Investigation of Inter- and Intratumoral Heterogeneity of Glioblastoma Using TOF-SIMS.

Glioblastoma (GBM) is one of the most aggressive human cancers with a median survival of less than two years. A distinguishing pathological feature of GBM is a high degree of inter- and intratumoral heterogeneity. Intertumoral heterogeneity of GBM has been extensively investigated on genomic, methylomic, transcriptomic, proteomic and metabolomics levels, however only a few studies describe intratumoral heterogeneity because of the lack of methods allowing to analyze GBM samples with high spatial resolution. Here, we applied TOF-SIMS (Time-of-flight secondary ion mass spectrometry) for the analysis of single cells and clinical samples such as paraffin and frozen tumor sections obtained from 57 patients. We developed a technique that allows us to simultaneously detect the distribution of proteins and metabolites in glioma tissue with 800 nm spatial resolution. Our results demonstrate that according to TOF-SIMS data glioma samples can be subdivided into clinically relevant groups and distinguished from the normal brain tissue. In addition, TOF-SIMS was able to elucidate differences between morphologically distinct regions of GBM within the same tumor. By staining GBM sections with gold-conjugated antibodies against Caveolin-1 we could visualize border between zones of necrotic and cellular tumor and subdivide glioma samples into groups characterized by different survival of the patients. Finally, we demonstrated that GBM contains cells that are characterized by high levels of Caveolin-1 protein and cholesterol. This population may partly represent a glioma stem cells. Collectively, our results show that the technique described here allows to analyze glioma tissues with a spatial resolution beyond reach of most of other omics approaches and the obtained data may be used to predict clinical behavior of the tumor.

Electronically excited states of membrane fluorescent probe 4-dimethylaminochalcone. Results of quantum chemical calculations.

Quantum-chemical calculations of ground and excited states for membrane fluorescent probe 4-dimethylaminochalcone (DMAC) in vacuum were performed. Optimized geometries and dipole moments for lowest-lying singlet and triplet states were obtained. The nature of these electronic transitions and the relaxation path in the excited states were determined; changes in geometry and charge distribution were assessed. It was shown that in vacuum the lowest existed level is of (n, π*) nature, and the closest to it is the level of (π, π*) nature; the energy gap between them is narrow. This led to an effective (1)(π, π*) →(1)(n, π*) relaxation. After photoexcitation the molecule undergoes significant transformations, including changes in bond orders, pyramidalization angle of the dimethylamino group, and planarity of the molecule. Its dipole moment rises from 5.5 Debye in the ground state to 17.1 Debye in the (1)(π, π*) state, and then falls to 2 Debye in the (1)(n, π*) state. The excited (1)(n, π*) state is a short living state; it has a high probability of intersystem crossing into the (3)(π, π*) triplet state. This relaxation path explains the low quantum yield of DMAC fluorescence in non-polar media. It is possible that (3)(π, π*) is responsible for observed DMAC phosphorescence.