Abstract
Simple SummaryIn Glioblastoma (GB), a malignant tumor of the central nervous system, diagnosis can currently only be obtained via tissue biopsy. In this study we were able to isolate GB derived extracellular vesicles (EVs) in the blood, after patients received 5-aminolevulinic acid (5-ALA) before surgery. This isolation is based on fluorescence caused by the accumulation of fluorescent protoporphyrin IX (PpIX) in these EVs. We show that these EVs contain various GB-related micro RNAs. While there are many ways in which our technique needs to be improved before being able to be implemented in the clinic, this study shows that detecting and analyzing circulating GB-derived EVs based on PpIX fluorescence is feasible. In the future, our technique could be developed to diagnose and monitor GB via blood samples instead of a brain biopsy.Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.
Highlights
In adult patients, glioblastoma (GB) is the most common primary malignancy of the central nervous system, with a dismal prognosis [1,2]
We show that administration of 5-aminolevulinic acid (5-ALA) induces accumulation of protoporphyrin IX (PpIX) in GB-derived Extracellular vesicles (EVs)
Administration of 5-ALA Leads to Accumulation of Fluorescent PpIX in EVs
Summary
Glioblastoma (GB) is the most common primary malignancy of the central nervous system, with a dismal prognosis [1,2]. Definitive diagnosis still requires tissue, which can only be acquired via (stereotactic) biopsy or neurosurgical resection of the tumor. In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. We assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
