the Expression of Long Non-Coding RNAs CCAT1 and CCAT2 in Prostate Cancer.

RNA methylation at position N6 in adenosine (m6A) and its associated methyltransferase complex (MTC) are involved in tumorigenesis. We aimed to explore m6A biological function for long non-coding RNAs (lncRNAs) in prostate cancer (PCa) and its clinical significance. m6A and MTC levels in PCa cells were characterized by ELISA and western blot. Putative m6A-regulated lncRNAs were identified and validated by lncRNA profiler qPCR array and bioinformatics analysis, followed by m6A/RNA co-immunoprecipitation. Impact of m6A depletion on RNA stability was assessed by Actinomycin D assay. The association of m6A-levels with PCa prognosis was examined in clinical samples. Higher m6A-levels and VIRMA overexpression were detected in metastatic castration-resistant PCa (mCRPC) cells (p < 0.05). VIRMA knockdown in PC-3 cells significantly decreased m6A-levels (p = 0.0317), attenuated malignant phenotype and suppressed the expression of oncogenic lncRNAs CCAT1 and CCAT2 (p < 0.00001). VIRMA depletion and m6A reduction decreased the stability and abundance of CCAT1/2 transcripts. Higher expression of VIRMA, CCAT1, and CCAT2 as a group variable was an independent predictor of poor prognosis (HR = 9.083, CI95% 1.911-43.183, p = 0.006). VIRMA is a critical factor sustaining m6A-levels in PCa cells. VIRMA downregulation attenuates the aggressive phenotype of PCa by overall reduction of m6A-levels decreasing stability and abundance of oncogenic lncRNAs.

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017.

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.

The Non-Coding Transcriptome of Prostate Cancer: Implications for Clinical Practice.

Prostate cancer (PCa) is the most common type of cancer and the second leading cause of cancer-related death in men. Despite extensive research, the molecular mechanisms underlying PCa initiation and progression remain unclear, and there is increasing need of better biomarkers that can distinguish indolent from aggressive and life-threatening disease. With the advent of advanced genomic technologies in the last decade, it became apparent that the human genome encodes tens of thousands non-protein-coding RNAs (ncRNAs) with yet to be discovered function. It is clear now that the majority of ncRNAs exhibit highly specific expression patterns restricted to certain tissues and organs or developmental stages and that the expression of many ncRNAs is altered in disease and cancer, including cancer of the prostate. Such ncRNAs can serve as important biomarkers for PCa diagnosis, prognosis, or prediction of therapy response. In this review, we give an overview of the different types of ncRNAs and their function, describe ncRNAs relevant for the diagnosis and prognosis of PCa, and present emerging new aspects of ncRNA research that may contribute to the future utilization of ncRNAs as clinically useful therapeutic targets.

A comprehensive repertoire of tRNA-derived fragments in prostate cancer.

Prostate cancer (PCa) is the most common cancer among men in developed countries. Although its genetic background is thoroughly investigated, rather little is known about the role of small non-coding RNAs (sncRNA) in this disease. tRNA-derived fragments (tRFs) represent a new class of sncRNAs, which are present in a broad range of species and have been reported to play a role in several cellular processes. Here, we analyzed the expression of tRFs in fresh frozen patient samples derived from normal adjacent prostate and different stages of PCa by RNA-sequencing. We identified 598 unique tRFs, many of which are deregulated in cancer samples when compared to normal adjacent tissue. Most of the identified tRFs are derived from the 5'- and 3'-ends of mature cytosolic tRNAs, but we also found tRFs produced from other parts of tRNAs, including pre-tRNA trailers and leaders, as well as tRFs from mitochondrial tRNAs. The 5'-derived tRFs comprise the most abundant class of tRFs in general and represent the major class among upregulated tRFs. The 3'-derived tRFs types are dominant among downregulated tRFs in PCa. We validated the expression of three tRFs using qPCR. The ratio of tRFs derived from tRNALysCTT and tRNAPheGAA emerged as a good indicator of progression-free survival and a candidate prognostic marker. This study provides a systematic catalogue of tRFs and their dysregulation in PCa and can serve as the basis for further research on the biomarker potential and functional roles of tRFs in this disease.

C/D-box snoRNA-derived RNA production is associated with malignant transformation and metastatic progression in prostate cancer.

Small nucleolar RNAs (snoRNAs) are dynamically regulated in different tissues and affected in disease. SnoRNAs are processed further to stable smaller RNAs. We sequenced the small RNA transcriptome of prostate cancer (PCa) at different PCa stages and generated a quantified catalogue of 3927 small non-coding RNAs (sncRNAs) detected in normal and malignant prostate tissue. From these, only 1524 are microRNAs. The remaining 2401 sncRNAs represent stable sncRNAs species that originate from snoRNA, tRNA and other sncRNAs. We show that snoRNA-derived RNAs (sdRNAs) display stronger differential expression than microRNAs and are massively upregulated in PCa. SdRNAs account for at least one third of all small RNAs with expression changes in tumor compared to normal adjacent tissue. Multiple sdRNAs can be produced from one snoRNA in a manner related to the conservation of structural snoRNA motifs. Q-PCR analysis in an independent patient cohort (n=106) confirmed the processing patterns of selected snoRNAs (SNORD44, SNORD78, SNORD74 and SNORD81) and the cancer-associated up-regulation of their sdRNAs observed in sequencing data. Importantly, expression of SNORD78 and its sdRNA is significantly higher in a subset of patients that developed metastatic disease demonstrating that snoRNA and sdRNAs may present as novel diagnostic and/or prognostic biomarkers for PCa.

FlaiMapper: computational annotation of small ncRNA-derived fragments using RNA-seq high-throughput data.

MOTIVATION: Recent discoveries show that most types of small non-coding RNAs (sncRNAs) such as miRNAs, snoRNAs and tRNAs get further processed into putatively active smaller RNA species. Their roles, genetic profiles and underlying processing mechanisms are only partially understood. To find their quantities and characteristics, a proper annotation is essential. Here, we present FlaiMapper, a method that extracts and annotates the locations of sncRNA-derived RNAs (sncdRNAs). These sncdRNAs are often detected in sequencing data and observed as fragments of their precursor sncRNA. Using small RNA-seq read alignments, FlaiMapper is able to annotate fragments primarily by peak detection on the start and end position densities followed by filtering and a reconstruction process. RESULTS: To assess performance of FlaiMapper, we used independent publicly available small RNA-seq data. We were able to detect fragments representing putative sncdRNAs from nearly all types of sncRNA, including 97.8% of the annotated miRNAs in miRBase that have supporting reads. Comparison of FlaiMapper-predicted boundaries of miRNAs with miRBase entries demonstrated that 89% of the start and 54% of the end positions are identical. Additional benchmarking showed that FlaiMapper is superior in performance compared with existing software. Further analysis indicated a variety of characteristics in the fragments, including sequence motifs and relations with RNA interacting factors. These characteristics set a good basis for further research on sncdRNAs. AVAILABILITY AND IMPLEMENTATION: The platform independent GPL licensed Python 2.7 code is available at: https://github.com/yhoogstrate/flaimapper.

Identification and diagnostic performance of a small RNA within the PCA3 and BMCC1 gene locus that potentially targets mRNA.

BACKGROUND: PCA3 is a long noncoding RNA (lncRNA) with unknown function, upregulated in prostate cancer. LncRNAs may be processed into smaller active species. We hypothesized this for PCA3. METHODS: We computed feasible RNA hairpins within the BMCC1 gene (encompassing PCA3) and searched a prostate transcriptome for these. We measured expression using qRT-PCR in three cohorts of prostate cancer tissues (n = 60), exfoliated urinary cells (n = 484 with cancer and n = 166 controls), and in cell lines (n = 22). We used in silico predictions and RNA knockup to identify potential mRNA targets of short transcribed RNAs. RESULTS: We predicted 13 hairpins, of which PCA3-shRNA2 was most abundant within the prostate transcriptome. PCA3-shRNA2 is located within intron 1 of PCA3 and appears regulated by androgens. Expression of PCA3-shRNA2 was upregulated in malignant prostatic tissues, exfoliated urinary cells from men with prostate cancer (13-273 fold change; t test P < 0.003), and closely correlated to PCA3 expression (r = 0.84-0.93; P < 0.001). Urinary PCA3-shRNA2 (C-index, 0.75-0.81) and PCA3 (C-index, 0.78) could predict the presence of cancer in most men. PCA3-shRNA2 knockup altered the expression of predicted target mRNAs, including COPS2, SOX11, WDR48, TEAD1, and Noggin. PCA3-shRNA2 expression was negatively correlated with COPS2 in patient samples (r = -0.32; P < 0.001). CONCLUSION: We identified a short RNA within PCA3, whose expression is correlated to PCA3, which may target mRNAs implicated in prostate biology. IMPACT: This short RNA is stable ex vivo, suggesting a role as a robust biomarker. We identify cytoplasmic enrichment of this RNA and potential targeting of mRNAs implicated in prostate carcinogenesis.