gms | German Medical Science

Objective: Previously we demonstrated the applicability of multiphoton microscopy (MF) to discrimination of glioma tissue and adjacent brain as well as intraoperative use of MF microscope ex vivo. Fluorescence lifetime imaging (FLIM), a modality of MF, is based on excitation of intrinsic autofluorophores. FLIM requires no tissue staining or markers and can performed on native living tissue. As many of the autofluorophores within the cells are molecules actively involved in cellular intrinsic processes, this technology might be used to detect cellular functional and metabolic states.

Method: 8 glioma initiating cell (GIC) lines and one non-stem cell line were used. Cultures were grown in Neurobasal medium supplemented with growth factors bFGF and EGF (10 and 20 ng/ml, respectively). For MF, cell suspensions were plated on ornithine coated coverslips. For differentiation inducing conditions, withdrawal of growth factors was used. Cell proliferation rate was evaluated using BrdU incorporation rate and immunofluorescence staining. Multiphoton microscopy was performed with MPTflex^™ Multiphoton Laser Tomograph (Jenlab, Germany) at an excitation wavelength of 760 nm. The FLIM data was post-processed and average exponential components for single cells were acquired. The association between autofluorescence lifetime of two exponential components, T1 and T2 as well as mean autofluorescence lifetime Tm and the BrdU incorporation rate were assessed.

Results: 6 out of 9 investigated cell lines demonstrated the ability to phenotypically differentiate. The differentiation was associated with marked reduction of the cell proliferation rate. MF discriminated phenotipically differentiated from undifferentiated glioma cells in culture. We found an inverse association of cell proliferation rate and autofluorescence lifetime. FLIM data showed prolonged autofluorescence lifetime in differentiated cells, the shortest fluorescence lifetime was observed in actively proliferating cell lines. Proliferation rates and autofluorescence lifetimes remained constant in differentiation incapable cells.

Conclusions: Different functional states of cultured glioma stem cells in vivo could be distinguished using MF. Given the speed of the analysis, experience in other clinical disciplines and the possibility of evaluating native cells, MF is a promising tool for identification of aggressive subpopulations of tumors in situ.