1. Introduction Conclusions
Treatment of tumour cells with 5-aza-2-deoxycytidine (DAC) for immune tumour therapy of Glioma in Fischer 344 rats
Bertil R.R. Persson,1,4 Per Engström, 1 Gustav Grafström,1,4 Catrin Bauréus-Koch,1,2,4
Bengt Widegren,3,4 and Leif G. Salford,2,4
1Medical Radiation Physics, 2Neurosurgery, 3Tumour Immunology, 4Rausing Laboratory, Biomedical Centre,
Lund University, LUND, Sweden
Correspondence to: Bertil R.R. Persson,
Introduction
Conclusions
The nucleoside analogue 5-aza-CdR, decitabine,(DAC) is a potent inhibitor of DNA methylation. It act as a de-methylating agent and have been used in the treatment of several types of neoplasm. Tumour cells treated with DAC up-regulate HLA class I antigens. In the present work we investigate if DAC treatment of tumour cell vaccine used for immunotherapy of glioma in Fischer-344 rats, affect the therapeutic effect. We also study the combination of immunotherapy with Pulse Electric Field (PEF) treatment and with Radiation Therapy.
Pulse Electric Field (PEF) treatment of tumours in combination with IFN-transfected tumour cells does not decrease the growth rate of N29 glioma tumours. But in the combination of PEF treatment and immunization with IFN-transfected cells grown in DAC medium the tumour growth rate decreased by about 50 % at the PEF treated tumour and there was a decrease of about 20% in tumour growth at the non-PEF treated tumour. But the DAC treatment of tumour cells seems to have no benefits in combination with radiation therapy.
Materials and Methods
Figure 1. Average tumour growth rate “TGR” for rats with subcutaneous tumours.
Results and Discussion
DAC -treatment of IFN transfected tumour cells
The DNA de-methylating agents 5-aza-2-deoxycytidine, and 5-aza-CdR (DAC) are cytosine analogues that covalently bind to DNA methyl-transferase and inhibits its activity which is causing de-methylation. The gene expressions, which may have been silenced by hyper-methylation of their promoters, are re-activated by DAC. Expression of HLA class I is significantly increased in DAC-treated glioma cells when compared to untreated cells, which makes DAC a potent immune stimulator.  
IFN transfected N29 cells were treated during 3 days in culture medium with 10M 5-aza-2-deoxycytidine (DAC). The medium was then replaced with fresh medium without DAC, and the cells were irradiated and used for immunization.
Subcutaneously inoculated N29-glioma tumour cells
N29-glioma cells were inoculated on both thighs of female Fischer-344 syngeneic rats to induce subcutaneous tumours.
For immunization the animals were given intra-peritoneal injections of IFN transfected N29 tumour cells or DAC treated IFN transfected N29-cells.
Treatment with Pulsed Electric Field
In the combined treatment the left tumour was PEF-treated once with 16 exponential pulses with an electric field strength of 1400 V/cm, and 1.0 ms duration (time constant). No anticancer drugs were given at any time. The following day and then once weekly for three weeks, the animals were given intra-peritoneal injections of irradiated, IFN transfected N29 tumour cells or DAC treated IFN transfected cells.
Radiotherapy of intracanial tumours.
Animals with intra-cranial tumours were treated on day 7 after the inoculation, with a single fraction RT(Co-60) of 15 Gy.
Immunization with DAC treated IFN-transfected tumour cells was given as intra-peritoneal injections 1 h before RT and then weekly for at most 3 weeks.
The treatment results were evaluated by daily measuring the size of tumour on both sides of the animals. Treatment with solely PEF in 32 animals resulted in a growth rate decrease of 30±6 % at the PEF exposed tumour. The effect at the non targeted tumour was 10±4 %. Treatment with IFN- secreting tumour cells resulted in a significant decrease of tumour growth rate on the right tumour of 20±2 % (p< 0.05). No significant effect (3±0.3%) was, however, observed on the left tumour. Immunization with DAC treated IFN secreting cells in 12 animals showed no significant decreased growth rate, on neither the left nor the right tumours.
By combining PEF+IFN-transfected cells no significant decrease in growth rate was achieved. But in the combination of PEF and IFN-transfected cells grown in DAC medium the tumour growth rate decreased by about 40 % at the PEF treated tumour and there was a decrease of about 10% in tumour growth at the non-PEF treated tumour which is about the same as for PEF treatment alone.
Immune therapy of rats with intracranial implanted N32 tumours by immunization with IFN- secreting syngeneic cells treated with DAC resulted in a slight (3%) but not significant increase in survival time. With just a single RT fraction of 15 Gy, however, there was a significant increase of 32% in the length of survival time of the rats with N32 tumours (p<0.02) and when RT was combined with immunotherapy using IFN transfected N32 cells the increase in survival was about 40%. RT with a single fraction of 15 Gy combined with immuni-zation with IFN- secreting syngeneic cells treated with DAC resulted in significant (p<0.01) 34% increased length of survival time for the N32 tumours, although no complete remissions.
Figure 2. Set-up for the PEF
treatment of the subcutaneous tumors.
Figure 3. Specific Therapeutic effect for rats with sub-cutaneous tumours: STE=(TGRControl-TGRExposed)/TGRcontrol
Figure 4 Survival plot of rats with cerebral N32 tumours
References
PERSSON, B. R. R Radiation Immune Modulation Therapy of Glioma, Advances in the Biology, Imaging and Therapies for Glioblastoma. In: CHEN, C. C. (ed.) InTech. (open access)
Lund University / Medical Radiation Physics/ Neuro-Surgery/ Rausing Laboratory