Optimizing the methods of whole lung cancer RNA-loaded dendritic cells

Abstract

Dendritic cells (DCs) are the unique antigen-presenting cells that can activate naive T lymphocytes. This function is critical for inducing specific immune response. DCs-based vaccines have been used broadly in immunotherapy for many carcinomas. Constructing vaccines by transfecting total tumor RNA into DCs can be done with a few tumor tissues and need not to identify tumor antigens, so it is especially suitable for lung cancer which lacks tumor-specific antigens but has great heterogenicity and weak immunogenicity. Currently, the best transfection stage and method are still indefinite. So, the objective of this study is to explore the best condition of transfecting total RNA extracted from lung cancer tissues into DCs. Ten patients with lung cancer were enrolled whose tumor tissues were CEA and MUC1 positive in immunohistochemical staining. Total tumor RNA were extracted by one-step method. Then DCs and T cells were separated and cultured from peripheral blood monocytes and the RNA was transfected into the DCs in different stages with different methods. CEA and MUC1 expression in the transfected DCs were measured by flow cytometry analysis and T cells' proliferation was examined by mixed lymphocyte reaction (MLR). The expression of CEA and MUC1 protein in immature DCs (11.33±2.64, 39.68±7.25) was remarkably higher than that in mature DCs (5.46±1.63, 27.17±4.16) after transfection with total RNA of lung cancer tissues (P < 0.01), and the DCs presented more powerful effects on T cell proliferation. The CEA and MUC1 expression on DCs were significantly higher in electroporation transfection group (20.53±3.64, 65.39± 9.33) than that in lipofection group (11.33±2.64, 39.68±7.25) and passive pulsing transfection group ( 0.91±0.27,18.53±3.26)(P < 0.01), and the DCs in electroporation transfection group presented more powerful effects on stimulating T cell proliferation than the other two groups did. Transfecting total tumor RNA into immature DCs by using electroporation is a good way to construct DCs-based vaccines for lung cancer and to achieve a higher activity to stimulate T cell proliferation.