The inhibition of proliferation and migration of glioma spheroids exposed to temozolomide is less than additive if combined with irradiation

Abstract

Abstract. The aim of this study was to investigate the effect of temozolomide (TZM) in combination with X-rays on proliferation and migration in human glioma spheroids. Multicellular spheroids were derived from GaMg and U87 cell lines. Spheroids were treated with various concentrations of TZM (5 μmol, 0.025 mmol, 0.05 mmol) and irradiation (RT). Proliferation and migration assays were performed. For GaMg spheroids, the proliferation inhibition was 30% (RT), 71%, 79%, 85% (for various TZM concentrations) and 78%, 83%, 90% following RT+TZM. For U87 spheroids, the inhibition of proliferation was 52% (RT), 62%, 78%, 88% (TZM), and 73%, 87%, 92% (RT+TZM). Inhibition of migration for GaMg was 30% (RT), 37%, 63%, 78% (TZM), and 56%, 75%, 84% (RT+TZM). For U87, migration inhibition was 29% (RT), 48%, 52%, 67% (TZM), and 62%, 67%, 73% (RT+TZM). Radiotherapy enhancement ratio (RER) of GaMg/U87 spheroid proliferation was 1.4/1.7 (5 μmol TZM), 1.3/1.8 (0.025 mmol TZM), and 1.4/1.4 (0.05 mmol TZM). RER for migration of GaMg/U87 was 2.2/1.9 (5 μmol TZM), 1.7/1.8 (0.025 mmol TZM), and 1.5/1.4 (0.05 mmol TZM). In terms of inhibition of proliferation and migration, irradiation can lead to an enhancement of the TZM effect in human glioma spheroids, which is less than additive. IntroductionGliomas are the most common primary human brain tumours and prognosis is poor, in particular with high-grade tumours such as glioblastoma multiforme (GBM). Intensive surgery followed by radiotherapy with or without adjuvant chemotherapy is considered as standard therapy for primary brain tumours (1). Treatment of brain tumours with adjuvant chemotherapy, mainly 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), has only resulted in minor improvements (2-4). Phase I-II clinical studies with temozolomide (TMZ) have demonstrated that this drug crosses the blood-brain barrier and has activity against malignant glioma (5-8).TMZ in the treatment of primary and recurrent malignant brain tumours is still a topic of clinical trials due to the promising pre-clinical and clinical reports. Recently, Stupp et al found a clinically meaningful increase, by a factor of 2.5, in the survival rate at two years, from 10% with radiotherapy alone to 27% with radiotherapy plus TZM in a randomized phase III trial. The addition of temozolomide to irradiation was associated with improvements in median progression-free survival (6.9 vs. 5.0 month) and overall survival (14.6 vs. 21.1 month) (9).Since radiotherapy in combination with TMZ might be assumed as gold standard in the treatment of brain malignancies in future (10), it is of interest to investigate further what the pre-clinical effects are on various human glioma cell lines. In a study with the human glioma cell line U373 treated as monolayer cultures, an additive effect was reported for TMZ and X-rays (11). In glioma cells lines U251 and D384, combinations of TMZ and X-rays showed additional as well as supra-additional cytotoxic effects (12).We have chosen spheroid...

Doxorubicin-mediated radiosensitivity in multicellular spheroids from a lung cancer cell line is enhanced by composite micelle encapsulation

“…We successfully established three-dimensional A549 multicellular spheroids Dose (Gy) Figure 6 Cell survival curves after treatments with radiation alone or combined with 2.5 µg/mL blank composite micelles (radiation alone + blank composite micelles) or doxorubicin (radiation alone + doxorubicin) or doxorubicin-loaded composite micelle (radiation alone + MIX, P , 0.05 for radiation alone + MIX versus radiation alone that are a more physiologically relevant model of lung cancer than monolayer cells, because they resemble the architecture of in vivo tumors 17,25,26 and can recapitulate in vivo processes which may affect drug performance, such as cell-cell contact, variations in the cell cycle, altered metabolism, and diffusion of nutrients, oxygen, or drugs. 17,25,27,28 Multicellular spheroids are relatively easy to produce and maintain in culture, and treatment-related changes in spheroid growth kinetics and tumor cell outgrowth are established and reproducible endpoints. 17,25,29 As a consequence of their particular architectural characteristics, multicellular spheroids have been demonstrated to be extremely useful for testing radiotherapy protocols.…”

“…14,15 However, the spheroid model is a three-dimensional cell culture system that more closely resembles in vivo tumor cell growth and is more physiologically relevant than monolayer cell growth. 16,17 Individual tumor cells growing under these conditions face different microenvironments depending on their position inside the three-dimensional framework of the spheroid.…”

Three-Dimensional In Vitro Models in Glioma Research - Focus on Spheroids

“…However, a study by Glicklis et al (Glicklis et al, 2004) has described that hepatocyte spheroids with diameters up to 100 μm have a good oxygenation status. Other studies by Fehlauer et al (Fehlauer et al, 2005;Fehlauer et al, 2006;Fehlauer et al, 2007) have reported that by using glioma spheroids with diameters of 200-250 μm, there are only few hypoxic cells and no central necrosis present.…”

“…The amount of formazan produced is directly proportional to the amount of LDH in the culture medium, which in turn is directly proportional to the number of dead or damaged cells (Korzeniewski & Callewaert, 1983). The size of spheroids after drug treatment has also been used as a measure of cell viability (Fehlauer et al, 2007;Johannessen et al, 2009;Khaitan et al, 2009;Yamaguchi et al, 2010). Khaitan et al (Khaitan et al, 2009) investigated the effect of the glycolytic inhibitor 2-deoxy-D-glucose on spheroids derived from a human glioma cell line by measuring the size of the spheroids after drug exposure.…”

“…This may be explained by effects of hypoxia in vitro on proliferation of the tumor cells, spheroid formation and expression of stem cell markers, suggesting that also this aspect should be taken into consideration (Heddleston et al, 2009;Kolenda et al, 2010;McCord et al, 2009;Soeda et al, 2009). Several studies have used the different types of spheroids mentioned above for investigating the effects of chemotherapy and radiation on the tumor cells and in particular on the tumor stem-like cells Bauman et al, 1999;Fehlauer et al, 2005;Fehlauer et al, 2006;Fehlauer et al, 2007;Genc et al, 2004;Gliemroth et al, 2003;Haas-Kogan et al, 1996;Johannessen et al, 2009;Kaaijk et al, 1997;Khaitan et al, 2009;Sunayama et al, 2010;Terzis et al, 1997;Terzis et al, 1998;Wakimoto et al, 2009;Wang et al, 2010). After such treatments cell viability and cell proliferation assays as well as secondary spheroid formation assays have been used to evaluate induced effects.…”