p19NR-Mel, HGF-Mel, and B16 cells were transduced with GFP reporter gene by the LVs described in Fig. 1. The GFP-positive cells were sorted and maintained in culture continuously for 32 to 35 days. During this period, cells were split and analyzed by FACS every three days. (A) Time-course change in GFP-positive percentages for p19NR-Mel cells (left panel), HGF-Mel cells (middle panel), and B16 cells (right panel). The key is placed next to the right panel; FerH stands for FerH-GFP labeling, and the same for other reporters thereafter in this figure. (B) The FACS histograms of GFP intensity distribution at three time points for p19NR-Mel cells (upper panels), HGF-Mel cells (middle panels), and B16 cells (lower panels). The key for the time points of FACS analysis is at the bottom; the last time points (bright green line) for p19NR-Mel, HGF-Mel and B16 cells were day 32, 33 and 34, respectively.

FerH-Luc/GFP and Pol2-Luc/GFP LVs were constructed by replacing the original GFP reporter gene with Luc/GFP fusion gene. HeLa cells were infected with different amounts of LV supernatant as described in Methods. The GFP and luciferase expression in the transduced cells were measured by the mean fluorescence intensity (MFI) by FACS, and bioluminescence intensity (BL) by luciferase assay, respectively. (A) The regression analysis of MFI and BL in FerH-Luc/GFP-transduced HeLa cells. The correlation coefficient (R²) is 0.995. (B) The regression analysis of MFI and BL in Pol2-Luc/GFP-transduced HeLa cells. The correlation coefficient (R²) is 0.982.

HGF-RMS rhabdomyosarcoma cells were labeled with FerH-Luc/GFP or Pol2-Luc/GFP and transplanted subcutaneously into FVB/N mice, as described in Methods, and grown for two weeks. (A) Size and BL intensity of the tumors labeled with Pol2-Luc/GFP (left panels) and FerH-Luc/GFP (right panels) during the same period. Changes in growth and BL intensity of the individual tumors are shown in the upper and lower panels, respectively. (B) The BL imaging of the tumors labeled with Pol2-Luc/GFP (left panels) and FerH-Luc/GFP (right panels) at day 6 and 13 after transplantation. (C) The BL/size ratio of individual tumors labeled with Pol2-Luc/GFP (left panel) and FerH-Luc/GFP (right panel), at day 6 and 13 after transplantation. The thick bar indicates the median of the values, and the whiskers mark the middle two quartiles. The significance is analyzed by Wilcoxon matched pairs test.

(A) The structures of the six pSico-based LVs adopted and developed in this study. The name of each vector is listed to the left of each structural scheme. All vectors contain self-inactivating (SIN) LTRs, the LV packaging site (Psi), the Rev-responsive element (RRE), a central polypurine tract (cPPT), and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). FerH, the hybrid SV40 enhancer (SV40e)-human ferritin promoter. Pol2, the RNA polymerase II gene promoter. GRP78, the hybrid SV40 enhancer (SV40e)-glucose regulation protein 78 gene promoter. GRP94, the hybrid SV40 enhancer (SV40e)-glucose regulation protein 94 gene promoter. CAG, the combination of CMV enhancer (CMVe)/chicken β-actin promoter (Act)/globin first intron (G-In). (B) The reporter promoter activities of the six LVs in HeLa and melanoma cells. The four cell lines were transduced with the LVs to reach a GFP-positive ratio of between 1% to 10%, and median fluorescence intensity was measured as the index of the promoter activity.

The GFP-positive labeled mouse melanoma cells described in Fig. 2 were grown in soft agar, which allowed colonies to form from single cells within three to four weeks. The LV-mediated reporter used to label the cells is denoted by the name of the promoter above each panel. For example, FerH stand for FerH-GFP. In each panel, the bright-field images and the corresponding GFP images are at the bottom and top, respectively. The yellow and blue bars mark scale of 250 and 50 μM, respectively. (A) Colonies derived from GFP-positive p19NR-Mel cells. The dashed lines mark the edge of the colonies in the GFP images. p19NR-Mel cells are unpigmented; therefore, for all bright field images, the dark area was caused by the shadowing as the bright light projected in the middle of the colonies. (B) Colonies derived from GFP-positive HGF-Mel cells. HGF-Mel cells are unpigmented, and the darker area in the center of each colony in the bright-field images is also the shadowing from the bright light. (C) Colonies derived from GFP-positive B16 cells. Some B16 cells became pigmented in each colony.

Tumors derived from B16BL6 cells were labeled with FerH-Luc/GFP or Pol2-Luc/GFP and then transplanted subcutaneously into C57BL/6 mice, as described in Methods. After three weeks, all tumors were resected, and one tumor with average BL was selected for transplantation into mice as the next passage. The B16BL6 tumors went through three passages in this study. (A) Size and BL intensity of the tumors labeled with Pol2-Luc/GFP (left panels) and FerH-Luc/GFP (right panels) in the first passage. Changes in growth and BL intensity of the individual tumors are shown in the upper and lower panels, respectively. (B) The representative FACS GFP-scatter profiles of the labeled B16BL6 tumors of the first passage. Upper panel, unlabeled control B16BL6 cells. Middle panel, Pol2-Luc/GFP-labeled tumor #7. Lower panel, FerH-Luc/GFP-labeled tumor #1. The tumor numbers correspond to those shown in the panels of (A). (C) The BL imaging of the tumors labeled with Pol2-Luc/GFP (left pictures) and FerH-Luc/GFP (right pictures), at day 14 after transplantation, in the three passages. (D) The BL/size ratio of individual tumors labeled with Pol2-Luc/GFP (left panel) and FerH-Luc/GFP (right panel), at day 14 after transplantation in each passage. The red bar indicates the median of the values, and the blue whiskers mark the middle two quartiles. The significance is analyzed by 1-way ANOVA, Kruskal-Wallis test.