Effects of irradiation and cisplatin on human glioma spheroids: inhibition of cell proliferation and cell migration

Abstract

Purpose: Investigation of cell migration and proliferation of human glioma cell line spheroids (CLS) and evaluation of morphology, apoptosis, and immunohistochemical expression of MIB-1, p53, and p21 of organotypic muticellular spheroids (OMS) following cisplatin (CDDP) and irradiation (RT).

Material and methods: Spheroids of the GaMg glioma cell line and OMS prepared from biopsy tissue of six glioblastoma patients were used. Radiochemosensitvity (5 microg/ml CDDP followed by RT) was determined using migration and proliferation assays on CLS. In OMS, histology and immunohistochemical studies of MIB-1, p53, and p21 expression were examined 24 and 48 h following treatment.

Results: Combination treatment led to a migration inhibition of 38% (CDDP 13%; RT 27%) and specific growth delay of 2.6 (CDDP 1.3; RT 2.1) in CLS. Cell cycle analysis after combination treatment showed an accumulation of cells in the G2/M phase. In OMS, apoptosis increased, cell proliferation decreased, and p53/p21 expression increased more pronounced following CDDP+RT. No morphological damage was observed.

Conclusion: CDDP can lead to enhancement of the RT effect in spheroids of both human glioma cell line spheroids and biopsy spheroids from glioblastoma specimens. The exerted effect is additive rather than synergistic.

Fig. 1

Tumour cell migration for GaMg cell line spheroids following RT, CDDP, and CDDP+RT. Each treatment group consisted of six spheroids. Changes at day 4 were significant (P<0.05)

Fig. 2

Volume growth of GaMg cell line spheroids following RT, CDDP, and CDDP+RT. Each treatment group consisted of six spheroids. Changes at day 20 were significant (P<0.05)

Fig. 3

Apoptosis and immunohistochemical expression of organotypic multicellular spheroids (OMS) of six patients (S13, S14, S17, S18, S19, S21) as determined 24 h (left column) and 48 h (right column) following RT, CDDP, and CDDP+RT compared to the untreated controls. From top, relative apoptotic index, relative expression of p53, p21, and relative cell proliferation (+, P<0.05)

Fig. 4

a Haematoxylin–Eosin stained section of a organotypic muticellular spheroid (OMS). b Immunohistochemical staining of p53. OMS showed a significant increased rate of apoptosis (a) and p53 protein expression (b) 24 h after irradiation and cisplatin treatment. Original magnification, ×40. c and d Immunohistochemical staining of Ki-67 (MIB-1). The number of proliferating cells was higher in control (c) compared to corresponding singe modality treated spheroids or to the corresponding spheroids treated with irradiation and cispaltin (d). Original magnification, ×10