Figure 1

EGFP-positive cancer cells can be identified against a background of millions of normal cells. (A–C) A rare cancer cell in a mixture of millions of normal cells is difficult to identify under bright-field microscopy and is time-consuming (A). Under fluorescent microscopy, EGFP-positive NV1066-infected cancer cells can be easily identified by means of green fluorescence (B). Overlap of fluorescent picture with bright-field identifies the cancer cell (C) for further studies. (Upper panelshows mixture of lung cancer cells and normal cells in bronchoalveolar lavage, and lower panel demonstrates a mixture of bladder cancer and normal cells in urine.) A rare cancer cell among millions of cells can be detected and separated out for further studies by flow cytometry. (D) NV1066 selective infection of cancer cells among a mixture of millions of normal cells is confirmed by coun-terstaining with immunohistochemistry. Human mesothelioma cancer cells were mixed with normal pleural cells (upper left panel) and were incubated with NV1066 for 18 h. Examination under fluorescence microscope identified cancer cells by expression of strong green fluorescence (upper right panel). These cancer cells express integrin (CD 51/61) surface antigen. Incubation with R-PE-conjugated mouse antihuman CD51/61 monoclonal antibody confirmed that EGFP expression is selective to only cancer cells (identified by red fluorescence, lower left panel). Overlap of fluorescent pictures with bright-field identifies cancer cells among normal cells (lower right panel). Live cells among the cell clumps were identified by nuclear Hoechst staining (blue). (E) A similar experiment repeated with human lung cancer cells mixed with normal bronchoalveolar cells and incubated with NV1066 confirms similar results as described in (D).