NK CELLS FROM GLIOMA-BEARING MICE TREATED WITH TEMOZOLOMIDE ARE ENRICHED FOR GENES RELATED TO MULTI-DRUG RESISTANCE AND CHEMOTAXIS

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Abstract

BACKGROUND: Chemotherapy can influence the immune response by inducing immunogenic death of tumor cells or modulating tumor microenvironment (Galluzzi et al. 2012). The limitations of chemotherapy and immunotherapy as single therapeutic modalities have generated considerable interest in combinatorial strategies (Zitvogel L et al 2008). Here we focused on understanding the molecular mechanisms directly induced by temozolomide (TMZ), the standard chemotherapeutic agent for glioblastoma, that can activate immune cells. METHODS: We studied the in vivo effects of TMZ on immune cells by treating mice 9 days after intracranial implantation of GL261 gliomas with intraperitoneal injections of TMZ (5 mg/kg) or vehicle (control mice) for 5 days. Mice were sacrificed 20 hours after treatment on days 1 to 5: tumor and peripheral blood were harvested and analyzed by flow cytometry. RESULTS: Trafficking of NK1.1+ CD3- NK cells in blood, but not of CD8+ T cells, and their homing ability into the brain significantly increased in TMZ-treated mice on day 12 after the second administration of TMZ. At this early time point TMZ led to a significant enrichment of CD11b(low) CD27(high) and CD11b(high) CD27(high) subsets. Microarray analysis revealed differentially expressed genes represented in different clusters of which chemotaxis, cell cycle, multidrug resistance and anti-apoptosis were predominantly up-regulated in blood-derived NK cells isolated from TMZ-treated compared to control mice. We focused validation experiments on genes related to multidrug resistance and chemotaxis. Chemotaxis was assayed using the in vitro transwell system confirming that the migration ability of NK cells from treated mice significantly increased by 3-fold compared with controls. Increased expression of three ATP transporter genes was confirmed by real time PCR (2.6-fold higher than controls, P = 0.006). Five days after the end of the treatment we found an enrichment of CD11(high) CD27(low), the most potent effector subset of NK cells (Chiossone et al 2009). In agreement, IFN-gamma production evaluated by flow cytometry and the in vitro cytotoxic activity of NK cells from TMZ-treated mice were higher than that of NK cells from control mice. CONCLUSIONS: Our results support the idea that NK cells can be resistant to chemotherapy (Lowdell 2003; MacDonald HR et al 1995). In particular at early time points TMZ leads to an enrichment of NK cells with migratory function, in the later phase TMZ leads to an increase of NK cells promoting cytotoxic ability. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

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