Triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro

Abstract

Background: Combined chemoradiation offers a promising therapeutic strategy for dogs with glioma. The alkylating agents temozolomide (TMZ) and lomustine (CCNU) penetrate the blood-brain barrier, and doses for dogs are established. Whether such combinations are clinically advantageous remains to be explored together with tumour-specific markers.

Objective: To investigate if triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro.

Methods: We evaluated the sensitising effect of CCNU alone and in combination with TMZ-irradiation in canine glioma J3T-BG cells and long-term drug-exposed subclones by using clonogenic survival and proliferation assays. Bisulphite-SEQ and Western Blot were used to investigate molecular alterations.

Results: TMZ (200 μM) or CCNU alone (5 μM) reduced the irradiated survival fraction (4 Gy) from 60% to 38% (p = 0.0074) and 26% (p = 0.0002), respectively. The double-drug combination reduced the irradiated survival fraction (4 Gy) more potently to 12% (p < 0.0001). After long-term drug exposure, both subclones show higher IC₅₀ values against CCNU and TMZ. For CCNU-resistant cells, both, single-drug CCNU (p = 0.0006) and TMZ (p = 0.0326) treatment combined with irradiation (4 Gy) remained effective. The double-drug-irradiation combination reduced the cell survival by 86% (p < 0.0001), compared to 92% in the parental (nonresistant) cell line. For TMZ-resistant cells, only the double-drug combination with irradiation (4 Gy) reduced the cell survival by 88% (p = 0.0057) while single-drug treatment lost efficacy. Chemoresistant cell lines demonstrated higher P-gp expression while MGMT-methylation profile analysis showed a general high methylation level in the parental and long-term treated cell lines.

Conclusions: Our findings indicate that combining CCNU with TMZ-irradiation significantly reduces canine glioma cell survival. Such a combination could overcome current challenges of therapeutic resistance to improve overall patient survival.

Keywords: O-6-methylguanine-DNA methyltransferase (MGMT); brain tumour; chemoradiation; dog; in vitro.