Figure 1.
Sensitivity of PLuc-G-U87 cells, Rluc-R-tTK-hAMSC and hAMSC and co-cultures comprising different proportions of Rluc-R-tTK-hAMSCs and PLuc-U87 cells to GCV.
Cells were grown during the indicated times and treated with either GCV (4 µg/ml) or PBS, as indicated. Number of viable cells was evaluated spectrophotometrically by standard 3-(4-5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS assay) (A, B), or by BLI (C), and expressed as percentage increase relative to cell number at day 0. Histograms show mean ± SEM, *p<0.05, **p<0.01 n = 4 for each group.
Figure 2.
The indicated numbers of Pluc-G-U87 (A) and Rluc-R-tTK-hAMSC cells (B) were stereotactically implanted, at the same coordinates later used for experiments in the brain of immunosupressed mice and imaged after luciferin or coelenterazine substrate administration, respectively. The number of light events (PHCs) recorded from the mice brains was plotted, after background subtraction, versus the number of implanted cells. R2 correlation coefficient. Data are presented as mean ±SEM, n = 4 in each group.
Figure 3.
In vivo optimization of tumor to therapeutic cell ratio.
Tumor and therapeutic cells mixed in proportions 1∶0, 1∶1, 1∶2 and 1∶4 (Pluc-G-U87∶Rluc-R-tTK-hAMSC) were implanted in the brain of SCID mice and treated at day 6 i.p. with either GCV or PBS as indicated and monitored by BLI at weekly intervals. (A) The graph shows in vivo changes in light production by Pluc expressing tumor cells resulting from GCV treatment. (B) Histogram showing PHC values at the end of the experiment (T = 35). Values were normalized relative to those at T = 0 according to the formula [(T5/T0)×100 = tumor variation (%)]. Data are shown as mean ± SEM, *P<0.05, compared to the 1∶0+GCV group, n = 5 for each group. (C) Representative pseudo-color BLI images showing evolution of tumor size during treatment of animals inoculated with the indicated proportions of Pluc-G-U87 to Rluc-R-tTK-hAMSC. Color bars represent light intensity levels from Pluc luciferase, ranging from low: blue to high: red. Luciferase images are superimposed on black and white with images of the same mouse.
Figure 4.
In vivo BLI of hAMSC mediated tumor therapy.
(A) Diagram illustrating experimental procedure. (B) Composite pseudo-color BLI images from mice implanted with 4×104 Pluc-G-U87 plus 1.6×105 Rluc-R-tTK-hAMSCs and treated with GCV (1∶4+GCV); 4×104 Pluc-G-U87 plus 1.6×105 Rluc-R-tTK-hAMSC and treated with PBS (1∶0+GCV), and with 4×104 Pluc-G-U87 only and treated with GCV. Images were acquired once per week. (C) Representative images of brain sections at day 49. Top: brain of a 1∶4+GCV treated mouse. Bottom: brain of a 1∶0+GCV mouse, arrow points to tumor area. (D) In vivo changes in light production by U87 tumors expressing Pluc, resulting from GCV treatment and tumor cell death. Light values, calculated from the recorded images, are represented as log10 mean ± SEM; n = 8; * p<0.05, compared to the 1∶0+GCV. (E) Representative pseudo-color images from Rluc-R-tTK-hAMSC and Pluc-G-U87 at day of implantation, T = −6 (1 and 3) and at initiation of GCV treatment, T = 0 (2 and 4). Luciferase images are superimposed on black and white images of the same mouse. The included numerical values indicate the cell numbers extrapolated from the “light vs cells" standard curve. Arbitrary color bars illustrate relative light intensities from PLuc and Rluc luciferases, lowest: blue and black; highest: red and blue, respectively.
Figure 5.
Effect of GCV on fluorescent Rluc-R-tTK-hAMSC at tumor implantation sites.
Left, representative images of HE stained brain sections; Right, representative fluorescence confocal microscope images showing implanted red fluorescent cells Rluc-R-tTK-hAMSC (arrow), and Pluc-G-U87 cells (green). (A) Control mouse (1∶4+PBS) at T = 0, (B) control mouse (1∶4+PBS) at T = 49, (C) treated mouse (1∶4+GCV) at day 49. Blue, Hoescht stained nuclei. Scale bar = 10 µm.
Figure 6.
Endothelial differentiation of hAMSCs in U87 tumors.
Double transduced hAMSCs expressing PECAM-promoter regulated PLuc-GFP and CMV-promoter regulated Rluc-R-tTK were mixed 4∶1 with unlabelled U87 glioma cells, implanted in the brain of mice and imaged at the indicated times. Mice were then inoculated with a 2,000,000 MW FITC-conjugated dextran and the brains harvested, fixed in formalin and prepared for microscopy. (A) Representative BLI images showing Rluc (left) and Pluc (right) activities at T = 0 (1 and 2) and T = 7 (3 and 4), respectively. Pseudo color images are superimposed on black and white dorsal images of recipient mice. Color bars illustrate relative light intensities of Pluc (right) and Rluc (left); low: blue and black; high: red and blue, respectively. (B) Histograms showing the change in the ratio of Pluc/Rluc activities during the indicated time period. Bars represent mean ± SEM of photon counts recorded in BLI images. * p<0.05, n = 4. (C) Representative laser confocal microscope images of the corresponding brain tumor slices showing hAMSCs expressing CMV-promoter regulated RFP (red) and PECAM-promoter regulated eGFP (green). Scale bar = 20 µm. (D) FITC-stained microvessel (green), associated with red fluorescent hAMSCs (red), also labeled by an anti human PECAM antibody (gray) Scale bar = 25 µm.
Figure 7.
Response of U87 tumors to direct implantation of therapeutic cells and GCV treatment.
(A) In vivo changes in light production by U87 tumors expressing Pluc, resulting from inoculation of Rluc-R-tTK-hAMSCs and GCV treatment. Dots represent PHCs values from individual animals (T: Treated mice; C: Control mice). The bars represent the average values from each group. Arrows indicate the inoculation of Rluc-R-tTK-hAMSCs. Light values, calculated from the recorded images, are represented as log10 of the mean ± SEM; n = 4; * p<0.05, compared to the control group. (B) Kaplan-Meyer graph sumarizing mice survival following the above treatment. Median survival, treated mice, 88,5 d; control mice, 51 d, p<0.05.