Search

Multiple biological factors, including age, sex, and genetics, influence Alzheimer’s disease (AD) risk. Of the 6.2 million Americans living with Alzheimer’s dementia in 2021, 3.8 million are women and 2.4 million are men. The strongest genetic risk factor for sporadic AD is apolipoprotein E-e4 (APOE-e4). Female APOE-e4 carriers develop AD more frequently than age-matched males and have more brain atrophy and memory loss. Consequently, biomarkers that are sensitive to biological risk factors may improve AD diagnostics and may provide insight into underlying mechanistic changes that could drive disease progression. Here, we have assessed the effects of sex and APOE-e4 on the miRNA cargo of cerebrospinal fluid (CSF) extracellular vesicles (EVs) in AD. We used ultrafiltration (UF) combined with size exclusion chromatography (SEC) to enrich CSF EVs (e.g., Flotillin+). CSF EVs were isolated from female and male AD or controls (CTLs) that were either APOE-e3,4 or -e3,3 positive (n = 7/group, 56 total). MiRNA expression levels were quantified using a custom TaqMan™ array that assayed 190 miRNAs previously found in CSF, including 25 miRNAs that we previously validated as candidate AD biomarkers. We identified changes in the EV miRNA cargo that were affected by both AD and sex. In total, four miRNAs (miR-16-5p, -331-3p, -409-3p, and -454-3p) were significantly increased in AD vs. CTL, independent of sex and APOE-e4 status. Pathway analysis of the predicted gene targets of these four miRNAs with identified pathways was highly relevant to neurodegeneration (e.g., senescence and autophagy). There were also three miRNAs (miR-146b-5p, -150-5p, and -342-3p) that were significantly increased in females vs. males, independent of disease state and APOE-e4 status. We then performed a statistical analysis to assess the effect of APOE genotype in AD within each sex and found that APOE-e4 status affects different subsets of CSF EV miRNAs in females vs. males. Together, this study demonstrates the complexity of the biological factors associated with AD risk and the impact on EV miRNAs, which may contribute to AD pathophysiology.

Complexity in mechanisms that drive cancer development and progression is exemplified by the transforming growth factor β (TGF-β) signaling pathway, which suppresses early-stage hyperplasia, yet assists aggressive tumors to achieve metastasis. Of note, several molecules, including mRNAs, non-coding RNAs, and proteins known to be associated with the TGF-β pathway have been reported as constituents in the cargo of extracellular vesicles (EVs). EVs are secreted vesicles delimited by a lipid bilayer and play critical functions in intercellular communication, including regulation of the tumor microenvironment and cancer development. Thus, this review aims at summarizing the impact of EVs on TGF-β signaling by focusing on mechanisms by which EV cargo can influence tumorigenesis, metastatic spread, immune evasion and response to anti-cancer treatment. Moreover, we emphasize the potential of TGF-β-related molecules present in circulating EVs as useful biomarkers of prognosis, diagnosis, and prediction of response to treatment in cancer patients.

In non-small cell lung cancer (NSCLC), identifying the presence of sensitizing and resistance epidermal growth factor receptor (EGFR) mutations dictates treatment plans. Extracellular vesicles (EVs) are emerging as abundant, stable potential liquid biopsy targets that offer the potential to quantify EGFR mutations in NSCLC patients at the RNA and protein level at multiple points through treatment. In this study, we present a systematic approach for serial mutation profiling of 34 EV samples from 10 metastatic NSCLC patients with known EGFR mutations through treatment. Using western blot and droplet digital PCR (ddPCR), sensitizing (exon 19 deletion, L858R) mutations were detected in EV-Protein, and both sensitizing and resistance (T790M) mutations were quantified in EV-RNA. EGFR mutations were detected in EV-Protein from four patients at multiple time points through treatment. Using EV-RNA, tumor biopsy matched sensitizing mutations were detected in 90% of patients and resistance mutations in 100% of patients. Finally, mutation burden in EV-RNA at each time point was compared to disease status, described as either stable or progressing. For 6/7 patients who were longitudinally monitored through treatment, EV mutation burden mirrored clinical trajectory. When comparing mutation detection between EV-RNA and ctDNA using ddPCR, EVs had a better detection rate for exon 19 deletions and the L858R point mutation. In conclusion, this study demonstrates that integrating EV analysis into liquid biopsy mutation screening has the potential to advance beyond the current standard of care “rule in” test. The multi-analyte testing allows future integration of EGFR mutation monitoring with additional EV-markers for a comprehensive patient monitoring biomarker.

Multiple biological factors, including age, sex, and genetics, influence Alzheimer's disease (AD) risk. Of the 6.2 million Americans living with Alzheimer's dementia in 2021, 3.8 million are women and 2.4 million are men. The strongest genetic risk factor for sporadic AD is apolipoprotein E-e4 (APOE-e4). Female APOE-e4 carriers develop AD more frequently than age-matched males and have more brain atrophy and memory loss. Consequently, biomarkers that are sensitive to biological risk factors may improve AD diagnostics and may provide insight into underlying mechanistic changes that could drive disease progression. Here, we have assessed the effects of sex and APOE-e4 on the miRNA cargo of cerebrospinal fluid (CSF) extracellular vesicles (EVs) in AD. We used ultrafiltration (UF) combined with size exclusion chromatography (SEC) to enrich CSF EVs (e.g., Flotillin+). CSF EVs were isolated from female and male AD or controls (CTLs) that were either APOE-e3,4 or -e3,3 positive (n = 7/group, 56 total). MiRNA expression levels were quantified using a custom TaqMan™ array that assayed 190 miRNAs previously found in CSF, including 25 miRNAs that we previously validated as candidate AD biomarkers. We identified changes in the EV miRNA cargo that were affected by both AD and sex. In total, four miRNAs (miR-16-5p, -331-3p, -409-3p, and -454-3p) were significantly increased in AD vs. CTL, independent of sex and APOE-e4 status. Pathway analysis of the predicted gene targets of these four miRNAs with identified pathways was highly relevant to neurodegeneration (e.g., senescence and autophagy). There were also three miRNAs (miR-146b-5p, -150-5p, and -342-3p) that were significantly increased in females vs. males, independent of disease state and APOE-e4 status. We then performed a statistical analysis to assess the effect of APOE genotype in AD within each sex and found that APOE-e4 status affects different subsets of CSF EV miRNAs in females vs. males. Together, this study demonstrates the complexity of the biological factors associated with AD risk and the impact on EV miRNAs, which may contribute to AD pathophysiology., 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 Sandau, McFarland, Smith, Galasko, Quinn and Saugstad.)

Complexity in mechanisms that drive cancer development and progression is exemplified by the transforming growth factor β (TGF-β) signaling pathway, which suppresses early-stage hyperplasia, yet assists aggressive tumors to achieve metastasis. Of note, several molecules, including mRNAs, non-coding RNAs, and proteins known to be associated with the TGF-β pathway have been reported as constituents in the cargo of extracellular vesicles (EVs). EVs are secreted vesicles delimited by a lipid bilayer and play critical functions in intercellular communication, including regulation of the tumor microenvironment and cancer development. Thus, this review aims at summarizing the impact of EVs on TGF-β signaling by focusing on mechanisms by which EV cargo can influence tumorigenesis, metastatic spread, immune evasion and response to anti-cancer treatment. Moreover, we emphasize the potential of TGF-β-related molecules present in circulating EVs as useful biomarkers of prognosis, diagnosis, and prediction of response to treatment in cancer patients., 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 Rodrigues-Junior, Tsirigoti, Lim, Heldin and Moustakas.)

In non-small cell lung cancer (NSCLC), identifying the presence of sensitizing and resistance epidermal growth factor receptor (EGFR) mutations dictates treatment plans. Extracellular vesicles (EVs) are emerging as abundant, stable potential liquid biopsy targets that offer the potential to quantify EGFR mutations in NSCLC patients at the RNA and protein level at multiple points through treatment. In this study, we present a systematic approach for serial mutation profiling of 34 EV samples from 10 metastatic NSCLC patients with known EGFR mutations through treatment. Using western blot and droplet digital PCR (ddPCR), sensitizing (exon 19 deletion, L858R) mutations were detected in EV-Protein, and both sensitizing and resistance (T790M) mutations were quantified in EV-RNA. EGFR mutations were detected in EV-Protein from four patients at multiple time points through treatment. Using EV-RNA, tumor biopsy matched sensitizing mutations were detected in 90% of patients and resistance mutations in 100% of patients. Finally, mutation burden in EV-RNA at each time point was compared to disease status, described as either stable or progressing. For 6/7 patients who were longitudinally monitored through treatment, EV mutation burden mirrored clinical trajectory. When comparing mutation detection between EV-RNA and ctDNA using ddPCR, EVs had a better detection rate for exon 19 deletions and the L858R point mutation. In conclusion, this study demonstrates that integrating EV analysis into liquid biopsy mutation screening has the potential to advance beyond the current standard of care "rule in" test. The multi-analyte testing allows future integration of EGFR mutation monitoring with additional EV-markers for a comprehensive patient monitoring biomarker., 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 © 2021 Purcell, Owen, Prantzalos, Radomski, Carman, Lo, Zeinali, Subramanian, Ramnath and Nagrath.)