Development of Combined PK/PD Studies of RAF/MEK/mTOR Inhibitors for the Treatment of Pediatric Low-Grade Astrocytomas by MALDI Mass Spectrometry Imaging

摘要

Pediatric low-grade astrocytomas (LGAs) acc ount for the majority of pediatric central nervous system neoplasms. A wealth of studies has evidenced disregulations of the MAPK pathway in tumorigenesis of such tumors. This pathway is a conserved signaling cascade involving protein kinases for signal transduction from the ce ll membrane to the nucleus to mediate biological functions such as cell growth, survival, and differentiation. This pathway has therefore become a significant target for the development of drugs for the treatment of pediatric LGAs. MALDI ma ss spectrometry imaging (MALDI MSI) has proven to be an effective method to reveal the two-dimensional spatial distribution of drugs, lipids and peptides in tissue, and opens the way to combine pharmacokinetic and pharmacodynamic studies of RAF/MEK/mTOR inhibitors. The int egrated approach has the potential to identify drug-responsive biom arkers for the developm ent and testing of novel therapeutic approaches, while monitori ng drug distribution at high spatial and spectral resolutions. This study supports the value of MALDI MSI in drug re-purposing, and for the development of new drugs fo r the treatment of brain cancers. RAF/MEK/mTOR inhibitors were prepared at final concentration s of 0.1, 1, 10 and, 100 μM in solvents precised below. Nude mice implanted intracranially with murine PVL311 neural stem cells (derived from embryonic brains of p53- null mice and transformed to express human B-RAF V600E point mutation and overexpress luciferase) were dosed by oral gavage with one of the selected RAF/MEK/ mTOR inhibitors at 300 mg/kg. The flash frozen mouse brains were sectioned at 12-μm thickness using a Microm HM550 cryostat and thaw mounted onto ITO-coated microscope sli des. Samples on the MALDI target slides were allowed to dry for 30 minutes in a desiccator before use for drug and lipid analyzes. For proteomics, mouse brain sections were firstly allowed to dry for 1 h in a desiccator and washed three times with 70% ethanol (2 min) and 100% ethanol (2 min) twice. ITO slides were then dried in a desiccator for 1 hour. 200 μL of trypsin (100 ng/μL) were then deposited usi ng an ImagePrep automated sprayer device (Bruker Daltonics, GmbH, Bremen, Germany). En zymatic digestion was performed by an incubation of the tissue secti ons at 37°C for 1.5 h at a relative humidity of 100%.