BACKGROUND: Pediatric high-grade glioma (pHGG) is a leading cause of cancer-related deaths in children, with universally poor outcomes. Current standard of care neuronavigation during surgery relies on preoperative MRI images that may not accurately reflect extent of tumor due to brain shift. In 2017, the FDA approved 5-aminolevulinic acid as an optical imaging agent for gliomas surgery in adults. Yet its inadequate tumor specificity resulted in false negatives. To address this, fluorescent antibodies were explored despite long incubation time and high production cost^1-3. However, their delivery across the blood-brain barrier (BBB) was limited due to their size, reducing detection sensitivity⁴. Affinity peptides are small molecules with fast binding kinetics and lower cost than antibodies^5-7. Therefore, we propose an alternative therapeutic approach for fluorescence-guided surgery and targeted therapy of pHGG with an EGFRwt/vIII-specific peptide delivered through BBB modulation.

METHOD: EGFRwt/vIII immunohistochemistry in 66 resected pHGG tissue was correlated with patient demographic and molecular characteristics. Flow cytometry determined in vitro binding affinity and kinetics for EGFRwt/vIII-positive pHGG cells by a near-infrared (NIR) labeled peptide identified through phage display against purified EGFR protein. EGFRwt/vIII-positive pHGG cells were genetically modified to constitutively express green fluorescent protein and luciferin (GFP+/luc+) for tracking and monitoring purposes. A non-targeting peptide with a scrambled sequence served as control. Mice with EGFRwt/vIII-positive orthotopic pHGG patient-derived xenografts received 100 mM targeting or control peptide via tail vein (n = 5). Intravital uptake of NIR peptide by pHGG was observed through a cranial window installed over the pHGG xenograft. Intratumoral delivery following transcranial magnetic resonance-guided focused ultrasound (MRgFUS, 0.25 MPa, 2 min) sonication was confirmed by dynamic contrast-enhanced (DCE) MRI and intratumoral peptide concentration determined by standard curve4. Fluorescent tumor-to-background ratio was evaluated via macroscopic and microscopic near-infrared imaging. Treatment efficacy was characterized both in vivo using Kaplan-Meier survival curves and ex vivo with apoptotic rate by TUNEL assay. The therapeutic significance of EGFRwt versus EGFRvIII was investigated in vitro and in vivo with corresponding knockdown of either biomarker.

RESULTS: EGFRwt/vIII expression detected pHGG with 63% sensitivity and 91% specificity (Figure 1), respectively, and was correlated with older age (³ 10 years), ependymal involvement, and EGFR-amplified IDH-wildtype tumors (Table 1). NIR-labeled EGFR-targeting peptide immunostained EGFRwt/vIII-positive pHGG cells in a dose-dependent manner (IC50 = 5.3 nM) with high affinity (Kd = 6.1 nM, R2 = 0.99) and fast kinetics (1/k = 2.7 hours, R2 = 0.98), Fig. 2. Intravital uptake of NIR-labeled peptide by pHGG xenograft was observed 500 μm beneath the brain surface (Fig. 3), which occurred rapidly within 5 minutes of injection and persisted for at least eight hours. Mice receiving EGFR-targeting peptide weekly for one month following xenograft implantation showed reduced tumor proliferation (37 ± 9% vs. 78 ± 7%) and increased apoptosis rate (4.1 ± 1.3% vs. 0.2 ± 0.1%), compared to those receiving the control peptide (Fig. 4). Following MRgFUS, intratumoral peptide uptake (23 ± 4.7% injected dose) led to 3.1 ± 0.2 tumor contrast (Fig. 5). Median survival was prolonged (29 vs. 17.5 days, P < 0.0001, n = 10) in pHGG-bearing mice receiving MRgFUS-delivered EGFR peptide for eight weeks, Fig. 6. EGFRvIII conferred greater growth advantage (P = 0.041 in vitro, P = 0.008 in vivo) and contributed more to therapeutic efficacy compared to EGFRwt (P = 0.0018), Fig. 7.

CONCLUSION: EGFRwt/vIII are promising pHGG-specific biomarkers whose expression can be stratified from demographic and molecular characteristics. NIR peptide co-targeting EGFRwt/vIII improved tumor contrast and produced antitumor activity, potentially benefiting 68% of pHGG population. Enhanced delivery of peptide probe via MRgFUS may accelerate its safe adoption for fluorescence-guided surgery and targeted treatment of childhood brain tumors.

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Study design and experiment workflow

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