Tracking of green fluorescent protein (GFP)-labeled LAK cells in mice carrying B16 melanoma metastases.

In adoptive immunotherapy, in vivo trafficking of adoptively transferred cells, including their accumulation at tumor sites, remains to be further investigated. Tracking of these cells by visualization is useful to clarify antitumor mechanisms and develop new modalities to enhance antitumor capacities. In the present study, an in vivo tracking study was performed using an adoptive transfer model of lymphokine- activated killer (LAK) cells induced from green mice into C57/BL6 mice with B16 melanoma metastases. Green mice are green fluorescent protein (GFP) transgenic mice originating from C57/BL6 mice. All of the tissues, except for erythrocytes and hair, express green fluorescence under excitation light. Although LAK cells in combination with IL-2 potently suppressed pulmonary metastases with survival prolongation, very few LAK cells accumulated in tumor tissues compared to those localized in the spleen, as visualized by fluorescent microscopy and quantitated by flow cytometry. The present method using transfer of green mice-derived cells into parental tumor-bearing mice is simple because there is no need for in vitro labeling and is feasible for the in vivo tracking of effector cells in an adoptive immunotherapy model. Adoptive immunotherapy (AIT) has been intensively studied using lymphokine-activated killer (LAK) cells, tumor- infiltrating lymphocytes (TIL) or cytotoxic T lymphocytes (CTL) in large numbers of preclinical and clinical studies, since the first report of AIT with LAK cells and interleukin- 2 (IL-2) in 1982 (1, 2). However, in contrast to the promising results obtained in animal studies, clinical results were generally disappointing. Although the therapeutic efficacy of AIT is evaluated by tumor regression or prolongation of survival, in vivo visualization of the trafficking of adoptively transferred cells and their accumulation into tumor tissues may contribute to elucidating antitumor mechanisms or developing new modalities to enhance antitumor capacities. Green fluorescent protein (GFP) is a protein derived from the jellyfish Aequorea victoria, which expresses green fluorescence under excitation light (3). Green mice are GFP-transgenic mice derived from C57/BL6 mice in which all the tissues, except for erythrocytes and hair, can express green fluorescence (4). Transfer of green mice-derived cells into C57/BL6 mice may enable their stable and long-term in vivo tracking (5). In the present study, we performed an in vivo tracking study using an adoptive transfer model of LAK cells induced from green mice into C57/BL6 mice with B16 melanoma metastases.