Abstract
Abstract INTRODUCTION The administration of 5-Aminolevunlinic acid (5-ALA) in the context of fluorescence-guided surgery is contingent on the highly selective accumulation of PpIX in tumor cells. PpIX is a precursor in the heme pathway that naturally exhibits fluorescent and phototoxic properties upon excitation. We have previously reported evidence of PpIX fluorescence in tumor-specific extracellular vesicles (EVs). Here, we explore the implications of exogenous 5-ALA on cellular function, EV characteristics, and gene expression. METHODS We have characterized a range of glioblastoma, meningioma, and healthy cell lines (Gli36 EGFRvIII, Gli36 EGFR WT, U87, CH-157, IOMM-Lee, and HBMVEC). At equal confluency, cells were dosed with 0.8 mM 5-ALA or mock dosed. Media was collected and processed to eliminate debris after 24 hours. Analysis of cells and EVs was conducted using Amnis ImageStream (IFC) and Nanoparticle Tracking Analysis. EV subpopulations were analyzed with IFC following antibody staining. RNA was extracted using Qiagen exoRNeasy and RNeasy. Libraries were prepared via Qiagen UPX 3’ Transcriptome kit and sequenced using Illumina MiSeq. Data analysis was carried out via GeneGlobe, CLC workbench, and MATLAB. RESULTS Following 5-ALA dosing, all tumor cells and their derived EVs exhibit significant levels of fluorescence. Analysis of EV subpopulations demonstrated a general decrease in tetraspanin-positive EVs following 5-ALA dosing. At 24 hours, Gli36 cell lines exhibited increased EV release post 5-ALA whereas U87 and meningioma lines resulted in a decreased EV release rate. This clustering is also reflected in EV size distributions and in cellular and EV differential gene expression analysis. Gene ontology analysis of common genes from mock and dosed EVs demonstrated high counts of genes controlling biological regulation as well as cellular and metabolic processes. CONCLUSION Collection and characterization of cancer specific EVs may be advantageous to liquid biopsy development.
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