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.

Centre for Molecular Medicine Central European Institute of Technology Masaryk University Kamenice 5 625 00 Brno Czech Republic

Department of Biology Faculty of Medicine Masaryk University Kamenice 5 625 00 Brno Czech Republic

Department of Comprehensive Cancer Care Faculty of Medicine Masaryk Memorial Cancer Institute Brno Czech Republic

Department of Gastroenterology and Digestive Endoscopy Masaryk Memorial Cancer Institute Brno Czech Republic

Department of Pharmacology and Toxicology Veterinary Research Institute Brno Czech Republic

Department of Surgery Faculty of Medicine University Hospital Brno Bohunice Masaryk University Brno Czech Republic

Department of Surgical Oncology Faculty of Medicine Masaryk Memorial Cancer Institute Brno Czech Republic

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Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–49. PubMed

Siegel RL, Wagle NS, Cercek A, Smith RA, Jemal A. Colorectal cancer statistics, 2023. CA Cancer J Clin. 2023;73:233–54. PubMed

Nguyen LH, Goel A, Chung DC. Pathways of colorectal carcinogenesis. Gastroenterology. 2020;158:291–302. PubMed PMC

Lin JS, Perdue LA, Henrikson NB, Bean SI, Blasi PR. Screening for colorectal cancer: updated evidence report and systematic review for the US preventive services task force. Jama - J Am Med Assoc. 2021;325:1978–97. PubMed

Boukouris S, Mathivanan S. Exosomes in bodily fluids are a highly stable resource of disease biomarkers. Proteomics - Clin Appl. 2015;9:358–67. PubMed PMC

Jeppesen DK, Fenix AM, Franklin JL, Higginbotham JN, Zhang Q, Zimmerman LJ, et al. Reassessment of exosome composition. Cell. 2019;177:428-445.e18. PubMed PMC

Théry C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7:1535750. PubMed PMC

Yáñez-Mó M, Siljander PRM, Andreu Z, Zavec AB, Borràs FE, Buzas EI, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles. 2015;4:1–60. PubMed PMC

Cesi G, Walbrecq G, Margue C, Kreis S. Transferring intercellular signals and traits between cancer cells: extracellular vesicles as “homing pigeons.” Cell Commun Signal. 2016;14:1–12. PubMed PMC

Whiteside TL. Tumor-derived exosomes and their role in cancer progression. Adv Clin Chem. 2016;74:103–41. PubMed PMC

Schulz-Siegmund M, Aigner A. Nucleic acid delivery with extracellular vesicles. Adv Drug Deliv Rev. 2021;173:89–111. PubMed

Fang Y, Fullwood MJ. Roles, functions, and mechanisms of long non-coding RNAs in cancer. Genomics Proteomics Bioinform. 2016;14:42–54. PubMed PMC

Derrien T, Johnson R, Bussotti G, Tanzer A, Djebali S, Tilgner H, et al. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 2012;22:1775–89. PubMed PMC

Yang Y, Yan X, Li X, Ma Y, Goel A. Long non-coding RNAs in colorectal cancer: novel oncogenic mechanisms and promising clinical applications. Cancer Lett. 2021;504:67–80. PubMed PMC

Liu T, Zhang X, Gao S, Jing F, Yang Y, Du L, et al. Exosomal long noncoding RNA CRNDE-h as a novel serum-based biomarker for diagnosis and prognosis of colorectal cancer. Oncotarget. 2016;7:85551–63. PubMed PMC

Vychytilova-Faltejskova P, Vilmanova S, Pifkova L, Catela Ivković T, Madrzyk M, Jugas R, et al. Optimized procedure for high-throughput transcriptome profiling of small extracellular vesicles isolated from low volume serum samples. Clin Chem Lab Med. 2023;62:157–67. PubMed

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3:RESEARCH0034. PubMed PMC

Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004;64:5245–50. PubMed

Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006;30:3221–32229. PubMed

Wang J, Yao Y, Wu J, Li G. Identification and analysis of exosomes secreted from macrophages extracted by different methods. Int J Clin Exp Pathol. 2015;8:6135–42. PubMed PMC

Schwarzmueller L, Bril O, Vermeulen L, Léveillé N. Emerging role and therapeutic potential of lncrnas in colorectal cancer. Cancers (Basel). 2020;12:1–21. PubMed PMC

Liu SJ, Dang HX, Lim DA, Feng FY, Maher CA. Long noncoding RNAs in cancer metastasis. Nat Rev Cancer. 2021;21:446–60. PubMed PMC

Drula R, Pardini B, Fu X, De Los Santos MC, Jurj A, Pang L, et al. 17β-estradiol promotes extracellular vesicle release and selective miRNA loading in ERα-positive breast cancer. Proc Natl Acad Sci U S A. 2023;120:e2122053120. PubMed PMC

Ricklefs FL, Maire CL, Reimer R, Dührsen L, Kolbe K, Holz M, et al. Imaging flow cytometry facilitates multiparametric characterization of extracellular vesicles in malignant brain tumours. J Extracell Vesicles. 2019;8:1588555. PubMed PMC

Tian F, Zhang S, Liu C, Han Z, Liu Y, Deng J, et al. Protein analysis of extracellular vesicles to monitor and predict therapeutic response in metastatic breast cancer. Nat Commun. 2021;12:2536. PubMed PMC

Odaka H, Hiemori K, Shimoda A, Akiyoshi K, Tateno H. CD63-positive extracellular vesicles are potential diagnostic biomarkers of pancreatic ductal adenocarcinoma. BMC Gastroenterol. 2022;22:1–10. PubMed PMC

Wang Y, Wu P, Lin R, Rong L, Xue Y, Fang Y. LncRNA NALT interaction with NOTCH1 promoted cell proliferation in pediatric T cell acute lymphoblastic leukemia. Sci Rep. 2015;5:13749. PubMed PMC

Ye M, Zhao L, Zhang L, Wu S, Li Z, Qin Y, et al. LncRNA NALT1 promotes colorectal cancer progression via targeting PEG10 by sponging microRNA-574-5p. Cell Death Dis. 2022;13:1–14. PubMed PMC

Piao HY, Guo S, Wang Y, Zhang J. Long noncoding RNA NALT1-induced gastric cancer invasion and metastasis via NOTCH signaling pathway. World J Gastroenterol. 2019;25:6508–26. PubMed PMC

Ma X, Mo M, Tan HJJ, Tan C, Zeng X, Zhang G, et al. LINC02499, a novel liver-specific long non-coding RNA with potential diagnostic and prognostic value, inhibits hepatocellular carcinoma cell proliferation, migration, and invasion. Hepatol Res. 2020;50:726–40. PubMed

Zhang D-D, Shi Y, Liu J-B, Yang X-L, Xin R, Wang H-M, et al. Construction of a Myc-associated ceRNA network reveals a prognostic signature in hepatocellular carcinoma. Mol Ther Nucleic Acids. 2021;24:1033–50. PubMed PMC

Chung IH, Lu PH, Lin YH, Tsai MM, Lin YW, Yeh CT, et al. The long non-coding RNA LINC01013 enhances invasion of human anaplastic large-cell lymphoma. Sci Rep. 2017;7:1–10. PubMed PMC

Wang W, Xu S, Di Y, Zhang Z, Li Q, Guo K, et al. Novel role of LINC01013/miR-6795-5p/FMNL3 axis in the regulation of hepatocellular carcinoma stem cell features. Acta Biochim Biophys Sin (Shanghai). 2021;53:652–62. PubMed

Deng Y, Hu H, Xiao L, Cai T, Gao W, Zhu H, et al. Identification of EMT-related lncRNAs as potential prognostic biomarkers and therapeutic targets for pancreatic adenocarcinoma. J Oncol. 2022;2022. PubMed PMC

Huang CS, Ho JY, Chiang JH, Yu CP, Yu DS. Exosome-derived LINC00960 and LINC02470 promote the epithelial-mesenchymal transition and aggressiveness of bladder cancer cells. Cells. 2020;9:1419. PubMed PMC