ABSTRACT
Title
Cytotoxic activity of human iNKT cells against ostosarcoma cells
Authors
S. Fallarini1, T. Paoletti1, G. Lombardi1
1DISCAFF Department, Faculty of Pharmacy, University of “Piemonte Orientale Amedeo Avogadro”, Novara, Italy
1DISCAFF Department, Faculty of Pharmacy, University of “Piemonte Orientale Amedeo Avogadro”, Novara, Italy
Abstract
Osteosarcoma (OS) is the most common primary solid tumor of bone in childhood and adolescence with 70-75% of cases occurring between the ages of 10 and 25. OS affects especially the long bones, where it starts intramedullary and grows toward the cortex (Ta et al., 2009).OS treatment typically consists of pre-operative chemotherapy, surgical resection, and post-operative chemotherapy. The standard drug protocol includes pre-operative and post-operative treatment with methotrexate, cisplatin, doxorubicin, and ifosfamide. Despite this aggressive treatment, however, 30-40% of children still die of OS, highlighting the need for new and more effective approaches (Ferrari et al., 2007). Recently, many investigators have focused on immunotherapy, alone or in combination with conventional drug treatment (Neyssa and Gorlick 2009), and its applicability to OS. Multiple immune strategies, including the administration of ex vivo expanded tumor-specific cytotoxic immune cells (passive or adoptive immunotherapy) or antigen-pulsed dendritic cells (active immunotherapy) have been explored (Mori et al., 2006).
The aim of our study was to evaluate in vitro weather human invariant natural killer T (iNKT) cells, a lymphocyte lineage with features of both T and NK cells, exert cytotoxic activities against OS cells, and to explore the possibility that iNKT cells potentiate drug cytotoxicity. For this purpose, iNKT cell cultures were prepared from human peripheral blood mononuclear cells (PBMC) by 10 ng/ml alpha-galactosylceramide(alpha-GalCer) + 40 U/ml IL-2 treatments for 14 days,and co-cultured (3.6 x 10-5 cells/well) with an human OS cell line (U2-OS) (0.6 x 105 cells/well) for 24 h. Afterwards, cell death was measured in U2-OS cells, fluorescently labeled with 1 mM calcein-AM (CAM), by flow cytometry (FACSVantage-SE®).
iNKT cell treatment induced a significant cytotoxicity in U2-OS cells, but not in human non-malignant monocytes. This antitumor activity is dependent on iNKT cells, since U2-OS cell death resulted significantly decreased when the same experiments were repeated in the presence of 1 mg/ml anti-Vα24Vβ11 (specific iNKT cell surface antigen). Furthermore, when U2-OS cells (0.6 x 105 cells/well) were exposed to increasing concentrations (10-1-102 μg/ml) of doxorubicin/cisplatin (24 h) or methotrexate (96 h) plus iNKT cells (3.6 x 10-5 cells/well) a potentiation of the chemotherapic-induced antitumor effect was measured. The EC50 calculated values were: 5.61 μg/ml, 10.7 μg/ml, 1.4 μg/ml and 1.5 μg/ml, 0.9 μg/ml, 0.2 μg/ml of cell death over controls in absence/presence of iNKT cells, for doxorucin, cisplatin, and methotrexate, respectively.
In conclusion, our study demonstrates that human iNKT cell treatment is able to kill tumor OS cells, but not human non-malignant monocytes. Furthermore, the antitumor effects of doxorubicin, cisplatin, and methotrexate against U2-OS cells can be potentiated by iNKT cell co-treatment.
References
Ta et al. (2009). Cancer Metastasis Rev.28: 247-263.
Ferrari et al. (2007). Curr Opin Oncol. 19:341-346.
Mori et al. (2006). Oncol Rep. 15:693-700.
Neyssa and Gorlick (2009). Cancer Biol Ther. 8:981-983.
The aim of our study was to evaluate in vitro weather human invariant natural killer T (iNKT) cells, a lymphocyte lineage with features of both T and NK cells, exert cytotoxic activities against OS cells, and to explore the possibility that iNKT cells potentiate drug cytotoxicity. For this purpose, iNKT cell cultures were prepared from human peripheral blood mononuclear cells (PBMC) by 10 ng/ml alpha-galactosylceramide(alpha-GalCer) + 40 U/ml IL-2 treatments for 14 days,and co-cultured (3.6 x 10-5 cells/well) with an human OS cell line (U2-OS) (0.6 x 105 cells/well) for 24 h. Afterwards, cell death was measured in U2-OS cells, fluorescently labeled with 1 mM calcein-AM (CAM), by flow cytometry (FACSVantage-SE®).
iNKT cell treatment induced a significant cytotoxicity in U2-OS cells, but not in human non-malignant monocytes. This antitumor activity is dependent on iNKT cells, since U2-OS cell death resulted significantly decreased when the same experiments were repeated in the presence of 1 mg/ml anti-Vα24Vβ11 (specific iNKT cell surface antigen). Furthermore, when U2-OS cells (0.6 x 105 cells/well) were exposed to increasing concentrations (10-1-102 μg/ml) of doxorubicin/cisplatin (24 h) or methotrexate (96 h) plus iNKT cells (3.6 x 10-5 cells/well) a potentiation of the chemotherapic-induced antitumor effect was measured. The EC50 calculated values were: 5.61 μg/ml, 10.7 μg/ml, 1.4 μg/ml and 1.5 μg/ml, 0.9 μg/ml, 0.2 μg/ml of cell death over controls in absence/presence of iNKT cells, for doxorucin, cisplatin, and methotrexate, respectively.
In conclusion, our study demonstrates that human iNKT cell treatment is able to kill tumor OS cells, but not human non-malignant monocytes. Furthermore, the antitumor effects of doxorubicin, cisplatin, and methotrexate against U2-OS cells can be potentiated by iNKT cell co-treatment.
References
Ta et al. (2009). Cancer Metastasis Rev.28: 247-263.
Ferrari et al. (2007). Curr Opin Oncol. 19:341-346.
Mori et al. (2006). Oncol Rep. 15:693-700.
Neyssa and Gorlick (2009). Cancer Biol Ther. 8:981-983.