Introduction
Glioblastoma is a highly aggressive form of brain cancer with a 5-year survival rate below 10%. Temozolomide (TMZ) is the main chemotherapy drug used globally for the treatment of glioblastoma, which when added to surgery and radiation therapy, has been demonstrated to extend survival. However, detailed spatial analysis of TMZ uptake across the blood-brain barrier and into tumors (and specific tumor regions) is currently lacking. Here, we apply a Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-2-MSI) approach utilizing post-ionization that offers enhanced TMZ detection in comparison to traditional MALDI-MSI. We have utilized this technique to study the distribution of TMZ within tumors from a glioblastoma patient-derived xenograft (PDX) mouse model. In addition, we apply untargeted MALDI-2-MSI to investigate metabolic perturbations occurring within the heterogeneous tumors.
Methods
Mice were orally dosed with 50 mg/kg TMZ and sacrificed at 30- or 90-minute post-dose. 12 µm coronal brain tissue sections were prepared onto indium tin oxide (ITO) slides and spray-coated with 2,4,6-Trihydroxyacetophenone (TMZ and positive mode metabolite analysis) or 1,5-Diaminonaphthalene (negative mode metabolites) using an HTX TM Sprayer.
MALDI-MSI was performed using a Q-Exactive HF Hybrid quadrupole Orbitrap mass spectrometer equipped with a MALDI/ESI injector elevated pressure source incorporating a primary Nd:YAG laser and post-ionization 266 nm solid-state laser. For MALDI-2 MSI acquisition, both the CryLas (primary) and Explorer (post-ionization) lasers were operated at 30 Hz repetition rate, with a delay time of ~20 ns and Explorer pulse energy of 5 µJ. Metabolites and lipids were identified using LipidMaps and HMDB databases with a 2 ppm tolerance.
Results
Comparative MSI analysis of TMZ spiked onto control mouse brain tissue sections using MALDI or MALDI-2 demonstrated a 6-fold decrease in the on-tissue limit of detection of TMZ with the use of post-ionization. Hence, MALDI-2 was used for the analysis of the TMZ-dosed tissues. TMZ was detected within the brain parenchyma and was heterogeneously distributed within the tumor at both 30- and 90-minute post-dose time points. The major TMZ metabolite, 3-methyl-(triazen-1-yl) imidazole-4-carboximide (MTIC), was not detected and further optimization of the method to increase analytical sensitivity is ongoing.
Metabolomic analysis revealed carnitine and acylcarnitines were significantly increased within the tumor in comparison to the parenchyma. Lipidomic analysis revealed that tumor regions were highly enriched in neutral triglyceride and cholesteryl ester lipids. Ceramide lipid species (ceramide, ceramide-1-phosphate, and sphingomyelin) were also enriched within the tumors, particularly within necrotizing regions. Free arachidonic acid and arachidonic acid-containing phospholipids were significantly increased within the tumors indicating the presence of substantial inflammation. In ongoing studies, metabolite and lipid ion maps will be registered with immunohistochemistry images performed on the same and/or adjacent tissue sections for detailed spatial pathway analysis.
Conclusion
We have developed a novel mass spectrometry imaging approach to visualize TMZ distribution within the tumor and parenchyma of a mouse PDX model. In addition, we have utilized our approach for high spatial resolution imaging of metabolic perturbations occuring within tumors.
