Therapeutic effects of Holmium-166 chitosan complex in rat brain tumor model

Abstract

This study examined the effectiveness of Holmium-166 (Ho-166) chitosan complex therapy for a malignant glioma. Cultured C6 glioma cells (100,000 in 5 microl) were injected into the caudate/putamen of 200-250 gram Wistar rats. Five days later, a Ho-166 chitosan complex was injected into the same site of the glioma injection. Four injection doses were administered: the control group received PBS 10 microl, group 1 received an injection of 100 microCi (10 microl), group 2 received an injection of 50 microCi (5 microl), and group 3 received an injection of 10 microCi (1 microl). The average tumor volume for each group was 1.385 mm3 for the control group, 0.036 mm3 for group 1, 0.104 mm3 for group 2, and 0.111 mm3 for group 3. Compared with the control group, the size of the tumors in groups 1, 2 and 3 was reduced by an average of 97.4%, 92.5% and 91.9%, respectively. The Kaplan-Meier survival curve of group 2 was the longest, followed by groups 3, group 1 and the control. The mean survival was 22.8, 59, 60, and 44.6 days for the control group and groups 3, 2 and 1, respectively. H-E staining revealed that group 2 yielded the best results in the destruction of the malignant glioma. TUNEL staining and immunohistochemical studies indicated apoptotic features. The Ho-166 chitosan complex proved to be effective in destroying the malignant glioma.

Fig. 1

Gross images for each group. In the control group, a successful tumor implant in the left putamen can be observed. In group 1, cavitation (arrows) can be observed in spots where the Ho-166 chitosan complex was over-administered. In groups 2 and 3, the implanted tumors are substantially reduced in size due to destruction by Ho-166 chitosan complex.

Fig. 2

Comparison of the tumor volume for each group. The average tumor volume for the control group was 1.385 mm³. For group 1, it was 0.036 mm³, for group 2, it was 0.104 mm³, and for group 3 it was 0.111 mm³. The ratio of the tumor volume against the control volume was 97.4% for group 1, 92.5% for group 2, and 91.1% for group 3, showing a gradual reduction.

Fig. 3

Survival curve for each group. Curves for groups 2 and 3 extended further than the control and group 1, and this extension was statistically significant (p < 0.001).

Fig. 4

H&E stain images. Under 100 × and 400 × magnification, malignant cerebral tumors with abundant nucleus and cytoplasm could be observed in the control group. In group 3, some tumor cells destroyed by Ho-166 chitosan complex could be observed, but the areas with active tumor cells were larger than in the other groups. In group 2, necrosis of the tumor cells were observed more extensively. In group 1, cavities (almost all cells completely destroyed with no structure) could be observed around the centers of administration (marked with arrow). Destruction spread even into normal tissues, and tumor cells showed the appearance of coagulation necrosis.

Fig. 5

TUNEL stain images. In all experimental groups with the Ho-166 chitosan complex administration, there were positive results with the nucleus being stained (arrowed). In group 3, positive reactions were observed even in the normal cells around a tumor cell where it appeared to be going through radionecrosis.

Fig. 6

Comparison of TUNEL-stain-positive rats. The average positive rates were 19.6% for group 3, 30.4% for group 2, 36.7% for group 1.

Fig. 7

Bax stain results. In group 1, extensive staining of the cytoplasm could be observed around the tumor destruction as the center. In group 2, similar observations were made. However, in group 3, there appeared to be barely any staining.