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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.

Introduction: Extracellular vesicles (EVs) have emerged as a very attractive source of cancer- derived RNA biomarkers for the early detection, prognosis and monitoring of various cancers, including prostate cancer (PC). However, biofluids contain a mixture of EVs released from a variety of tissues and the fraction of total EVs that are derived from PC tissue is not known. Moreover, the optimal biofluid—plasma or urine—that is more suitable for the detection of EV- enclosed RNA biomarkers is not yet clear.Methodology: In the current study, we performed RNA sequencing analysis of plasma and urinary EVs collected before and after radical prostatectomy, and matched tumor and normal prostate tissues of 10 patients with prostate cancer.Results and Discussion: The most abundant RNA biotypes in EVs were miRNA, piRNA, tRNA, lncRNA, rRNA and mRNA. To identify putative cancer-derived RNA biomarkers, we searched for RNAs that were overexpressed in tumor as compared to normal tissues, present in the pre-operation EVs and decreased in the post-operation EVs in each RNA biotype. The levels of 63 mRNAs, 3 lncRNAs, 2 miRNAs and 1 piRNA were significantly increased in the tumors and decreased in the post-operation urinary EVs, thus suggesting that these RNAs mainly originate from PC tissue. No such RNA biomarkers were identified in plasma EVs. This suggests that the fraction of PC-derived EVs in urine is larger than in plasma and allows the detection and tracking of PC-derived RNAs.

Increasing evidence suggests that regular physical exercise not only reduces the risk of cancer but also improves functional capacity, treatment efficacy and disease outcome in cancer patients. At least partially, these effects are mediated by the secretome of the tissues responding to exercise. The secreted molecules can be released in a carrier-free form or enclosed into extracellular vesicles (EVs). Several recent studies have shown that EVs are actively released into circulation during physical exercise. Here, we for the first time investigated the effects of exercise-induced EVs on the progression of cancer in an F344 rat model of metastatic prostate cancer. Although we did not observe a consistent increase in the circulating EV levels, RNA sequencing analysis demonstrated substantial changes in the RNA content of EVs collected before and immediately after forced wheel running exercise as well as differences between EVs from runners at resting state and sedentary rats. The major RNA biotype in EVs was mRNA, followed by miRNA and rRNA. Molecular functions of differentially expressed RNAs reflected various physiological processes including protein folding, metabolism and regulation of immune responses triggered by the exercise in the parental cells. Intravenous administration of exercise-induced EVs into F344 rats with orthotopically injected syngeneic prostate cancer cells PLS10, demonstrated reduction of the primary tumor volume by 35% and possibly—attenuation of lung metastases. Hence, our data provide the first evidence that exercise-induced EVs may modulate tumor physiology and delay the progression of cancer.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Brokāne, Bajo-Santos, Zayakin, Belovs, Jansons, Lietuvietis, Martens-Uzunova, Jenster and Linē.)

Introduction: Extracellular vesicles (EVs) have emerged as a very attractive source of cancer- derived RNA biomarkers for the early detection, prognosis and monitoring of various cancers, including prostate cancer (PC). However, biofluids contain a mixture of EVs released from a variety of tissues and the fraction of total EVs that are derived from PC tissue is not known. Moreover, the optimal biofluid-plasma or urine-that is more suitable for the detection of EV- enclosed RNA biomarkers is not yet clear. Methodology: In the current study, we performed RNA sequencing analysis of plasma and urinary EVs collected before and after radical prostatectomy, and matched tumor and normal prostate tissues of 10 patients with prostate cancer. Results and Discussion: The most abundant RNA biotypes in EVs were miRNA, piRNA, tRNA, lncRNA, rRNA and mRNA. To identify putative cancer-derived RNA biomarkers, we searched for RNAs that were overexpressed in tumor as compared to normal tissues, present in the pre-operation EVs and decreased in the post-operation EVs in each RNA biotype. The levels of 63 mRNAs, 3 lncRNAs, 2 miRNAs and 1 piRNA were significantly increased in the tumors and decreased in the post-operation urinary EVs, thus suggesting that these RNAs mainly originate from PC tissue. No such RNA biomarkers were identified in plasma EVs. This suggests that the fraction of PC-derived EVs in urine is larger than in plasma and allows the detection and tracking of PC-derived RNAs., Competing Interests: IR-S was employed by Genera Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bajo-Santos, Brokāne, Zayakin, Endzeliņš, Soboļevska, Belovs, Jansons, Sperga, Llorente, Radoviča-Spalviņa, Lietuvietis and Linē.)

Increasing evidence suggests that regular physical exercise not only reduces the risk of cancer but also improves functional capacity, treatment efficacy and disease outcome in cancer patients. At least partially, these effects are mediated by the secretome of the tissues responding to exercise. The secreted molecules can be released in a carrier-free form or enclosed into extracellular vesicles (EVs). Several recent studies have shown that EVs are actively released into circulation during physical exercise. Here, we for the first time investigated the effects of exercise-induced EVs on the progression of cancer in an F344 rat model of metastatic prostate cancer. Although we did not observe a consistent increase in the circulating EV levels, RNA sequencing analysis demonstrated substantial changes in the RNA content of EVs collected before and immediately after forced wheel running exercise as well as differences between EVs from runners at resting state and sedentary rats. The major RNA biotype in EVs was mRNA, followed by miRNA and rRNA. Molecular functions of differentially expressed RNAs reflected various physiological processes including protein folding, metabolism and regulation of immune responses triggered by the exercise in the parental cells. Intravenous administration of exercise-induced EVs into F344 rats with orthotopically injected syngeneic prostate cancer cells PLS10, demonstrated reduction of the primary tumor volume by 35% and possibly-attenuation of lung metastases. Hence, our data provide the first evidence that exercise-induced EVs may modulate tumor physiology and delay the progression of cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sadovska, Auders, Keiša, Romanchikova, Silamiķele, Kreišmane, Zayakin, Takahashi, Kalniņa and Linē.)