Introduction: 5-aminolevulinic acid (5-ALA) is a well-known agent metabolized by GBM into the fluorophore protoporphyrin IX (PPIX) for fluorescence-guided surgery. A prospective, Phase II clinical trial was undertaken to define if patients with GBM tumors, who were administered 5-ALA, could shed circulating serum microparticles containing PPIX-derived fluorophore as a novel tool to endogenously label, track, and quantify tumor-derived microparticles.
Methods: Serum samples from GBM patients (n=31) undergoing surgery were collected prior to, and at different time points up to 48 hours following oral dosing with 5-ALA (20mg/kg). Microparticles were isolated by gel filtration and characterized using Nanoparticle Tracking Analysis (NTA) and BCA for microparticle size/number and protein content. Endogenous fluorescence from the microparticles was assessed using NTA in the fluorescence detection mode (lex = 405 nm, lem > 430 nm). Western blot analysis for canonical protein and CPOX converting enzyme (forming PPIX) were utilized.
Results: Microparticles (mode diameter of 50-100 nm) expressing Tenascin-C, CD63, and CD9 are present at a concentration of ~ 10^11 particles/mL of serum (protein content = 283.5 + 47 microgram/ml of sera). Multiple microparticle phenotypes based upon size (~ 20 nm to ~ 200 nm) were observed under fluorescence mode implying capture of the cytosolic fluorophore during biogenesis of at least two major populations of shed microparticles (Fig. 1). Microparticles from GBM patients administered 5-ALA contain a fluorescent species unique to PPIX that is observed in a small 5x10^8 (~ 0.1%) fraction of the total number of microparticles after dosing suggesting that cellular cytosol may circulate with shed microparticles.
Conclusions: Upon oral administration, 5-ALA is taken by tumor cells, enzymatically modified to PPIX, visualized during GBM resection, and shed back into the blood as circulating microparticles within hours of dosing. This direct measure of tumor function affords potential diagnostic opportunities for the early detection of tumor recurrence using a “liquid biopsy” procedure.
Patient Care: Isolation and detection of microparticles shed from GBM tumor cells into the blood may permit better treatment monitoring of patients with earlier detection of tumor recurrence and differentiation from pseudoprogression.
Learning Objectives: 1. Understand the use of 5-ALA for targeting of GBM tumors in patients.
2. Understand the isolation of PPIX-loaded microparticles in the serum shed from GBM tumors.
2. Understand the role of microparticles shed from GBM tumors in the serum as a method for treatment monitoring of patients.