A Multimodal NMR study of Apoptosis Induced by HSV-tk Gene Therapy in a Rat Experimental Glioma Model

In vivo nuclear magnetic resonance (NMR) techniques are widely used nowadays, both in experimental and clinical cancer research. Advantages of NMR include the ability to reveal abnormal tissue types non-invasively, and to provide a wealth of information from cancer tissue in situ, which can be used to assess the degree of malignancy and treatment responses. This study was devised to search for endogenous NMR detectable biomarkers for apoptotic cell death in a rat BT4C glioma model treated with Herpes Simplex virus thymidine kinase gene (HSV-tk) ganciclovir (GCV) gene therapy. To this end, multimodal NMR methods in vivo, ex vivo, and in vitro were used. Altered microenvironments of water were observed in the apoptosing BT4C glioma in vivo, as revealed with diffusion MR imaging (MRI). Increases both in extracellular volume and net water content, as well as decrease in intracellular space was also detected in the gene therapy treated apoptosing gliomas. These changes preceded or coincided with the collapse of tumour cell count determined by histology and were expressed before a decline in the tumour volume observed in T2 weighted MR images. Therefore, diffusion MRI proved to be a sensitive imaging method for the detection of a tumour’s response to gene therapy –induced apoptosis prior to tumour shrinkage. A Carr-Purcell (CP) T2 MRI method was introduced for imaging of rat brain tumours. CP T2 MRI contrast at a 4.7 T magnetic field strength, acquired with a short (6.1 ms) interpulse interval, highlighted tissue changes associated with gene therapy -induced cytotoxicity. This was also evident well before any cell loss or decrease in tumour growth rate could be observed. This MRI contrast is expected to provide valuable information about early cytotoxic tumour responses and might be useful in clinics. Ex vivo high resolution magic angle spinning (HRMAS) NMR spectroscopy revealed that none of the chemical species containing a choline moiety contributing to the in vivo choline H NMR signal at 3.2 ppm, were found to decrease with cell density early in the apoptosing glioma. In contrast, the concentration of taurine resonating close to 3.2 ppm followed the cell density and this may explain the decrease in the intensity of the choline peak in vivo in early phase of tumour apoptosis. Accumulation of polyunsaturated fatty acids (PUFA) was found to be the most significant contributor to the increase in H NMR lipid resonances during the apoptotic cell death. Multidimensional NMR analyses of tissue samples ex vivo and extracts in vitro showed that H NMR detectable PUFAs were mainly 18:1 and 18:2 fatty acids. This argues that membrane breakdown products may be the source for the H NMR detected PUFAs. The present results show that several potential endogenous biomarkers for apoptosis can be revealed with NMR methods in vivo. These results expand our understanding of the biomolecular and biophysical changes behind the NMR data in the apoptotic BT4C glioma. They also show the ability of in vivo NMR techniques to reveal apoptosis in the experimental glioma prior to tumour growth arrest or shrinkage. It is believed that NMR methods used here may provide a way of visualising treatment response in early phase of therapy. Some or all of these may also have uses in the clinical environment, thus allowing for the more efficient management of cancer. National Library of Medicine Classification: WN 185, QZ 380, QU 375,QZ 52 Medical Subject Headings: diagnostic imaging; neoplasms; glioma; magnetic resonance imaging; magnetic resonance spectroscopy; simplexvirus; thymidine kinase; gene therapy; ganciclovir; apoptosis; biological markers; diffusion; fatty acids, unsaturated; choline; taurine; rats