NV1020, an oncolytic herpes simplex virus type 1, can destroy colon cancer cells by selectively replicating within these cells, while sparing normal cells. NV1020 is currently under investigation in a clinical phase I/II trial as an agent for the treatment of colon cancer liver metastases, in combination with conventional chemotherapeutic agents such as 5-fluorouracil (5-FU), SN38 (the active metabolite of irinotecan), and oxaliplatin. To study the synergy of NV1020 and chemotherapy, cytotoxicity and viral replication were evaluated in vitro by treating various human and murine colon carcinoma cell lines, using a colorimetric viability assay, a clonogenic assay, and a plaque-forming assay. In vivo experiments, using a subcutaneous syngeneic CT-26 tumor model in BALB/c mice, were performed to determine the efficacy of combination therapy. In vitro studies showed that the efficacy of NV1020 on human colon carcinoma cell lines HT-29, WiDr, and HCT-116 was additively or synergistically enhanced in combination with 5-FU, SN38, or oxaliplatin. The sequence of application was not important and effects were still apparent after a 21-day incubation period. Three intra-tumoral treatments with NV1020 (1 x 10(7) plaque-forming units), followed by three subcutaneous treatments with 5-FU (50 mg/kg), resulted in substantially higher inhibition of tumor growth and prolongation of survival compared with monotherapies (NV1020/5-FU vs. NV1020, p = 0.027). On WiDr cells, reduced replication of NV1020, in combination with 5-FU, indicated that additive and synergistic effects of combination therapy must be independent from viral replication. These results suggest that NV1020, in combination with chemotherapy, is a promising therapy for treating patients with metastatic colorectal cancer of the liver. We hypothesize that infection of cells with NV1020 sensitizes the infected cells for the cytotoxic effect of the chemotherapeutics.

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...

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.

Multicellular tumor spheroids have been used to examine aspects of combined modality treatment since they often recreate the in vivo tumor environment much more closely than other models. The radioenhancement by gemcitabine (dFdC) on human glioma spheroids derived from cell lines (CLS) and biopsy tissue, grown as organotypic multicellular spheroids (OMS), was studied. CLS of GaMg and U87 and OMS of four glioblastoma patients were used. Radiochemosensitvity 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. Cell death (ethidium homodimer) was studied using a fluorescence cell viability assay. In CLS, combination treatment led to migration inhibition in GaMg and U87 of 85% and 62% (dFdC 46% and 52%, RT 21% and 43%) and proliferation inhibition of 83% and 85%, respectively. Following dFdC + RT in OMS (% of cases), apoptosis and p21 expression increased (50%), p53 expression increased (75%) and cell proliferation decreased (75%). Only minor morphological damage was observed. Confocal laser scanning microscopy identified an increased dead cell core after dFdC + RT (50%). In conclusion, dFdC can lead to an additively radioenhancement in CLS and individual OMS.

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