P15.03.B MOLECULARLY TARGETED PROTEASE-ACTIVATED PROBES FOR THE VISUALIZATION OF GLIOBLASTOMA: A COMPARISON WITH 5-ALA
Authors: Konecna D , Vymola P , Ternerova N , Vymolova B , Garcia-Borja E , Mateu R , Sroubek F , Pankrac J , Widen J , Bogyo M , Netuka D , Sedo A , Busek P 
Published: September 2023

Abstract

BACKGROUND
Surgery is the main treatment option for glioblastoma. However, due to its highly infiltrative growth, it is difficult to distinguish the tumor from normal brain tissue. The aim of this work was to evaluate the ability of recently developed molecularly targeted near-infrared (NIR) protease-activated probes to be specifically activated in brain tumor tissue and to compare the most promising candidate with the FDA approved 5-aminolevulinic acid (5-ALA).

MATERIAL AND METHODS
The cysteine cathepsin-cleavable probes 6QC-ICG and 6QC-Cy5 (single-substrate), the cysteine cathepsin and caspase 3-cleavable dual, AND-GATE substrate probes AG2-FNIR and AG2-Cy5 (double-substrate) were administered intravenously to mice with orthotopic tumors. Activation of the probes was further evaluated in glioma cell lines, patient-derived glioma stem-like cells, M2-polarized macrophages in vitro, and biopsy material from patients with glioblastoma ex vivo. Tumor to normal brain tissue fluorescence ratio (TNR) was quantified in tissue sections using preclinical and clinical visualization platforms. Fluorescence in tissue homogenates and cell suspensions was quantified by spectrofluorimetry.

RESULTS
6QC-ICG was activated in glioma cells and macrophages in vitro and the resulting fluorophore accumulated intracellularly. In experimental glioblastomas, single- and double-substrate probes visualized tumor tissue with a minimum signal generated in healthy brain. TNR was highest for 6QC-ICG and AG2-FNIR. Nevertheless, the signal intensity was higher for 6QC-ICG. Using xenograft and syngeneic mouse models, we confirmed the ability of 6QC-ICG to specifically visualize glioma tissue based on image analysis and spectrofluorimetric measurement. Furthermore, we confirmed that 6QC-ICG was activated in human glioblastoma tissue using ex vivo biopsy material and could be visualized on a clinical visualization platform. Finally, a comparison with 5-ALA in animals co-administered with both compounds revealed a higher TNR for 6QC-ICG in experimental glioblastomas.

CONCLUSION
6QC-ICG is activated by glioma cells and tumor-associated macrophages leading to a high contrast between tumor and non-tumorous brain tissue that is superior to that of the current standard 5-ALA. In addition to their well-defined mechanism of action, the protease-activated probes using NIR fluorophores such as ICG offer the advantage of low absorption and scattering of the NIR light and lower tissue autofluorescence. Our results thus suggest the potential of 6QC-ICG to become a targeted agent for the intraoperative detection of glioblastoma.
Supported by National Institute for Cancer Research (LX22NPO5102), Centre for Tumour Ecology (CZ.02.1.01/0.0/0.0/16_019/0000785), EATRIS-CZ (LM2023053), Cooperatio Program-,,Oncology and Haematology", Ministry of Health of the Czech Republic (NV190300501).