Volume 15, Issue 11, Pages 1982-1990 (November 2007) Human Matrix Metalloproteinase-8 Gene Delivery Increases the Oncolytic Activity of a Replicating Adenovirus  Jin Cheng, Harald Sauthoff, YaoQi Huang, David I Kutler, Sofia Bajwa, William N Rom, John G Hay  Molecular Therapy  Volume 15, Issue 11, Pages 1982-1990 (November 2007) DOI: 10.1038/sj.mt.6300264 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Virus diffusion across a collagen matrix. The bases of 24-well plates were pre-seeded with 293 cells. After 24 hours' incubation, inserts containing Adβ-gal virus (multiplicity of infection of 5) diluted in 0.3 ml of minimum essential medium were placed onto the wells. The inserts had a 3-μm pore size and were pre-coated with collagen I. The control insert had no coating. After 24 hours' infection, the ability of the virus to spread through the insert was analyzed. In situ staining of cells for β-galactosidase (β-gal) is shown. (a) Control wells allowed free diffusion of the virus. (b) Collagen I markedly reduced the proportion of cells expressing β-gal compared with (a) control, and this can be restored to control levels by (c) pre-treatment with collagenase. Molecular Therapy 2007 15, 1982-1990DOI: (10.1038/sj.mt.6300264) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Construction and activity of AdMMP8. (a) Schematic of AdMMP8, an E1-deleted non-replicating adenovirus expressing the human matrix metalloproteinase-8 (MMP-8) full-length complementary DNA under the control of a cytomegalovirus (CMV) promoter. (b) A549 cells infected with AdMMP8 express MMP-8 messenger RNA as detected by real-time polymerase chain reaction (lane 3). The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a control. Controls include non-infected cells (lane 1) and control vector–infected cells (lane 2). (c) Medium from cultures of infected cells was separated on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) glycine gel for immunoblotting with a goat polyclonal anti-MMP-8 antibody. Evidence of MMP-8 protein expression is seen in the AdMMP8-infected group in lane 3. (d) Supernatants from AdMMP8-infected cells show collagen-degrading activity (lane 3), as shown on 10% SDS-PAGE glycine with 0.1% gelatin gel (zymogen gel). Adcon, AdMMP8-infected control virus; LITR, left inverted terminal repeat; RITR, right inverted terminal repeat. Molecular Therapy 2007 15, 1982-1990DOI: (10.1038/sj.mt.6300264) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Effects of matrix metalloproteinase-8 (MMP8)–expressing cells on fibrillar collagen. Fibrillar collagen inserts conditioned by A549 cells infected with AdMMP8, but not Adcon-infected cells, facilitate diffusion of an adenoviral reporter construct. Adcon, control virus; AdMMP8, non-replicating adenoviral vector that expresses MMP-8; β-gal, β-galactosidase. Molecular Therapy 2007 15, 1982-1990DOI: (10.1038/sj.mt.6300264) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Effects of AdMMP8 on cell survival and viral replication. (a) Viability of A549 cells infected with AdMMP8. A549 cells were infected with AdMMP8 or Adcon at the multiplicities of infection (MOIs) shown. Control cells remained uninfected. Cell viability was measured using a WST-1 assay and is presented as a percentage of uninfected cell control values. AdMMP8 did not modulate cell viability compared with control virus. The experiment was performed on four occasions and the titers measured in duplicate for each experiment. (b) Replication of wild-type adenovirus in the presence of AdMMP8. A549 cells were infected with Adwt300 at an MOI of 1 alone (gray) or in combination with AdMMP8 (solid) or control virus Adcon (white) in a dose-response as shown. Total viral yield was no different in the AdMMP8 dose-response compared with the Adcon dose-response. The experiment was performed twice and the titer measured in duplicate. Mean values are shown. AdMMP8, non-replicating adenoviral vector that expresses matrix metalloproteinase-8. Molecular Therapy 2007 15, 1982-1990DOI: (10.1038/sj.mt.6300264) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 AdMMP8 in combination with Adwt300 reduces tumor growth. (a) A Kaplan–Meir analysis and log-rank test of animals bearing A549 lung cancer xenografts. Significant differences were seen between AdMMP8/Adwt300-injected and Adwt300/Adcon-injected animals (P = 0.008) on the basis of the time to a threefold increase in tumor volume (n= 6 each group). (b) A Kaplan–Meir analysis and log-rank test of animals bearing BxPC-3 pancreatic cancer xenografts. Significant differences were seen between AdMMP8/Adwt300-injected and Adwt300/Adcon-injected animals (P = 0.002) on the basis of the time to a threefold increase in tumor volume (n= 7 each group). (c) Tumor growth curve for A549 lung cancer. At day 26, tumors injected with Adwt300/AdMMP8 were approximately one-fifth the size of vehicle-injected control group tumors (508 ± 158 versus 2,530 ± 770) and one-third the size of Adwt300/AdCon-injected group tumors (508 ± 158 versus 1,436 ± 627). (d) Tumor growth curve for BxPC-3 pancreatic cancer. At day 44, Adwt300/AdMMP8 group tumors were one-seventh of the size of vehicle-treated control group tumors (200 ± 42 versus 1,488 ± 363) and approximately one-quarter the size of Adwt300/Adcon-injected group tumors (200 ± 42 versus 772 ± 196). Adcon, control virus; AdMMP8, non-replicating adenoviral vector that expresses matrix metalloproteinase-8. Molecular Therapy 2007 15, 1982-1990DOI: (10.1038/sj.mt.6300264) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Matrix metalloproteinase-8 (MMP8) messenger RNA (mRNA) and protein expression and collagenase activity in tumor lysates. (a) In vivo mRNA expression. Total RNA extracted from the fresh tumor tissues was used as template for real-time polymerase chain reaction of MMP-8. Four of six mice tumors in the Adwt300/AdMMP8 virus group were positive at 42 days from the time of injection; three of six mice tumors in the Adwt300/Adcon group were positive at day 26, when these animals with rapidly growing tumors were killed. The positive control is A549 cells infected with AdMMP8 in vitro (lane 1); the negative control is from a vehicle-injected tumor (lane 2). The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as a control. (b) MMP-8 protein expression is persistent. An MMP-8 enzyme-linked immunosorbent assay was performed on tumor lysates obtained from BxPC-3 pancreatic xenografts harvested between days 49 and 52. Compared with the control group, significantly higher levels of MMP-8 protein were detectable in the tumors that received AdMMP8/Adcon (P = 0.0001) or AdMMP8/Adwt300 (P = 0.0001). There was no difference between the AdMMP8/Adcon and AdMMP8/Adwt300 groups. (c) Collagenase activity can be detected in tumors infected with both AdMMP8 and Adwt300. All of the BxPC-3 tumor lysates were subject to zymography, apart from one tumor in the Adwt300/AdMMP8 group that was too small for further analysis. A clear band of collagenase activity is seen in the Adwt300/AdMMP8 group (lanes 5 and 6), which is at the same size as the positive control band (lane 9). Low background levels of activity are seen in the other groups. One representative blot of three is shown, but all of the AdMMP8/Adwt300 tumors (n = 6) showed activity on zymography, whereas none of the AdMMP8/Adcon group (n = 7) showed activity. Adcon, control virus; AdMMP8, non-replicating adenoviral vector that expresses MMP-8. Molecular Therapy 2007 15, 1982-1990DOI: (10.1038/sj.mt.6300264) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Collagen expression and viral protein expression within xenograft tumors. Immunohistochemistry (IHC) for adenoviral capsid proteins (a, c, e, g) and Masson's trichrome staining for collagen (b, d, f, g) in aligned serial sections. (a) As anticipated, no evidence of viral protein expression was seen in the control uninfected A549 xenograft tumors. (b) Abundant and dense collagen bands staining blue were seen in the trichrome stained control uninfected tumors. (c) The presence of adenovirus as detected by IHC was seen in the Adwt300/AdMMP8 injected tumors (many brown staining cells apparent in upper right quadrant of image, insert shows enlarged image). (d) This was associated with collagen degradation and extensive necrosis. (e) In the Adwt300/AdCon virus-injected tumors replicating adenoviral spread was inefficient, only a few cell in these tumors were infected on the basis of IHC for viral proteins (insert shows enlarged image) and (f) abundant collagen bands were present in necrotic and surrounding areas. However, AdMMP8/AdCon injected tumors showed (g) no adenoviral staining and (h) little evidence of collagen degradation. Adcon, control virus; AdMMP8, non-replicating adenoviral vector that expresses MMP-8. Molecular Therapy 2007 15, 1982-1990DOI: (10.1038/sj.mt.6300264) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 8 Intensity of collagen expression within xenograft tumors. The amounts of collagen within necrotic areas were scored by two blinded observers by ranking the intensity of collagen staining for all the tumors. A significant reduction in collagen within the Adwt300/AdMMP8 tumors compared with Adwt300/Adcon was seen (P < 0.0001). The AdMMP8/Adcon group also scored with less collagen than the vehicle control group (P = 0.0005) and Adwt300/Adcon group (P < 0.0001). Adcon, AdMMP8-infected control virus; AdMMP8, non-replicating adenoviral vector that expresses MMP-8. Molecular Therapy 2007 15, 1982-1990DOI: (10.1038/sj.mt.6300264) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions