RNA Content Research Articles

Effect of breast tissue density on cell-free orphan non-coding RNAs secreted by breast cancers.

3065 Background: Early detection of breast cancer is crucial for improved patient outcomes but cannot always be achieved through mammography for the more than 40% of women with dense breast tissue (BI-RADS C & D). Breast density masks the appearance of tumors, reducing mammographic sensitivity, which can result in false negatives and delayed diagnosis. Orphan non-coding RNA (oncRNAs) are a novel category of small RNAs that are present in tumors and largely absent in healthy tissue. We have recently demonstrated that oncRNAs secreted from breast cancer cells into the bloodstream enable early detection of breast cancer with high sensitivity and specificity. Here, we examine whether these oncRNAs are unaffected by breast tissue density, making them an effective blood-based biomarker in women with dense breast tissue. Methods: We prospectively collected serum samples and demographic/baseline characteristics from women being screened for breast cancer and analyzed samples from patients with documented breast cancer. We first assayed the cell-free small RNA content of every sample at an average depth of 50 million 50-bp single-end reads. Reads were then annotated using our proprietary RNA database to quantify oncRNA burden (expressed as the count of oncRNA reads per million mapped, unique reads). A previously developed oncRNA-based AI model for breast cancer detection was applied to calculate an oncRNA score for every sample. We compared both the oncRNA burden and the oncRNA score between breast density groups of fatty breast tissue (BI-RADS A & B) vs. dense breast tissue (BI-RADS C & D) using the Mann-Whitney (MW) U test and the Student’s t-test. Results: Of 68 women with breast cancer, 25 women had fatty breast tissue and 43 had dense breast tissue. Cohort characteristics were similar between women with fatty and dense breast tissue (cohort mean age: 58.9±11.6yr, BMI: 30.11±7.57, 74% Caucasian/White, 18% Hispanic, and 9% Black. In the overall cohort, all cancer stages were represented (I: n=36, II: n=18, III: n=10, IV: n=4), T-stage (T1: n=36, T2: n=23, T3: n=4, T4: n=5). The proportion of early stage (I/II) patients is comparable between women in the two breast density groups: fatty breast tissue, (21/25 = 84%) and dense breast tissue (33/43 = 77%). We did not observe a statistically significant difference in oncRNA burden (mean 56,452 counts per million or CPM vs 59,429 CPM in fatty vs. dense tissue, MW p=0.52; t=-0.59, p=0.56) nor in oncRNA score (mean 0.496 vs 0.554 in fatty vs. dense tissue, MW p=0.42; t=-0.79, p=0.43) between breast density groups. Conclusions: Taken together, our results indicate that unlike mammograms, the use of oncRNAs for detecting breast cancer are not influenced by breast density. Further research will utilize oncRNA score in determining breast cancer early detection in women with dense breast tissue.

Distinctive characteristics of extracellular vesicles from feline adipose and placenta stromal cells unveil potential for regenerative medicine in cats.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) are promising avenues in regenerative medicine, offering unique immunomodulatory and regenerative properties with lower immunogenicity. This study delves into the distinctive features of EVs extracted from feline adipose-derived MSCs (ASCs) and placenta-derived MSCs (PMSCs). The tissues were collected from 11 female cats aged between 4 and 7 years old. EVs extracted from MSCs from discarded fetal membranes from 7 female cats and SC adipose tissue from 11 cats. We comprehensively explored morphological characteristics, mitochondrial density, surface markers, and pro- and anti-inflammatory mediators, uncovering notable differences between ASCs and PMSCs. Morphologically, ASCs exhibit a spindle-shaped form in contrast to the spherical morphology of PMSCs. Proliferation and clonogenic potential assessments reveal the faster proliferation and robust clonogenic nature of ASCs, suggesting their potential vital role in regenerative processes. Surface marker expression analysis indicates a significantly higher expression of multipotency-associated markers in ASCs, suggesting their superior proregenerative potential. Phenotyping of EVs demonstrates distinctive features, with CD9 expression suggesting varied EV secretion patterns. Notably, PMSCs exhibit superior CD81 expression, indicating their potential as preferred donors of mitochondria. Pro- and anti-inflammatory mediators analyzed at mRNA and microRNA levels reveal higher RNA content in EVs compared to source cells, emphasizing the potential of EVs in directing regenerative processes. Differential microRNA expression in EVs derived from ASCs hints at their regulatory roles in anti-inflammatory and immunometabolic processes. This study lays a foundation for understanding the nuances between ASCs and PMSCs, which is crucial for harnessing the full therapeutic potential of MSCs and their EVs in tissue repair and regeneration.

Deep proteome profiling of reticulated platelets in patients with chronic coronary syndrome using mass cytometry

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Else Körner Fresenius Stiftung (EKFS) Background Immature or reticulated platelets (RPs), a subgroup of larger and RNA-rich platelets, which are newly released by megakaryocytes from the bone marrow into blood, have gained rising interest because of the further decryption of RPs’ prothrombotic biology and the potential as biomarkers for predicting adverse events in cardiovascular disease. Purpose The biological features of RPs in patients with chronic coronary syndrome (CCS) are largely unknown. With this project, we aimed to perform the first high resolution proteomic characterisation of reticulated platelets using mass cytometry(CyTOF). Method Two samples of thrombocytes from peripheral blood (non-stimulated, TRAP-stimulated) were prepared from each patient with a custom-made CyTOF-antibody panel of 20 antibodies targeting essential transmembrane proteins(anti-CD9, anti-CD29, anti-CD31, anti-CD36-, anti-CD40, anti-CD41, anti-CD42a, anti-CD42b-, anti-CD47, anti-CD61, anti-CD62P-, anti-CD63, anti-CD69, anti-CD107a, anti-CD154, anti-GPVI, anti-GPIIbIIa complex, anti-Par1, anti-PEAR-1 and the negative control anti-CD3 coupled with different metal isotopes). RPs and mature platelets (MPs) were detected based on their RNA content. Earth mover’s distance (EMD) was assessed as a measure of differential expression. Results CyTOF detected an upregulation of important transmembrane receptors in RPs compared to MPs in quiescent platelets (Fig.1A and B). The collagen receptor GPVI (EMD 33.95), the collagen integrin receptor unit ITGB1 (EMD 32.37), the adhesion protein CD9 (EMD 31.78) and the von Willebrand receptor unit CD42b (EMD 30.08) were upregulated in RPs which all serve as crucial regulators of platelet activation and aggregation. RPs show higher median signal intensities of typical activation markers compared to MPs upon TRAP stimulation (Fig.1C). Conclusion We provide the first proteomic characterization of RPs’ pro-thrombotic profile offering the first biomolecular explanation of their hyperactivity and detrimental role in CCS patients.

Unraveling the transcriptomic landscape of platelets: RPs vs. MPs in CCS patients

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): Else Kröner Fresenius Stiftung Background Reticulated platelets (RPs), characterized by youth, hyper-reactivity, and RNA abundance, signify pro-thrombotic potential and serve as predictors for inadequate antiplatelet therapy response post-myocardial infarction. Their pivotal role in chronic coronary syndrome (CCS) and high on-treatment platelet reactivity highlights their significance as promising biomarkers for adverse cardiovascular events in diverse pathological settings. Purpose We aimed to compare for the first time the transcriptomic profiles of RPs and MPs in CCS patients. Methods Using fluorescent activated cell sorting (FACS), RPs and MPs were isolated based on RNA content from peripheral blood of 19 CCS patients. RNA assessment involved the Tapestation 4200 platform (Agilent), and totalRNA libraries were sequenced on a NextSeq 500 Illumina platform. RNA sequencing analysis, with a cut-off of p <0.005 and a log2fc >1, and alternative splicing event detection using MAJIQ, were performed. Circular RNA (circRNA) analysis employed CIRCexplorer. Results Total RNA-sequencing identified 1589 differentially expressed genes, with 1100 transcripts upregulated in RPs and 489 enriched in MPs (Figure 1 A, B and D). Notably, collagen receptor GP6 (log2FC 1.12, p=6.89x10-41), thrombin receptor PAR4 (F2RL3, log2FC 1.1, p=3.54*10-21) and Von Willebrand Factor (log2FC 1.2, p=1.26*10-38) transcripts were significantly enriched in RPs. Gene Ontology identified a higher enrichment of relevant biological categories in RPs compared to MPs, such as "platelet activation", "platelet degranulation" and "blood coagulation" (Figure 1C). These findings are consistent with our previously established discovery and support the hypothesis that RPs in cardiovascular disease are hyper-reactive and pro-thrombotic due to their different RNA content compared to other platelets. Several splicing events were detected as differentially regulated: there is an upregulation of an alternative splicing on the collagen receptor transcript GP6 in RPs (Figure 1E); by the transcript of the G-protein GNAQ, one alternative splicing event was found upregulated in RPs, whilst two others were upregulated in MPs. Moreover, we found an enrichment of the total level of circular RNAs in MPs as compared to RPs (Figure 1F). Several previously undescribed circular RNAs were discovered to be enriched in RPs, as shown in Figure 1F. Conclusion This groundbreaking transcriptomic profiling of RPs and MPs in CCS patients elucidates the biological basis for RPs' hyperreactivity, emphasizing the differential enrichment of platelet activation transcripts. The pronounced upregulation of prothrombotic signaling in RPs provides insight into their hyperactivity and correlation with cardiovascular events. These findings illuminate a novel therapeutic niche in CCS patients, although further investigations are essential to comprehend the detrimental role of RPs in coronary artery disease.

Abstract PO2-13-08: Cell-free orphan noncoding RNAs and AI enable early detection of invasive breast cancer and ductal carcinoma in-situ

Abstract Background Earlier detection of breast cancer through mammography screening has reduced disease-specific mortality; however, confounding issues such as technical challenges, breast density, and tumor size can result in false negatives and ultimately later stage diagnosis. Next generation liquid biopsy has the potential to complement mammography and enable earlier detection for more women. We have previously demonstrated high sensitivity and specificity for early detection of invasive breast cancer (IBC) by utilizing a novel category of cancer-associated small RNAs, termed orphan noncoding RNAs (oncRNAs), through a liquid biopsy platform. Here, we further improve the ability to detect breast cancer in a larger, multi-source cohort through an AI-driven approach and demonstrate potential for detection of ductal carcinoma in-situ (DCIS). Methods We utilized The Cancer Genome Atlas (TCGA) small RNA-seq database to discover a library of 20,538 oncRNAs, through a female-specific analysis, that were significantly enriched among 1,103 breast tumors compared to 349 normal tissue samples spanning multiple tissue sites. The diagnostic performance of these oncRNAs were assessed in an independent cohort of archived serum samples from 663 female individuals, sourced from Indivumed (Hamburg, Germany), Proteogenex (Inglewood, CA), and MT Group (Los Angeles, CA), including 279 breast cancer patients of various stages (221 IBC and 58 DCIS; mean age: 57.0 ± 13.8 years; ever-smoker: 25.8%) and 304 age-matched controls (mean age: 58.5 ± 13.9 years; ever-smoker: 23.4%) without breast cancer. All samples were collected between 2010–2022 at time of diagnosis for breast cancer patients. We sequenced the small RNA content of these samples at an average depth of 25.28 ± 9.37 million 50-bp single-end reads. We detected 18,025 (87.8%) unique breast cancer-specific oncRNA species within at least one sample from the study cohort. We then trained a generative AI model using 5-fold cross-validation to predict cancer status for all samples. Results Our oncRNA-based model achieved an overall AUC of 0.95 (95% CI, 0.93–0.97) for prediction of IBC versus cancer-free controls with a sensitivity of 0.87 (0.82–0.91) at 90% specificity. We observed high sensitivities, also at 90% specificity, across all tumor stages and tumor sizes (Table 1). Sensitivities for the earliest stage and smallest tumor size were 0.87 (0.78–0.93) and 0.81 (0.61–0.93) for Stage I (n=83) and T1a–b ( >1mm to ≤10mm; n=26), respectively. Additionally, in a small single-source cohort, we also saw high model accuracy and sensitivity for DCIS, which we aim to confirm in additional cohorts. While our overall cancer cohort primarily consisted of individuals with luminal breast cancer, our model had high sensitivities across all breast cancer subtypes at 0.90 (0.84–0.94), 0.73 (0.59–0.85), and 0.86 (0.42–1.0) for luminal (n=181), HER2 positive (n=49), and triple negative (n=7), respectively. Conclusions We further demonstrate the potential utility of oncRNAs as a blood-based biomarker using an AI algorithm for sensitive and accurate early detection of breast cancer in a large cohort. Additionally, we have shown that this oncRNA-based assay performs well in detecting small, early-stage invasive breast tumors, with potential to detect precursors of breast cancer. Table 1: Model sensitivity in breast cancer by tumor stage and size For each tumor stage and size, as defined by the AJCC 7th Edition breast cancer staging system, sensitivity and 95% Pearson-Clopper confidence intervals (CI) are reported at 90% specificity for the number of samples (N). Citation Format: Noura Tbeileh, Taylor Cavazos, Mehran Karimzadeh, Jeffrey Wang, Alice Huang, Dung Ngoc Lam, Seda Kilinc, Jieyang Wang, Xuan Zhao, Andy Pohl, Helen Li, Lisa Fish, Kimberly Chau, Marra Francis, Lee Schwartzberg, Patrick Arensdorf, Hani Goodarzi, Fereydoun Hormozdiari, Babak Alipanahi. Cell-free orphan noncoding RNAs and AI enable early detection of invasive breast cancer and ductal carcinoma in-situ [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-13-08.

Myotube growth is associated with cancer-like metabolic reprogramming and is limited by phosphoglycerate dehydrogenase and pyruvate kinase M2

Skeletal myofiber size and oxidative metabolism are inversely related. Glycolytic fast type myofibers are larger than high high oxidative, slow type myofibers. Paradoxically, despite their lower capacity for protein synthesis (lower myonucluar density and RNA content), glycolytic, low oxidative are more responsive to an anabolic stimulus. We hypothesized that glycolytic myofibers may make use of a Warburg-like metabolic reprogramming in response to an anabolic stimulus. The Warburg effect links growth and glycolysis in tumor cells to generate glycolytic intermediates for synthesis of nucleotides and amino. The aim of this study was to test whether a similar ‘glycolysis-for-anabolism’ metabolic reprogramming also occurs in post-mitotic, hypertrophying myofibers. For this purpose, we first induced C2C12 myotube hypertrophy with IGF-1. We then added 14C glucose to the differentiation medium and measured radioactivity in isolated protein and RNA to establish whether 14C had entered anabolism. We found that especially protein became radioactive, suggesting a glucose → glycolytic intermediates → non-essential amino acid(s) → protein series of reactions, the rate of which was increased by IGF-1. Next, to investigate the importance of glycolytic flux and non-essential amino acid synthesis for myotube hypertrophy, we exposed C2C12 and primary mouse myotubes to the glycolysis inhibitor 2-Deoxy-D-glucose (2DG). We found that inhibiting glycolysis lowered C2C12 and primary myotube size. Similarly, siRNA silencing of PHGDH and PKM2, key enzymes of the Warburg-like metabolic reprogramming, reduced C2C12 and primary myotube growth; whereas retroviral PHGDH overexpression increased C2C12 myotube size. Together these results suggest that glycolysis is important for hypertrophying myotubes, which reprogramme their metabolism to facilitate anabolism, similar to cancer cells. Brendan M. Gabriel was supported by fellowships from the Novo Nordisk Foundation (NNF19OC0055072) & the Wenner-Gren Foundation, an Albert Renold Travel Fellowship from the European Foundation for the Study of Diabetes, and an Eric Reid Fund for Methodology from the Biochemical Society. Abdalla D. Mohamed was funded initially by Sarcoma UK (grant number SUK09.2015), then supported by funding from Postdoctoral Fellowship Program (Helmholtz Zentrum München, Germany), and currently by Cancer Research UK. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Skeletal muscle preservation in arctic ground squirrels during hibernation season

Reduced skeletal loading leads to muscle atrophy in humans and most mammals. By contrast, hibernating mammals demonstrate limited loss of skeletal muscle mass and strength by the end of winter after being physically inactive for several months. The present study objective was to establish potential underlying processes for the lack of muscle loss during the hibernation season of arctic ground squirrels (AGS). Our hypothesis was that early in the the hibernation season, protective mechanisms of AGS are not yet mobilized and muscles show signs of atrophy, while compensatory processes for preventing muscle loss appear at a later time point. Quadriceps muscles of juvenile male AGS were collected shortly before hibernation, and on 2, 6, 10-12 and 16-22 weeks during hibernation. Pre-hibernating animals were used as controls. Fiber cross-sectional area (CSA) and fiber type composition were determined with immunohistochemistry. Amount of total RNA, ribosomal RNA content and expression levels of genes involved in 2 pathways of protein degradation, ubiquitination and autophagy, were analyzed by real-time PCR, to evaluate molecular mechanisms of muscle maintenance. One-Way ANOVA was used to statistically analyze the group differences. We found that CSA was not different between the groups (P > 0.05). CSA frequency distribution curves did not reveal any shift to a smaller or larger size range. No difference was detected in myofiber composition between the hibernation groups compared to the control. Total RNA and ribosomal RNA content were not significantly different between the groups during hibernation. Muscle atrophy marker FBXO32 (Atrogin-1, MAFBX) and autophagy related genes MAP1LC3A and BECN1 did not show different level of expression between the groups. Only another muscle atrophy marker TRIM63 (MURF-1, Iris Ring Finger Protein) was significantly over-expressed at the time point of 2 weeks of hibernation. These results indicate that throughout the hibernation season, AGS preserve muscle fiber CSA showing limited signs of muscle atrophy only during the first weeks of hibernation, and yet-to-be determined processes exist to suppress protein degradation in AGS muscles during hibernation. The work was supported by Center of Biomedical Research Excellence under grant number [P20GM130443]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Impact of chronic muscle use and disuse on innate immune signaling in skeletal muscle

Mitochondria exhibit high levels of adaptability to various stimuli such as chronic muscle use (i.e. exercise) or disuse, such as immobilization or denervation. These changes in the mitochondrial network ultimately affect the health and functioning of skeletal muscle, which contributes to whole-body metabolism, locomotion, and postural stability. It is now acknowledged that mitochondrial dysfunction can activate inflammatory pathways through the recognition of damage-associated molecular patterns (DAMPs). The objective of this study was to examine how changes in mitochondria brought about by either chronic muscle inactivity or chronic exercise training affect innate immune system activation. We hypothesized that an inverse relationship between mitochondrial content and innate immune signaling would be evident in skeletal muscle. To investigate this, skeletal muscle from the hindlimbs of mice was collected after 7 days of sciatic muscle denervation, or after 4.5 weeks of endurance swim training involving 240 minutes of daily exercise. We analyzed the expression of the NLRP3 inflammasome, innate immune system signaling proteins, mitochondrial markers, and mitochondrial DNA (mtDNA) content using western blotting, cytochrome C oxidase (COX) activity, ELISA and qPCR. Following 7 days of denervation, there was a significant decrease in both COX-IV protein and COX activity, indicating a reduction in mitochondrial content. This was accompanied by an increase (p<0.05) in NLRP3, procaspase-1, gasdermin-D (GSDMD) and its active N-terminal fragment, GSDMD-N, as well as a notable increase in IL-1β. In addition, other possible converging pathways were also upregulated including cGAS-STING, and gasdermin-E. In comparison, following exercise training, there was a significant increase in mitochondrial content, apparent through increases in COX-IV protein, COX activity and mitochondrial RNA content. This was associated with decreases in procaspase-1 and caspase-1 protein expression alongside reduced STING activation. These findings highlight an inverse relationship between mitochondrial content and the activation of several inflammatory pathways that possibly converge upon NLRP3 inflammasome activation and pyroptotic cell death, leading to detriments in skeletal muscle health. This work aims to further the understanding of innate immune signaling pathways within muscle which can potentially highlight therapeutic targets to regulate its activation under divergent metabolic conditions. This work was supported by funds from the Natural Science and Engineering Research Council (NSERC). Priyanka Khemraj is the holder of an Ontario Graduate Scholarship. David A. Hood is the holder of a Canadian Research Chair in Cell Physiology. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Utility of bronchoscopically obtained frozen cytology pellets for next-generation sequencing

BackgroundNext-generation sequencing (NGS) is essential for lung cancer treatment. It is important to collect sufficient tissue specimens, but sometimes we cannot obtain large enough samples for NGS analysis. We investigated the yield of NGS analysis by frozen cytology pellets using an Oncomine Comprehensive Assay or Oncomine Precision Assay.MethodsWe retrospectively enrolled patients with lung cancer who underwent bronchoscopy at Kobe University Hospital and were enrolled in the Lung Cancer Genomic Screening Project for Individualized Medicine. We investigated the amount of extracted DNA and RNA and determined the NGS success rates. We also compared the amount of DNA and RNA by bronchoscopy methods. To create the frozen cytology pellets, we first effectively collected the cells and then quickly centrifuged and cryopreserved them.ResultsA total of 132 patients were enrolled in this study between May 2016 and December 2022; of them, 75 were subjected to frozen cytology pellet examinations and 57 were subjected to frozen tissue examinations. The amount of DNA and RNA obtained by frozen cytology pellets was nearly equivalent to frozen tissues. Frozen cytology pellets collected by endobronchial ultrasound-guided transbronchial needle aspiration yielded significantly more DNA than those collected by transbronchial biopsy methods. (P < 0.01) In RNA content, cytology pellets were not inferior to frozen tissue. The success rate of NGS analysis with frozen cytology pellet specimens was comparable to the success rate of NGS analysis with frozen tissue specimens.ConclusionsOur study showed that frozen cytology pellets may have equivalent diagnostic value to frozen tissue for NGS analyses. Bronchial cytology specimens are usually used only for cytology, but NGS analysis is possible if enough cells are collected to create pellet specimens. In particular, the frozen cytology pellets obtained by endobronchial ultrasound-guided transbronchial needle aspiration yielded sufficient amounts of DNA.Trial registrationThis was registered with the University Medical Hospital Information Network in Japan (UMINCTR registration no. UMIN000052050).

Modulating the mesenchymal stromal cell microenvironment alters exosome RNA content and ligament healing capacity.

Although mesenchymal stromal cell (MSC) based therapies hold promise in regenerative medicine, their clinical application remains challenging due to issues such as immunocompatibility. MSC-derived exosomes are a promising off-the-shelf therapy for promoting wound healing in a cell-free manner. However, the potential to customize the content of MSC-exosomes, and understanding how such modifications influence exosome effects on tissue regeneration remain underexplored. In this study, we used an in vitro system to compare the priming of human MSCs by 2 inflammatory inducers TNF-α and CRX-527 (a highly potent synthetic TLR4 agonist that can be used as a vaccine adjuvant or to induce anti-tumor immunity) on exosome molecular cargo, as well as on an in vivo rat ligament injury model to validate exosome potency. Different microenvironmental stimuli used to prime MSCs in vitro affected their exosomal microRNAs and mRNAs, influencing ligament healing. Exosomes derived from untreated MSCs significantly enhance the mechanical properties of healing ligaments, in contrast to those obtained from MSCs primed with inflammation-inducers, which not only fail to provide any improvement but also potentially deteriorate the mechanical properties. Additionally, a link was identified between altered exosomal microRNA levels and expression changes in microRNA targets in ligaments. These findings elucidate the nuanced interplay between MSCs, their exosomes, and tissue regeneration.

Investigation of long non-coding RNAs in extracellular vesicles from low-volume blood serum specimens of colorectal cancer patients.

Colorectal cancer (CRC) is the second most prevalent cancer type worldwide, which highlights the urgent need for non-invasive biomarkers for its early detection and improved prognosis. We aimed to investigate the patterns of long non-coding RNAs (lncRNAs) in small extracellular vesicles (sEVs) collected from low-volume blood serum specimens of CRC patients, focusing on their potential as diagnostic biomarkers. Our research comprised two phases: an initial exploratory phase involving RNA sequencing of sEVs from 76 CRC patients and 29 healthy controls, and a subsequent validation phase with a larger cohort of 159 CRC patients and 138 healthy controls. Techniques such as dynamic light scattering, transmission electron microscopy, and Western blotting were utilized for sEV characterization. Optimized protocol for sEV purification, RNA isolation and preamplification was applied to successfully sequence the RNA content of sEVs and validate the results by RT-qPCR. We successfully isolated sEVs from blood serum and prepared sequencing libraries from a low amount of RNA. High-throughput sequencing identified differential levels of 460 transcripts between CRC patients and healthy controls, including mRNAs, lncRNAs, and pseudogenes, with approximately 20% being lncRNAs, highlighting several tumor-specific lncRNAs that have not been associated with CRC development and progression. The validation phase confirmed the upregulation of three lncRNAs (NALT1, AL096828, and LINC01637) in blood serum of CRC patients. This study not only identified lncRNA profiles in a population of sEVs from low-volume blood serum specimens of CRC patients but also highlights the value of innovative techniques in biomolecular research, particularly for the detection and analysis of low-abundance biomolecules in clinical samples. The identification of specific lncRNAs associated with CRC provides a foundation for future research into their functional roles in cancer development and potential clinical applications.

Principles and Limitations of miRNA Purification and Analysis in Whole Blood Collected during Ablation Procedure from Patients with Atrial Fibrillation.

Background: MicroRNA (miRNA) have the potential to be non-invasive and attractive biomarkers for a vast number of diseases and clinical conditions; however, a reliable analysis of miRNA expression in blood samples meets a number of methodological challenges. In this report, we presented and discussed, specifically, the principles and limitations of miRNA purification and analysis in blood plasma samples collected from the left atrium during an ablation procedure on patients with atrial fibrillation (AF). Materials and Methods: Consecutive patients hospitalized in the First Department of Cardiology for pulmonary vein ablation were included in this study (11 with diagnosed paroxysmal AF, 14 with persistent AF, and 5 without AF hospitalized for left-sided WPW ablation-control group). Whole blood samples were collected from the left atrium after transseptal puncture during the ablation procedure of AF patients. Analysis of the set of miRNA molecules was performed in blood plasma samples using the MIHS-113ZF-12 kit and miScript microRNA PCR Array Human Cardiovascular Disease. Results: The miRNS concentrations were in the following ranges: paroxysmal AF: 7-23.1 ng/µL; persistent AF: 4.9-66.8 ng/µL; controls: 6.3-10.6 ng/µL. The low A260/280 ratio indicated the protein contamination and the low A260/A230 absorbance ratio suggested the contamination by hydrocarbons. Spectrophotometric measurements also indicated low concentration of nucleic acids (<10 ng/µL). Further steps of analysis revealed that the concentration of cDNA after the Real-Time PCR (using the PAXgene RNA Blood kit) reaction was higher (148.8 ng/µL vs. 68.4 ng/µL) and the obtained absorbance ratios (A260/A280 = 2.24 and A260/A230 = 2.23) indicated adequate RNA purity. Conclusions: Although developments in miRNA sequencing and isolation technology have improved, detection of plasma-based miRNA, low RNA content, and sequencing bias introduced during library preparation remain challenging in patients with AF. The measurement of the quantity and quality of the RNA obtained is crucial for the interpretation of an efficient RNA isolation.

Abstract 5031: Identification of predictive/prognostic biomarkers in metastatic castration-resistant prostate cancer patients using plasma cell-free total RNA-sequencing

Abstract Background Metastatic castration-resistant prostate cancer (mCRPC) is a heterogeneous disease that is still lacking an optimal patient selection strategy and a validated sequence of therapies. Therefore, there is an urgent need to identify non-invasive biomarkers to guide treatment choice in a perspective of personalized medicine. Plasma cell-free RNA profiling has emerged as a tool to investigate potential liquid biopsy biomarkers. In this study, we aimed to evaluate a method to capture total RNA content deriving from plasma and to identify potential biomarkers for mCRPC. Methods Plasma samples of 41 mCRPC patients treated with first line Abiraterone or Enzalutamide and 7 healthy donors were collected and stored from 2015 to 2022. Plasma cell-free RNA was purified using miRNeasy Serum/Plasma Advanced Kit (Qiagen) with DNase treatment. Bioanalyzer (Agilent) was used for the evaluation of RNA content and integrity profile of the samples. Sequencing libraries were generated using SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara Bio). An average of 80 millions reads pairs were sequenced on Novaseq 6000 (Illumina) using 100 bpx2 approach. After demultiplexing, reads were trimmed and aligned on hg19. Sequencing metrics were evaluated using Picard. Kallisto was used for gene expression analysis. Results A median of 10x109 bases per sample were aligned on hg19, of which a mean of 45% were located on coding or UTR regions. Storage time was investigated as a potential variable for RNA content and sequencing metrics, but no difference related to the year of storage was found. Unsupervised principal component analysis confirmed the absence of batch effects (including storage and sequencing analysis). PC1 seemed to reflect the different tumor RNA content of each sample, in which healthy donors and cancer patients were distinguished. Higher androgen receptor (AR) gene expression was found in mCRPC patients compared to healthy donors. Conclusions Despite enormous interest in extracellular nucleic acids, RNA-sequencing methods for total RNA content outside cells are rare and not standardized. In this study, we found a method to identify plasma non-invasive RNA biomarkers in mCRPC samples, retrieving coding and non-coding RNAs. Case series enlargement and differential gene expression analyses are ongoing. Citation Format: Giorgia Gurioli, Milena Urbini, Alessandra Virga, Chiara Casadei, Emilio Francesco Giunta, Nicole Brighi, Michela Cortesi, Michele Zanoni, Sara Bravaccini, Giovanni Martinelli, Paola Ulivi, Ugo De Giorgi. Identification of predictive/prognostic biomarkers in metastatic castration-resistant prostate cancer patients using plasma cell-free total RNA-sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5031.

Abstract 2747: Chaperonin-containing TCP-1 promotes the release of exosomes from prostate and breast cancer cells

Abstract Coping with cancer recurrence and assessing metastatic risk are challenges for cancer patients that could be addressed with an effective diagnostic predictor. Exosomes, a subpopulation of extracellular vesicles released by tumor cells, can promote distal metastatic site development and could have promising diagnostic applications. However, the mechanisms controlling exosome secretion by cancer cells remain to be determined, which impedes clinical development. The actions of protein-folding chaperones could facilitate tumor exosome formation, payload loading, and extracellular release. Protein folding complexes link the expression of tumor-promoting oncogenes with the proteins driving oncogenic cancer behavior. Of these, the eukaryotic type II chaperonin, Chaperonin-Containing TCP-1 (CCT), folds many of the oncoproteins (e.g., KRAS, MYC, CDKs, STAT3, etc.) responsible for cancer growth and invasion. CCT is a complex machine composed of eight subunits, each encoded by a unique gene (cct1-8), which folds proteins in an ATP-dependent fashion. We and others showed that CCT is upregulated in breast, lung, and prostate cancer cells as well as neuroblastoma, leading to alterations in apoptosis, migration, cell morphology, and proliferation. Using T47D breast cancer and PC3 and 22rV1 prostate cancer cells, we exogenously expressed or depleted the second subunit of the CCT complex (CCT2). With these cells, we isolated exosomes from culture media by membrane-based affinity binding and examined exosomal RNA and protein content. In exosomes from prostate and breast cancer cells, we found abundant CCT2 subunit mRNA and protein in CD63+ exosomes ranging from 50 to 120 microns in size. When CCT2 was exogenously expressed in breast and prostate cancer cells, CCT2 mRNA and protein were higher in exosomes than other CCT subunits, and CCT2 was randomly distributed throughout the size range of exosomes. Of interest, an increase in CD63+ small exosomes (50-75 microns) was evident in cancer cells exogenously expressing CCT2 compared to mock-transfected and parental controls, suggesting a role for CCT2 in the exosomal secretory pathway. FACS-sorted CD63+ exosomes subjected to mass spectrometry yielded distinct protein content based on CCT2 expression. These findings are the first report of CCT2 driving exosome secretion in cancer cells and suggest that exosomes containing CCT2 RNA and protein are associated with metastatic potential. Exosomal CCT2 thus represents a promising diagnostic indicator for a liquid biopsy approach to monitor cancer recurrence and metastasis. Citation Format: Annette R. Khaled, Lam Truong, Colten Frank, Carolyn Dang, James Velazquez, Priya Gopalan, Sally Litherland. Chaperonin-containing TCP-1 promotes the release of exosomes from prostate and breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2747.

Abstract 3678: Beyond detection: AI-based classification of breast cancer invasiveness using cell-free orphan non-coding RNAs

Abstract Background: Approximately 1 in 8 women will be impacted by breast cancer in their lifetimes. Earlier detection of breast cancer through screening has improved survival. Liquid biopsies have the potential to complement existing screening methods by enabling earlier detection and differentiating invasive cancer (IBC) from ductal carcinoma in-situ (DCIS). We previously demonstrated high sensitivity and specificity for early detection of IBC by using a blood-based liquid biopsy platform to analyze a novel category of cancer-associated small RNAs, termed orphan RNAs (oncRNAs). Here, we developed a test that could not only detect the presence of cancer, but also classify the invasiveness of breast cancer. Methods: We utilized The Cancer Genome Atlas (TCGA) small RNA profiles to discover a library of 20,538 oncRNAs that were significantly enriched among 1,103 breast tumors compared to 349 controls from normal tissues spanning multiple tissue sites, limited to female samples. The diagnostic performance of these oncRNAs was assessed in an independent cohort of serum samples from 708 women, including 380 breast cancer patients (221 IBC and 159 DCIS; mean age: 58.0 ± 13.4 years) and 328 age-matched controls (mean age: 58.4 ± 13.7 years). We sequenced the small RNA content from 1 ml of serum from these patients at an average depth of 21.6 million 50-bp single-end reads. We detected 19,736 (96%) of the breast cancer-specific oncRNA library within at least one sample in this cohort. We then trained a multi-class generative AI model using 5-fold cross-validation to predict IBC, DCIS, and absence of breast cancer (IBC or DCIS). Results: Our oncRNA-based generative AI model achieved an overall AUC of 0.95 (95% CI: 0.94-0.97) for prediction of breast cancer versus cancer-free controls. At 90% specificity, overall model sensitivity is 90.0% (86.5%-92.8%). For DCIS and stage I IBC, the model has a sensitivity of 88.1% (82.0%-92.7%) and 90.4% (81.9%-95.8%) respectively, both at 90% specificity. In the second step, restricting to samples flagged as cancer, we observed an overall AUC of 0.9 (0.87-0.93) and sensitivity of 62.4% (55.3%-69.1%) at 90% specificity for discriminating against invasive breast cancer. Conclusions: We have demonstrated the potential utility of oncRNAs as the foundation for a liquid biopsy platform for sensitive and accurate early detection of breast cancer. Our liquid biopsy assay has the potential to complement standard of care by not only detecting breast cancer but also differentiating IBC from DCIS. Citation Format: Mehran Karimzadeh, Taylor B. Cavazos, Nae-Chyun Chen, Noura K. Tbeileh, David Siegel, Amir Momen-Roknabadi, Jennifer Yen, Jeremy Ku, Selina Chen, Diana Corti, Alice Huang, Dang Nguyen, Rose Hanna, Ti Lam, Seda Kilinc, Philip Murzynowski, Jieyang Wang, Xuan Zhao, Andy Pohl, Babak Behsaz, Helen Li, Lisa Fish, Kim H. Chau, Marra S. Francis, Laura J. Van't Veer, Laura J. Esserman, Patrick A. Arensdorf, Hani Goodarzi, Fereydoun Hormozdiari, Babak Alipanahi. Beyond detection: AI-based classification of breast cancer invasiveness using cell-free orphan non-coding RNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3678.

Effects of Eribulin on the RNA Content of Extracellular Vesicles Released by Metastatic Breast Cancer Cells.

Extracellular vesicles (EVs) are small lipid particles secreted by almost all human cells into the extracellular space. They perform the essential function of cell-to-cell communication, and their role in promoting breast cancer progression has been well demonstrated. It is known that EVs released by triple-negative and highly aggressive MDA-MB-231 breast cancer cells treated with paclitaxel, a microtubule-targeting agent (MTA), promoted chemoresistance in EV-recipient cells. Here, we studied the RNA content of EVs produced by the same MDA-MB-231 breast cancer cells treated with another MTA, eribulin mesylate. In particular, we analyzed the expression of different RNA species, including mRNAs, lncRNAs, miRNAs, snoRNAs, piRNAs and tRNA fragments by RNA-seq. Then, we performed differential expression analysis, weighted gene co-expression network analysis (WGCNA), functional enrichment analysis, and miRNA-target identification. Our findings demonstrate the possible involvement of EVs from eribulin-treated cells in the spread of chemoresistance, prompting the design of strategies that selectively target tumor EVs.

Transcriptomic Signatures of Neuronally Derived Extracellular Vesicles Reveal the Presence of Olfactory Receptors in Clinical Samples from Traumatic Brain Injury Patients.

Traumatic brain injury (TBI) is defined as an injury to the brain by external forces which can lead to cellular damage and the disruption of normal central nervous system functions. The recently approved blood-based biomarkers GFAP and UCH-L1 can only detect injuries which are detectable on CT, and are not sensitive enough to diagnose milder injuries or concussion. Exosomes are small microvesicles which are released from the cell as a part of extracellular communication in normal as well as diseased states. The objective of this study was to identify the messenger RNA content of the exosomes released by injured neurons to identify new potential blood-based biomarkers for TBI. Human severe traumatic brain injury samples were used for this study. RNA was isolated from neuronal exosomes and total transcriptomic sequencing was performed. RNA sequencing data from neuronal exosomes isolated from serum showed mRNA transcripts of several neuronal genes. In particular, mRNAs of several olfactory receptor genes were present at elevated concentrations in the neuronal exosomes. Some of these genes were OR10A6, OR14A2, OR6F1, OR1B1, and OR1L1. RNA sequencing data from exosomes isolated from CSF showed a similar elevation of these olfactory receptors. We further validated the expression of these samples in serum samples of mild TBI patients, and a similar up-regulation of these olfactory receptors was observed. The data from these experiments suggest that damage to the neurons in the olfactory neuroepithelium as well as in the brain following a TBI may cause the release of mRNA from these receptors in the exosomes. Hence, olfactory receptors can be further explored as biomarkers for the diagnosis of TBI.

Exosomal small RNA profiling in first-trimester maternal blood explores early molecular pathways of preterm preeclampsia.

Preeclampsia (PE) is a severe obstetrical syndrome characterized by new-onset hypertension and proteinuria and it is often associated with fetal intrauterine growth restriction (IUGR). PE leads to long-term health complications, so early diagnosis would be crucial for timely prevention. There are multiple etiologies and subtypes of PE, and this heterogeneity has hindered accurate identification in the presymptomatic phase. Recent investigations have pointed to the potential role of small regulatory RNAs in PE, and these species, which travel in extracellular vesicles (EVs) in the circulation, have raised the possibility of non-invasive diagnostics. The aim of this study was to investigate the behavior of exosomal regulatory small RNAs in the most severe subtype of PE with IUGR. We isolated exosomal EVs from first-trimester peripheral blood plasma samples of women who later developed preterm PE with IUGR (n=6) and gestational age-matched healthy controls (n=14). The small RNA content of EVs and their differential expression were determined by next-generation sequencing and further validated by quantitative real-time PCR. We also applied the rigorous exceRpt bioinformatics pipeline for small RNA identification, followed by target verification and Gene Ontology analysis. Overall, >2700 small RNAs were identified in all samples and, of interest, the majority belonged to the RNA interference (RNAi) pathways. Among the RNAi species, 16 differentially expressed microRNAs were up-regulated in PE, whereas up-regulated and down-regulated members were equally found among the six identified Piwi-associated RNAs. Gene ontology analysis of the predicted small RNA targets showed enrichment of genes in pathways related to immune processes involved in decidualization, placentation and embryonic development, indicating that dysregulation of the induced small RNAs is connected to the impairment of immune pathways in preeclampsia development. Finally, the subsequent validation experiments revealed that the hsa_piR_016658 piRNA is a promising biomarker candidate for preterm PE associated with IUGR. Our rigorously designed study in a homogeneous group of patients unraveled small RNAs in circulating maternal exosomes that act on physiological pathways dysregulated in preterm PE with IUGR. Therefore, our small RNA hits are not only suitable biomarker candidates, but the revealed biological pathways may further inform us about the complex pathology of this severe PE subtype.

Bisphenol A Negatively Impacts Human Sperm MicroRNA and Protein Profiles

AbstractBisphenol A (BPA), a widely prevalent endocrine disruptor, has detrimental effects on human health and male reproduction. Elevated BPA levels have been linked to reduced sperm quality and production, while paternal exposure may result in poor reproductive outcomes and offspring health. For many years, the effects of BPA exposure in human reproduction were mainly attributed to the deregulation of the hypothalamic–pituitary–gonadal (HPG) axis. In the past decades, other mechanisms of action have been proposed, and studies highlighting epigenetic alterations linked to BPA in sperm emerged. Nevertheless, the impact of BPA on human sperm miRNA and protein profiles and its implications for fertilization and post-fertilization events remain unexplored. This study aimed to investigate the association between BPA concentration in seminal plasma and seminal quality and to examine the alterations in human sperm miRNA and protein expression among men with varying BPA levels in seminal plasma. BPA levels were measured in seminal plasma from 102 Portuguese men, and it was detected in 88% of the samples. No correlation was observed between BPA levels and age or seminal parameters. The small RNA content of 15 normozoospermic samples was evaluated using small RNA sequencing, revealing that 15 miRNAs exhibited a correlation with BPA levels. Gene ontology analysis of their target genes indicated their involvement in embryonic development and response to stress. The proteome of 20 normozoospermic human sperm samples, categorized into four groups based on BPA levels, was accessed by liquid chromatography–mass spectrometry. Data are available via ProteomeXchange with the identifier PXD047280. Sixty-two differentially expressed proteins were identified across groups. The biological processes most affected in samples with higher BPA levels were “protein sumoylation” and “cytoplasmic translation”. The altered miRNAs and proteins identified in spermatozoa from men environmentally exposed to BPA represent potential biological markers of exposure to this endocrine disruptor and may help elucidate cases of idiopathic male infertility, fertilization failure, and abnormal embryo development.

Extracellular vesicles produced by irradiated endothelial or Glioblastoma stem cells promote tumor growth and vascularization modulating tumor microenvironment

BackgroundGlioblastoma (GBM) is the most lethal primary brain tumor in adult, characterized by highly aggressive and infiltrative growth. The current therapeutic management of GBM includes surgical resection followed by ionizing radiations and chemotherapy. Complex and dynamic interplay between tumor cells and tumor microenvironment drives the progression and contributes to therapeutic resistance. Extracellular vesicles (EVs) play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.MethodsIn this study, we isolated by ultracentrifugation EVs from GBM stem-like cell (GSC) lines and human microvascular endothelial cells (HMVECs) exposed or not to ionizing irradiation. After counting and characterization, we evaluated the effects of exposure of GSCs to EVs isolated from endothelial cells and vice versa. The RNA content of EVs isolated from GSC lines and HMVECs exposed or not to ionizing irradiation, was analyzed by RNA-Seq. Periostin (POSTN) and Filamin-B (FLNB) emerged in gene set enrichment analysis as the most interesting transcripts enriched after irradiation in endothelial cell-derived EVs and GSC-derived EVs, respectively. POSTN and FLNB expression was modulated and the effects were analyzed by in vitro assays.ResultsWe confirmed that ionizing radiations increased EV secretion by GSCs and normal endothelial cells, affected the contents of and response to cellular secreted EVs. Particularly, GSC-derived EVs decreased radiation-induced senescence and promoted migration in HMVECs whereas, endothelial cell-derived EVs promoted tumorigenic properties and endothelial differentiation of GSCs. RNA-Seq analysis of EV content, identified FLNB and POSTN as transcripts enriched in EVs isolated after irradiation from GSCs and HMVECs, respectively. Assays performed on POSTN overexpressing GSCs confirmed the ability of POSTN to mimic the effects of endothelial cell-derived EVs on GSC migration and clonogenic abilities and transdifferentiation potential. Functional assays performed on HMVECs after silencing of FLNB supported its role as mediator of the effects of GSC-derived EVs on senescence and migration.ConclusionIn this study, we identified POSTN and FLNB as potential mediators of the effects of EVs on GSC and HMVEC behavior confirming that EVs play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.