Nanosized extracellular vesicles (EVs) exosomes serve as reliable sources of biomarkers. However, identifying cancer biomarkers through exosome multiomic molecular information mapping is challenging due to the heterogeneous populations of exosomes derived from different cell types.
Study: Nanopom-pom-prepared exosomes enable highly specific detection of cancer biomarkers. Image credit: Meletios Verras/Shutterstock.com
In an article published in a journal Communication biology, researchers presented novel three-dimensional (3D) nanographene immunomagnetic particles for the specific capture and release (defined by a marker) of an intact exosome. The designed new 3D structured nanographene particles had the unique morphology of flower pom poms.
In addition, the capture and release of intact exosomes was performed using photo-click chemistry. This approach to isolating exosomes has enabled the identification of cancer biomarkers with increased sensitivity and specificity. Multi-omic exosome analysis to identify cancer biomarkers was performed using tissue fluids obtained from a bladder cancer patient.
Nanographene immunomagnetic particles prepared by exosomes showed distinct in vivo biodistribution, indicating an integral quality with high viability. This technique based on nanographene immunomagnetic particles is a facile approach applicable to various biological fluids. Modification of the developed method facilitates scale-up, enrichment and high-throughput isolation of exosomes.
What are cancer biomarkers?
Despite continuous efforts to develop cancer biomarkers, only a few are clinically approved by the USFDA, including progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2/neu), cancer antigen 125 (CA -125) and prostate specific antigen (PSA).
EVs are an emerging source of biomarkers explored to discover a wide variety of cancer biomarkers to achieve cancer diagnosis, drug targeting and delivery, and immunotherapy. Small exosome-type EVs and their molecular components are associated with various physiological functions and disease pathologies. Exosomes are secreted from tumor cells and are enriched with tumor markers. As a result, their presence increases in the plasma and ascites of patients diagnosed with cancer.
Body fluids contain different types of EVs. Thus, cell-specific homogeneous exosome populations are not achievable. Cell-secreted membrane vesicles (EVs) are heterogeneous in origin, present in multiple subpopulations in a wide size range of 30 to 1000 nanometers. Thus, due to the heterogeneity of vesicles and their overlapping size ranges, the exact cellular origin of exosomes cannot be identified to understand disease pathogenesis.
Existing purification methods used to recover extracellular materials (EM) with vesicular or non-vesicular molecules include ultracentrifugation (UC)-based precipitation and precipitation polymer kits. However, these approaches are not scalable and populations of exosomes originating from different cell types or EV subtypes cannot be distinguished. Therefore, the study of cancer biomarkers from exosomes derived from cancer cells is challenging.
Schematic representation of the production of nano pom poms. b TEM and SEM images showing the unique 3D morphology of nanoscale flower poms compared to commercial immunomagnetic beads. c Immunogold nanoparticle staining TEM image of captured exosomes fully covering the surface of Nano pom-poms. Captured EVs are confirmed with antiCD63 gold nanoparticles. The inset shows captured individual exosomes in the ~100 nm size range with three bound gold nanoparticles (~10 nm). d Nanoparticle tracking analysis of isolated exosomes NanoPoms with a much narrower size distribution compared to UC-isolated EVs. e Nanoparticle tracking analysis of the size of exosomes isolated by NanoPoms (n = 4 independent experiments, mean ± SD) compared to ExoEasy isolation (n = 4 independent experiments, mean ± SD), which showed reproducible and smaller sizes of exosomes from the NanoPoms preparation. f SEM images showing dense exosomes are captured covering the surface of Nano pom-poms and can be completely released via photo-cleavage on demand. Once released, intact exosomes can be harvested for subsequent multi-omics analysis including DNA sequencing, next-generation RNA, western blotting and proteomic analysis, as well as in vivo studies. © He, N., Thippabhotla, S., Zhong, C., Greenberg, Z., Xu, L., Pessetto, Z., Godwin, AK. et al. (2022)
Nanographene exosomes towards specific detection of cancer biomarkers
This study demonstrated a novel approach for the specific capture and release of intact exosomes that utilizes 3D structured nanographene immunomagnetic particles. These nanographene immunomagnetic particles have a flower pom poms-like morphology, and the exosomes capture and release mechanisms were based on photo-click chemistry.
Intact exosomes for this study were isolated from a variety of biological fluids, including human urine and blood, cow’s milk, and cell culture medium. In addition, isolation of exosomes via nanographene immunomagnetic particles facilitated efficient identification of cancer biomarkers with high sensitivity and specificity compared to biomarkers isolated using immunomagnetic beads.
The in vivo biodistribution was tested for exosomes released from nanographene immunomagnetic particles. The results showed distinct patterns of biodistribution with insignificant changes in the surface properties of exosomes, suggesting the potential of immunomagnetic nanographene particles in therapeutic development.
Exosomes derived from tissue fluids (urine and plasma) from bladder cancer were compared with exosomes derived from tumor tissues using next-generation sequencing (NGS) of miRNA, global proteome and somatic DNA mutations to achieve invasive and ultrasensitive bladder diagnosis. cancer.
The results revealed that compared to ultracentrifugation or bead isolation approaches, exosomes isolated from nanographene immunomagnetic particles showed improved sensitivity and specificity to detect urological tumor biomarkers.
The effect of external stimuli such as light release was observed on exosome isolation through a nanographene immunomagnetic particle-based approach in which miRNA profiles were compared in the presence and absence of the light release process. The light release process showed specificity by only releasing captured exosomes. These observations confirmed the quality and integrity of nanographene immunomagnetic particles prepared exosomes as a robust and easy method.
What did the study find?
To summarize, the isolation of exosomes by the nanographene immunomagnetic particle method has been applied to various biological fluids, including human urine and blood, cell culture medium, and cow’s milk. This method of isolation was simple and did not require an additional ultracentrifugation process.
The 3D structure of the nanographene immunomagnetic particles and the controlled capture-release process using specific markers helped prepare homogeneous exosome subpopulations that could enrich for cancer biomarkers.
NGS and droplet digital polymerase chain reaction (ddPCR) demonstrated that DNA isolation from exosomes prepared by nanographene immunomagnetic particles could be enriched for DNA mutations in bladder cancer-related tumors and aid in the identification of cancer biomarkers.
He, N., Thippabhotla, S., Zhong, C., Greenberg, Z., Xu, L., Pessetto, Z., Godwin, AK. et al. (2022) Nano pom-pom-prepared exosomes enable highly specific detection of cancer biomarkers. Communication biology. https://doi.org/10.1038/s42003-022-03598-0
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