Characterization of plasma-derived protoporphyrin-IX-positive extracellular vesicles following 5-ALA use in patients with malignant glioma

• Published in: EBioMedicine Vol. 48; pp. 23 - 35
• Main Authors: Jones, Pamela S., Yekula, Anudeep, Lansbury, Elizabeth, Small, Julia L., Ayinon, Caroline, Mordecai, Scott, Hochberg, Fred H., Tigges, John, Delcuze, Bethany, Charest, Alain, Ghiran, Ionita, Balaj, Leonora, Carter, Bob S.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-10-2019; Elsevier
• Subjects: 5-ALA; Adult; Aged; Aminolevulinic Acid; Animals; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Extracellular vesicles; Extracellular Vesicles - metabolism; Female; Fluorescence guided surgery; Fluorescent Dyes - administration & dosage; Glioma - diagnosis; Glioma - metabolism; Glioma - surgery; Humans; Imaging flow cytometry; Levulinic Acids - administration & dosage; Liquid biopsy; Magnetic Resonance Imaging; Male; Malignant gliomas; Mice; Mice, Transgenic; Middle Aged; Optical Imaging - methods; Photosensitizing Agents - metabolism; Protoporphyrins - metabolism; Research paper; Surgery, Computer-Assisted; HBMVEC; Imaging flow cytometry; IFC; Extracellular vesicles; Fluorescence guided surgery; PpIX; ISX; GBM; FGS; Liquid biopsy; Malignant gliomas; sEVs; 5-ALA
• ISSN: 2352-3964; 2352-3964
• DOI: 10.1016/j.ebiom.2019.09.025

Malignant gliomas are rapidly progressive brain tumors with high mortality. Fluorescence guided surgery (FGS) with 5-aminolevulinic acid (5-ALA) provides fluorescent delineation of malignant tissue, which helps achieve maximum safe resection. 5-ALA-based fluorescence is due to preferential accumulation of the fluorophore protoporphyrin-IX (PpIX) in malignant glioma tissue. Additionally, gliomas cells release extracellular vesicles (EVs) which carry biomarkers of disease. Herein, we performed animal and human studies to investigate whether 5-ALA dosed glioma cells, in vitro and in vivo, release PpIX positive EVs in circulation which can be captured and analyzed.
We used imaging flow cytometry (IFC) to characterize PpIX-positive EVs released from 5-ALA-dosed glioma cells, glioma-bearing xenograft models, as well as patients with malignant glioma undergoing FGS.
We first show that glioma cells dosed with 5-ALA release 247-fold higher PpIX positive EVs compared to mock dosed glioma cells. Second, we demonstrate that the plasma of glioma-bearing mice (n = 2) dosed with 5-ALA contain significantly higher levels of circulating PpIX-positive EVs than their pre-dosing background (p = 0.004). Lastly, we also show that the plasma of patients with avidly fluorescent tumors (n = 4) undergoing FGS contain circulating PpIX-positive EVs at levels significantly higher than their pre-dosing background (p = 0.00009) and this rise in signal correlates with enhancing tumor volumes (r 2  = 0.888).
Our findings highlight the potential of plasma-derived PpIX-positive EV-based diagnostics for malignant gliomas, offering a novel liquid biopsy platform for confirming and monitoring tumor status.