JNCI Journal of the National Cancer Institute

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• Supplementary Fig. 1 - Transduction efficiency and viral spread of relaxin-expressing adenovirus in tumor spheroids. A) Xenograft tumor spheroids from mice. We prepared tumor spheroids from U343, U87MG, C33A, and A549 xenograft tumors in mice, as indicated. Spheroids were transduced with adenoviruses dl-lacZ, which did not express relaxin (columns 1), or dl-lacZ-RLX, which expressed relaxin (columns 2), at 1 x 108, 1 x 109, and 1 x 1010 viral particles (v.p.) per 50 μL of culture medium. Three days after infection, spheroids were processed to visualize β-galactosidase expression (blue) and then sectioned. Viral penetration and transduction efficiency in the central areas of the spheroids were then assessed by light microscopy. In each panel, columns to the left show whole spheroids (stereoscopic light microscope; original magnification = x38) and columns to the right show sections through the spheroid midpoint (original magnification = x 40). B) Primary human tumor spheroids. Primary human tumors from a patient with breast cancer and a patient with colon cancer were transduced with 1 x 1010 v.p. of dl-lacZ or dl-lacZ-RLX adenovirus, as described above. Three days after infection, tumor tissues were processed to visualize β-galactosidase expression (stereoscopic light microscope; original magnification = x38). Results are representative of three independent experiments, with triplicate samples for each experiment.
• Supplementary Fig. 2 - Transduction efficiency and viral spread of relaxin-expressing adenoviruses. These adenoviruses used green fluorescent protein (GFP) as the reporter gene. A) C33A tumor xenograft. The xenograft established in nude mice was injected three times with dl-GFP (that do not express relaxin) or dl-GFP-RLX (that express relaxin) (each at 5 x 1010 viral particles in 50 μL per injection). Three days after the last injection of adenoviruses, tumors were harvested and photographed with an inverted fluorescent microscope. In the tumor treated with dl-GFP, we observed a narrow path of fluorescence that appeared to follow the needle track when we injected the viruses. In contrast, in the tumor treated with dl-GFP-RLX, fluorescence was distributed through the tumor mass. B) Tumor spheroids prepared from a human patient with breast cancer. Spheroids were transduced with either dl-GFP or dl-GFP-RLX (each with 1 x 109 v.p.). Three days later, the spheroids were photographed with an inverted fluorescent microscope. In A and B, increased gene transfer, as shown by green fluorescence, was achieved with relaxin-expressing adenoviruses than with the control adenovirus. Representative micrographs are shown of three independent experiments (three replicates for each experiment), all with similar results. (Original magnifications = x10, x40.)
• Supplementary Fig. 3 - Relaxin and the induction of apoptosis. A) Percentage of sub-G1 cells as a measure of apoptosis. A549 or U343 cells were transfected by use of Lipofectamine with an expression plasmid encoding relaxin (pSP72∆E3-RLX) and its cognate control plasmid (pSP72∆E3) or were treated with Lipofectamine alone, as described by the manufacturer (Gibco BRL). Forty-eight hours after transfection, the sub-G1 cell population was assessed by flow cytometry. For A549 cell cultures, control Lipofectamine cultures had 2.35% sub-G1 cells, pSP72∆E3 (3 μg) had 1.74%, and pSP72∆E3-RLX (3 μg) had 10.96% (difference between pSP72∆E3 3 μg and pSP72∆E3-RLX 3 μg = 9.22%, 95% CI = 6.51% to 11.93%; P = .001). For 549 cell cultures, pSP72∆E3 (5 μg) had 5.11% sub-G1 cells and pSP72∆E3-RLX (5 μg) had 50.43% (difference between pSP72∆E3 5 μg and pSP72∆E3-RLX 5 μg = 45.32%, 95% CI = 41.29% to 49.35%; P<.001). For U343 cell cultures, control Lipofectamine had 5.80% sub-G1 cells, pSP72∆E3 (3 μg) had 3.45%, and pSP72∆E3-RLX (3 μg) had 12.79% (difference between pSP72∆E3 3 μg and pSP72∆E3-RLX 3 μg = 9.34%, 95% CI = 5.66% to13.02%; P = .002). For U343 cell cultures, pSP72∆E3 (5 μg) had 2.35% sub-G1 cells and pSP72∆E3-RLX (5 μg) had 50.43% (difference between pSP72∆E3 5 μg and pSP72∆E3-RLX 5 μg = 48.08%, 95% CI = 40.63% to 55.53%; P<.001). Plasmid-mediated relaxin expression statistically significantly increased the size of the sub-G1 population in U343 and A549 cell cultures. Results are representative of three independent experiments (all with similar results), with each point the mean and 95% CI of triplicate samples. B) Micrographs of cultures corresponding to results in panel A. A representative field from three independent experiments (all with similar results) is shown. (Original magnifications = x200).