**3.1.1 Animals and tumour cell lines**

Fischer-344 rats were maintained by continuous, single-line brother to sister mating in the laboratory at Lund. During the experiments rats of both sexes, females weighing around 190 g and males 370 g respectively, were housed in a climate controlled cabinet. Otherwise they were kept in Macralon cages provided with food pellets and water *ad libitum*. All experimental animal procedures were approved by the Animal Ethical Committee in Malmö/Lund (Lunds tingsrätt, Box 75, 22100 Lund Sweden).

All cells were maintained in culture flasks (Nunc, Denmark) and harvested by treatment with trypsin/EDTA. The culture medium was antibiotic-free RPMI-1640 medium supplemented with 5-10% foetal calf serum, L-glutamine (2 mM), HEPES (10 mM), pyruvate (0.5 mM) and NaHCO3 (11 mM). The cell-cultures were regularly checked for contaminating microbes by staining with the fluorescent dye Hoechst 32 258 and examined with fluorescent microscopy. If *Mycoplasma* infection was indicated the cultures were discharged or treated with *Mycoplasma Removal Agent* (Hoechst, Germany) twice with 7 days interval, and repeatedly confirmed free of infection.

The tumour cells (N29 or N32) used for immunization were interferon-gamma (IFN-) gene modified to enhance secretion of IFN. The cells were cultured for one week, washed twice, and suspended in serum free medium (IMDM-0) to a cell density of 2104 cells/ml. Just before immunization the cells were transferred from the culture flasks to 15 ml centrifuge test tubes (Nanclon) and stored on melting ice to prevent the cells to grow during the procedure. Irradiation of the cells was performed during 20 minutes at room temperature to an absorbed dose of 70 Gy by using a 137Cs gamma-ray source (*Gammacell 2000;* Mølsgaard Medical, Risø, Denmark) (Siesjö, et al. 1996; Sjögren, et al. 1996; Visse, et al. 1999).

#### **3.1.2 Inoculation and treatment of intracerebrally tumours**

Inoculation was performed by injecting 5 000 tumour cells in 5 l nutrient solution into the head of Fischer 344 rats, using a stereotactic technique with a Hamilton syringe. To avoid extra-cranial tumour growth, the injection site was cleaned with 70% ethanol after injection and the borehole was sealed with wax. The animals were arranged into 6 groups, which included: controls, RT with either 5 or 15 Gy, immunization with IFN- gene modified tumour cell, and RT with either 5 or 15 Gy combined with immunization (Table 1).

Animals were given a single radiation treatment using a 60Co radiotherapy unit (Siemens Gammatron S) with a source-skin distance (SSD) of 50 cm and the maximum absorbed dose rate 0.65-0.70 Gy/min. The radiation field size was collimated to cover the brain. The adsorbed dose of either 5 or 15 Gy was measured both by an dose-meter diode and TLD

Radiation Immune Modulation Therapy of Glioma 369

3 Radiation 15 Gy 8 6 6 4 Immunization 6 7 6

Immunization 8 7

Table 1. Number of animals in the groups of various treatments used in the experiments with either N29 or N32 tumours. The various experiments A, B and C respectively, were

The rats were examined daily and when the animals developed symptoms, they were

None of the rats, which were inoculated with N32 tumour cells, survived longer than 30 days. But in the group inoculated with N29 tumour cells, surviving animals could be observed for more than 170 days. In this group of animals with N29 tumours, re-challenge was performed with 2105 N29 glioma cells in 200 l, administered just under the skin in the thigh of the hind leg. Fourteen out of the originally 46 rats, and 4 extra control rats with no

In Table 2 are given the fractions of animals intracerebrally implanted with N29 tumour cells, which were surviving more than 170 days: Controls; IFNcell immunization (IMU IFN), single fraction radiation therapy (RT with either 5 or 15 Gy), and their combinations (IMU IFN+ RT with either 5 or 15 Gy). RT and first immunization was performed at 7 days after inoculation. Immunizations were then repeated for at least two more times at days 21 and 35. In the 2nd column of Table 2 are given the numbers of animals survived more than 170 days, versus the number in each group of animals with intra cerebral N29 tumour. In the 3rd column is given the number of tumours appeared, relative to the number of animals that

In the last column of Table 2 is given the number of re-challenged animals without tumour versus the original number in each group. Those animals, which resisted re-challenge, seem

Following symptoms of the rats were used as signs of progressing tumour growth:

euthanatized and the brains were stained for histopathological examination.

**3.1.3 Survival of rats with intracerebrally implanted N29 tumours** 

2 Radiation 5 Gy 8 7

Number of N29 Animals Experiment A

treatment 6 9 3

Immunization 8 7 6

Number of N32 Animals Experiment B

Number of N32 Animals Experiment C

Group

No. Treatment

<sup>1</sup>Controls with no

<sup>5</sup>Radiation 5 Gy +

<sup>6</sup>Radiation 15 Gy +

keeping their heads turned to one side,

 shaggy fur and reddening of the eyes and nose.

previous treatment were inoculated.

were re- challenged, including the 4 extra controls.

to have been cured from their primary glioma.

performed at different occasions

 rotating or losing weight, unwillingness to move,

dose meter. A sheet of tissue equivalent bolus, 5 mm thick, was placed over the head for radiation build up.

Fig. 2. Radiation therapy was performed at day 7 after inoculation with the animals anesthetized with 5% chloral hydrate given intraperitoneally (i.p.) or Ketalar/Rompun, 0.55 ml per 100g. The animals were given a single radiation exposure using a 60Co radiotherapy unit (Siemens Gammatron S) at a source-skin distance (SSD) of 50 cm with a maximum absorbed dose rate of 0.70 Gy/min. The radiation field (1 cm2) was collimated to cover the brain (Fig. 2). The delivered adsorbed dose of either 5 or 15 Gy was measured both by an dose-meter diode and a Lithium fluoride (LiF) TLD chip placed next to the tumour in the field under the bolus.

The animals were immunized by intraperitoneally administration of 3 x 106 IFN- gene modified N29 or N32 tumour cells, which immediately before had been irradiated with 70 Gy 134Cs gamma-radiation. The first immunization was performed within one hour after the radiotherapy session at day 7. In the rats still alive it was repeated at least two more times at days 21 and 35.

dose meter. A sheet of tissue equivalent bolus, 5 mm thick, was placed over the head for

Fig. 2. Radiation therapy was performed at day 7 after inoculation with the animals

0.55 ml per 100g. The animals were given a single radiation exposure using a 60Co

anesthetized with 5% chloral hydrate given intraperitoneally (i.p.) or Ketalar/Rompun,

radiotherapy unit (Siemens Gammatron S) at a source-skin distance (SSD) of 50 cm with a maximum absorbed dose rate of 0.70 Gy/min. The radiation field (1 cm2) was collimated to cover the brain (Fig. 2). The delivered adsorbed dose of either 5 or 15 Gy was measured both by an dose-meter diode and a Lithium fluoride (LiF) TLD chip placed next to the tumour in

The animals were immunized by intraperitoneally administration of 3 x 106 IFN- gene modified N29 or N32 tumour cells, which immediately before had been irradiated with 70 Gy 134Cs gamma-radiation. The first immunization was performed within one hour after the radiotherapy session at day 7. In the rats still alive it was repeated at least two more times

radiation build up.

the field under the bolus.

at days 21 and 35.


Table 1. Number of animals in the groups of various treatments used in the experiments with either N29 or N32 tumours. The various experiments A, B and C respectively, were performed at different occasions

Following symptoms of the rats were used as signs of progressing tumour growth:


The rats were examined daily and when the animals developed symptoms, they were euthanatized and the brains were stained for histopathological examination.

None of the rats, which were inoculated with N32 tumour cells, survived longer than 30 days. But in the group inoculated with N29 tumour cells, surviving animals could be observed for more than 170 days. In this group of animals with N29 tumours, re-challenge was performed with 2105 N29 glioma cells in 200 l, administered just under the skin in the thigh of the hind leg. Fourteen out of the originally 46 rats, and 4 extra control rats with no previous treatment were inoculated.

## **3.1.3 Survival of rats with intracerebrally implanted N29 tumours**

In Table 2 are given the fractions of animals intracerebrally implanted with N29 tumour cells, which were surviving more than 170 days: Controls; IFNcell immunization (IMU IFN), single fraction radiation therapy (RT with either 5 or 15 Gy), and their combinations (IMU IFN+ RT with either 5 or 15 Gy). RT and first immunization was performed at 7 days after inoculation. Immunizations were then repeated for at least two more times at days 21 and 35. In the 2nd column of Table 2 are given the numbers of animals survived more than 170 days, versus the number in each group of animals with intra cerebral N29 tumour. In the 3rd column is given the number of tumours appeared, relative to the number of animals that were re- challenged, including the 4 extra controls.

In the last column of Table 2 is given the number of re-challenged animals without tumour versus the original number in each group. Those animals, which resisted re-challenge, seem to have been cured from their primary glioma.

Radiation Immune Modulation Therapy of Glioma 371

the survival time by 60% (p=0.04). Radiation therapy alone with 5 Gy, however, did not significantly increased the survival time. But immunization combined with 5 Gy radiation therapy resulted in a significantly increased survival time with 87% (p=0.003). Radiation therapy alone with 15 Gy did not significantly increased the survival time. But 15 Gy RT

**<sup>8</sup> RT 5 Gy + Imu**

**0 20 40 60 80 100 120 140 160**

Fig. 3. Survival plot of intra cerebral implanted N29 tumours: Controls (Lower panel), immunization with syngeneic N29 tumour cells (2nd panel); radiation therapy (3rd panel) and

The pooled results of the two experimental series (B and C in Table 1) with rats implanted with N32 tumours are displayed in Table 4. The results are given in terms of the mean survival time and weight of tumour at the time of death for each group animals. None of the rats with N32

The survival of all rats with implanted N32 tumours were followed during 30 days and the

For the N32 tumours given a single fraction radiation therapy with 15 Gy resulted in significant increase of survival time with about 20% (p<0.001). The combination of 15 Gy single fraction radiation therapy with immunization of IFN- secreting syngeneic cells resulted in increased survival time by about 40% (p<0.001), although there were no complete remissions. But neither immunization with IFN- secreting syngeneic cells alone, nor radiation therapy with a single fraction of 5 Gy alone, or in combination with

There is no significant difference in the weight of tumours in the different groups. Although the average growth rate of the N32 tumours treated with 5 Gy radiation therapies combined

**Time after inoculation / days**

**RT 15 Gy** 

combined with immunization increased the survival time with 45% (p=0.03).

*Number of living rats with N29 tumours in brain* 

**Controls**

combinations of radiation therapy and immunization (upper panel).

**3.1.4 Survival of rats with intracerebrally implanted N32 tumours** 

tumours survived more than 30 days and thus no re-challenging could be done.

results in the various groups of rats with different treatments are displayed in Fig. 4.

immunization, resulted in any increase in survival time of the N32 tumours in rats.

with immunization was decreased by 30% compared with the controls.

**Immune-therapy only**

**RT 5 Gy** 

**RT 15 Gy + Imu**


\*) p=0.03; +) extra controls

Table 2. The fraction of living rats in the various groups with different treatments, followed during 170 days after inoculation of N29 tumour cells in their brain, number of tumours after re-challenge, and fraction of cure.

By using Fisher exact probability test the results show that treatment with 5 Gy radiation therapy combined with immunization resulted in significantly increased number of survivals versus controls (p = 0.03). But neither immunization alone nor radiation therapy alone with single fractions of 5 or 15 Gy resulted in any significant therapeutic effect versus the controls.

The combination of radiation therapy with immunization compared with radiotherapy alone, however, resulted in significant survival fraction at both 5 Gy and 15 Gy, with pvalues <0.01\*\* and p <0.05\* respectively.

The number of living rats in the various groups with different treatments, followed during 170 days after inoculation of N29 tumour cells in their brain, is displayed in Fig. 3 for each group respectively.

In Table 3 is given the median survival time and the p-values of two-sided non-parametric Mann-Whitney test versus the control. Immunization with N29 cells significantly increased


Table 3. Number of rats; mean survival time and tumour weight at time of death of rats with intra cerebrally implanted N29 tumours treated one week after inoculation, with IFN cell immunization, radiation therapy (RT) and their combination. Immunization (IMU-IFN) was repeated for at least two more times at days 21 and 35. The rats were observed during up to 170 days after inoculation.

**Controls** 1/6 5/(1+4+) 0 **IMU IFN 3x** 2/6 1/2 1/6 **RT 5 Gy** 0/8 - 0 **RT 15 Gy** 2/8 2/2 0 **IMU IFN 3x + RT 5 Gy 6/8\*) 4/6 2/8 IMU IFN 3x + RT 15 Gy** 3/8 2/3 1/8

Table 2. The fraction of living rats in the various groups with different treatments, followed during 170 days after inoculation of N29 tumour cells in their brain, number of tumours

By using Fisher exact probability test the results show that treatment with 5 Gy radiation therapy combined with immunization resulted in significantly increased number of survivals versus controls (p = 0.03). But neither immunization alone nor radiation therapy alone with single fractions of 5 or 15 Gy resulted in any significant therapeutic effect versus the controls. The combination of radiation therapy with immunization compared with radiotherapy alone, however, resulted in significant survival fraction at both 5 Gy and 15 Gy, with p-

The number of living rats in the various groups with different treatments, followed during 170 days after inoculation of N29 tumour cells in their brain, is displayed in Fig. 3 for each

In Table 3 is given the median survival time and the p-values of two-sided non-parametric Mann-Whitney test versus the control. Immunization with N29 cells significantly increased

> **Median survival time (days)**

**Control** 6 82 46 0.39 0.22

**RT 5 Gy** 8 46 14 NS 0.25 0.25 **RT 15 Gy** 8 93 35 NS 0,24 0.14

**IMU IFN + RT 15 Gy** 8 119 35 P=0.03 \* 0.01 0.01 Table 3. Number of rats; mean survival time and tumour weight at time of death of rats with intra cerebrally implanted N29 tumours treated one week after inoculation, with IFN cell immunization, radiation therapy (RT) and their combination. Immunization (IMU-IFN) was repeated for at least two more times at days 21 and 35. The rats were observed during

**IMU IFN** 6 132 44 P=0.04 \* 0

**IMU IFN + RT 5 Gy 8 153 31 P=0.003 \*\* 0** 

**Fraction of animals with tumour in the re challenged survivors** 

> **Mann-Whitney 2-tailed versus Control**

**Tumour weight** 

**g** 

**Fraction of Cured animals** 

**Fraction of Animals Survived >170 d**

**Type of treatment** 

\*) p=0.03; +) extra controls

group respectively.

**Type of treatment** 

up to 170 days after inoculation.

after re-challenge, and fraction of cure.

values <0.01\*\* and p <0.05\* respectively.

**Num. Rats** 

the survival time by 60% (p=0.04). Radiation therapy alone with 5 Gy, however, did not significantly increased the survival time. But immunization combined with 5 Gy radiation therapy resulted in a significantly increased survival time with 87% (p=0.003). Radiation therapy alone with 15 Gy did not significantly increased the survival time. But 15 Gy RT combined with immunization increased the survival time with 45% (p=0.03).

Fig. 3. Survival plot of intra cerebral implanted N29 tumours: Controls (Lower panel), immunization with syngeneic N29 tumour cells (2nd panel); radiation therapy (3rd panel) and combinations of radiation therapy and immunization (upper panel).

#### **3.1.4 Survival of rats with intracerebrally implanted N32 tumours**

The pooled results of the two experimental series (B and C in Table 1) with rats implanted with N32 tumours are displayed in Table 4. The results are given in terms of the mean survival time and weight of tumour at the time of death for each group animals. None of the rats with N32 tumours survived more than 30 days and thus no re-challenging could be done.

The survival of all rats with implanted N32 tumours were followed during 30 days and the results in the various groups of rats with different treatments are displayed in Fig. 4.

For the N32 tumours given a single fraction radiation therapy with 15 Gy resulted in significant increase of survival time with about 20% (p<0.001). The combination of 15 Gy single fraction radiation therapy with immunization of IFN- secreting syngeneic cells resulted in increased survival time by about 40% (p<0.001), although there were no complete remissions. But neither immunization with IFN- secreting syngeneic cells alone, nor radiation therapy with a single fraction of 5 Gy alone, or in combination with immunization, resulted in any increase in survival time of the N32 tumours in rats.

There is no significant difference in the weight of tumours in the different groups. Although the average growth rate of the N32 tumours treated with 5 Gy radiation therapies combined with immunization was decreased by 30% compared with the controls.

Radiation Immune Modulation Therapy of Glioma 373

in Fischer-344 rats. In the rats, which were inoculated with N32 tumour cells, the combination of 15 Gy single fraction radiation therapy with immunization of IFN- secreting syngeneic cells resulted in increased survival time by about 40% (p<0.001). But none of these rats survived longer than 30 days. In the group inoculated with N29 tumour cells and treated with 5 Gy RT combined with immunization the survival time was significantly increased by 87% (p=0.003), and 75% of the animals survived for more than 170 days. The difference in response of N29 and N32 cell lines indicate that there is difference in immune

**3.2 The Hungarian experience of single fraction RT and Immunization with (GM-CSF,** 

In Hungary a study was performed in a mouse glioma (Gl261) brain tumour model with single fraction radiotherapy combined with administration of cytokine-producing cancer cell vaccines (Lumniczky, et al. 2002). Their brain tumour bearing mice were treated with various cytokine producing vaccines made by in vitro transduction of Gl261 tumour cells with different genes such as: IL-4, IL-6, IL-7, GM-CSF, TNF. Immunotherapy alone with vaccines producing either IL-4 or GM-CSF resulted in complete remission in 20–40% of the mice. By combining immunotherapy using (GM-CSF, IL-4, IL-12) producing vaccines with local tumour radiotherapy (single fraction 6 Gy X-ray radiations) about 80–100% of the glioma-bearing mice were cured. The high efficiency of the combined treatment was maintained even under suboptimal conditions when neither of the individual modalities alone cured any of the mice (Lumniczky, et al. 2002). Their results are in good agreement the survival rate of 75% (p<0.05) achieved in the Lund study of N29 tumours in rats treated with IFN- secreting vaccine combined with 5 Gy single fraction RT (B. R. R. Persson, et al.

**3.3 The U.S. experience of radiation therapy combined with vaccination of mice with** 

The cytotoxic T lymphocyte-associated protein CTLA-4 is involved in the immune regulatory mechanisms that control the early stage of the T cell response. It has previously been demonstrated that blockade of the CTLA-4 protein enhance anti-tumour responses both in experimental systems and in clinical trials (Chambers, et al. 2001; Egen, et al. 2002). In a mouse model of the poorly immunogenic metastatic mouse mammary carcinoma 4T1, however, neither anti-tumour response nor survival-time was affected by using an anti-CTLA-4 monoclonal antibody for blocking the CTLA-4 protein. But anti-CTLA-4 monoclonal antibody administration combined with one 12 Gy fraction of radiation therapy, inhibited the growth of the primary irradiated tumour. Also the survival-time of the mice was significantly increased by this combined treatment (Demaria, et al. 2004; Demaria, et al.

Another investigation of the effects of systemic CTLA-4 blockade with monoclonal antibody (9H10) to CTLA-4 employed in a mice model with well-established glioma, showed that CTLA-4 blockade confers long-term survival in 80% of treated mice (Fecci, et al. 2007). Thus the combination of local RT with CTLA-4 blockade might be applied as radio-immune-

**3.3.1 Combining radiation therapy with blockade of the CTLA-4 pathway** 

response in different clones of glioma.

**Glioma or mammary carcinoma** 

2003; Demaria, et al. 2005b).

modulating therapeutic strategy also against glioma.

2010).

**IL-4, IL-12) in a mouse glioma (Gl261) brain tumour model** 

Fig. 4. Survival plot of intra-cerebral implanted N32 tumours: Controls (Lower panel); Immunization with syngeneic N32 tumours cells (2nd panel); radiation therapy (3rd panel), and a combination of radiation therapy and immunization (upper panel).


Table 4. Number of rats, mean survival time, and the significance of Mann-Whitney 2-tailed test versus control is shown in columns 2-4. In the last column is given the tumour weight at time of death of intra cerebral N32 tumours treated with syngeneic IFN transfected tumour cells (IMU IFN), radiation therapy (RT) and their combination (RT + IMU IFN).
