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.

Strategies for labelling of exogenous and endogenous extracellular vesicles and their application for in vitro and in vivo functional studies.

This review presents a comprehensive overview of labelling strategies for endogenous and exogenous extracellular vesicles, that can be utilised both in vitro and in vivo. It covers a broad spectrum of approaches, including fluorescent and bioluminescent labelling, and provides an analysis of their applications, strengths, and limitations. Furthermore, this article presents techniques that use radioactive tracers and contrast agents with the ability to track EVs both spatially and temporally. Emphasis is also placed on endogenous labelling mechanisms, represented by Cre-lox and CRISPR-Cas systems, which are powerful and flexible tools for real-time EV monitoring or tracking their fate in target cells. By summarizing the latest developments across these diverse labelling techniques, this review provides researchers with a reference to select the most appropriate labelling method for their EV based research.

From fish to cells: Establishment of continuous cell lines from embryos of annual killifish Nothobranchius furzeri and N. kadleci.

There is a growing need of alternative experimental models that avoid or minimize the use of animals due to ethical, economical, and scientific reasons. Surprisingly, the stable embryonic cell lines representing Nothobranchius spp., emerging vertebrate models in aging research, regenerative medicine, ecotoxicology, or genomics, have been not derived so far. This paper reports establishment and deep characterization of ten continuous cell lines from annual killifish embryos of N. furzeri and N. kadleci. The established cell lines exhibited mostly fibroblast- and epithelial-like morphology and steady growth rates with cell doubling time ranging from 27 to 40 h. All cell lines retained very similar characteristics even after continuous subcultivation (more than 100 passages) and extended storage in liquid nitrogen (∼3 years). The cytogenetic analysis of the cell lines revealed a diploid chromosome number mostly equal to 38 elements (i.e., the native chromosome count for both killifish species), with minor but diverse line/passage-specific karyotype changes compared to the patterns observed in non-cultured N. furzeri and N. kadleci somatic cells. Based on transcriptional analysis of marker genes, the cell lines displayed features of an undifferentiated state without signs of senescence even in advanced passages. We confirmed that the cell lines are transfectable and can form viable 3-D spheroids. The applicability of the cell lines for (eco)toxicological surveys was confirmed by assessing the effect of cytotoxic and growth inhibitory agents. Properties of established Nothobranchius embryonic cell lines open new possibilities for the application of this model in various fields of life sciences including molecular mechanisms of aging, karyotype (in)stability or differences in lifespan.

An Independent Validation Study of Candidate microRNAs as Predictive Biomarkers for Bevacizumab-based Therapy in Patients With Metastatic Colorectal Cancer.

BACKGROUND/AIM: The monoclonal antibody bevacizumab is a standard drug used in combination with oxaliplatin (FOLFOX) or irinotecan (FOLFIRI) based chemotherapy in the first or second-line treatment of metastatic colorectal cancer (mCRC). Our previous study identified and subsequently validated 4 microRNAs in a small group of patients as predictors of the therapeutic response to bevacizumab combined with chemotherapy. The aim of this follow-up study is to confirm the predictive ability of these tissue miRNAs in a larger independent cohort of mCRC patients. PATIENTS AND METHODS: The retrospective study included 92 patients with generalized-radically inoperable tumors treated with the combined therapy of bevacizumab/FOLFOX in a standard regimen. RESULTS: Expression levels of candidate miRNA biomarkers (miR-92b-3p, miR-3156-5p, miR-10a-5p and miR-125a-5p) were determined in tumor tissue specimens and statistically evaluated. MiR-92b-3p and miR-125a-5p were confirmed to be associated with radiological response according to RECIST criteria (p=0.005 and 0.05, respectively) and to be up-regulated in responders to bevacizumab/FOLFOX therapy. Higher levels of miR-92b-3p were also significantly associated with extended progression-free survival (p=0.024). CONCLUSION: We have successfully confirmed miR-92b-3p to be up-regulated in tumor tissue of mCRC patients with good response to bevacizumab/FOLFOX therapy.

Histopathology of laboratory-reared Nothobranchius fishes: Mycobacterial infections versus neoplastic lesions.

Short-lived killifishes of the genus Nothobranchius Peters, 1868 (Cyprinodontiformes) are considered promising model organisms for biomedical research on ageing and tumorigenesis. We conducted histopathological analysis of 411 adult individuals from three Nothobranchius species to study details on spontaneous age-related neoplastic lesions. Light microscopy based on H&E and toluidine blue-stained sections revealed (a) non-proliferative liver changes with pronounced vacuolation of hepatocytes; (b) proliferation of kidney haemopoietic tissue contributing to excretory system damage; (c) proliferation of splenic mononuclear haemoblasts accompanied by reduced erythropoiesis; (d) proliferation of mononuclear cell aggregates in the liver parenchyma; and (e) rare occurrence of hepatocellular adenomas. Ziehl-Neelsen (ZN) staining revealed that the proliferative lesions are a host defence response to mycobacterial infections manifested by activation of the mononuclear phagocytic system and atypical granulomatous inflammatory reaction. 16S rRNA analysis identified three species of Mycobacterium in our samples. Our findings turn attention to lesions which mimic neoplasms by their gross appearance and question the light microscopic interpretation of lesions unless differential ZN staining is included. Beyond the limitations of our morphological approach, the intensity of mycobacterial infections is a challenging opportunity for research into the molecular-genetic background of the mononuclear phagocytic system reaction in Nothobranchius killifish.

MiR-215-5p Reduces Liver Metastasis in an Experimental Model of Colorectal Cancer through Regulation of ECM-Receptor Interactions and Focal Adhesion.

Background: Growing evidence suggests that miR-215-5p is a tumor suppressor in colorectal cancer (CRC); however, its role in metastasis remains unclear. This study evaluates the effects of miR-215 overexpression on the metastatic potential of CRC. Methods: CRC cell lines were stably transfected with miR-215-5p and used for in vitro and in vivo functional analyses. Next-generation sequencing and RT-qPCR were performed to study changes on the mRNA level. Results: Overexpression of miR-215-5p significantly reduced the clonogenic potential, migration, and invasiveness of CRC cells in vitro and tumor weight and volume, and liver metastasis in vivo. Transcriptome analysis revealed mRNAs regulated by miR-215-5p and RT-qPCR confirmed results for seven selected genes. Significantly elevated levels of CTNNBIP1 were also observed in patients' primary tumors and liver metastases compared to adjacent tissues, indicating its direct regulation by miR-215-5p. Gene Ontology and KEGG pathway analysis identified cellular processes and pathways associated with miR-215-5p deregulation. Conclusions: MiR-215-5p suppresses the metastatic potential of CRC cells through the regulation of divergent molecular pathways, including extracellular-matrix-receptor interaction and focal adhesion. Although the specific targets of miR-215-5p contributing to the formation of distant metastases must be further elucidated, this miRNA could serve as a promising target for CRC patients' future therapeutic strategies.

Spontaneous adenocarcinoma of the gas gland in Nothobranchius fishes.

Nothobranchius fishes (Cyprinodontiformes), known for their genetically encoded extremely compressed lifespan, are considered an excellent vertebrate model for the research of aging. Unlike the rapid accumulation of data concerning their biology, ecology and genome, knowledge of their age-related diseases, including tumours, is still very limited. This Note reports spontaneous neoplastic lesions in the swim bladder gas glands of Nothobranchius furzeri, N. kadleci and N. orthonotus. Based on light and transmission electron microscopy, the neoplastic proliferation of gas gland cells was classified as adenocarcinoma. There was a concurrent proliferation of haemopoietic cells in the kidney interstitium in all individuals diagnosed with this type of primary neoplasia.