Relaxin expression from tumor-targeting adenoviruses and its intratumoral spread, apoptosis induction, and efficacy.
BACKGROUND
The use of oncolytic adenoviruses as cancer gene therapy is limited by their uneven penetration and distribution in tumors. We investigated whether the expression of the cell matrix-degradative protein relaxin by adenovirus could improve adenovirus distribution and penetration in tumors.


METHODS
We generated relaxin-expressing, replication-incompetent (dl-lacZ-RLX) and -competent (Ad-deltaE1B-RLX) adenoviruses by inserting a relaxin gene into the E3 adenoviral region. Controls were parental adenoviruses (dl-lacZ and Ad-deltaE1B) and phosphate-buffered saline (PBS) (vehicle). Replication-incompetent viruses, which do not lyse cells, were used to assess transduction efficiency. Viral spread in tumor spheroids, made by dissecting tumor tissue into homogeneous fragments, was assessed by reporter gene (i.e., lacZ) expression. Tumor growth inhibition was assessed by injecting adenoviruses into xenograft tumors in athymic mice (n = 8 or 9). Overall survival was assessed by the Kaplan-Meier method. Extracellular matrix was examined with Masson's trichrome staining. Therapeutic efficacy was evaluated by assessing spontaneous pulmonary metastasis in the B16BL6 melanoma mouse model and growth inhibition of orthotopically implanted hepatoma (n = 4-6). All statistical tests were two-sided.


RESULTS
In tumor spheroids and established solid tumors in vivo, transduction with dl-lacZ-RLX, compared with parental virus or vehicle, elicited higher transduction efficiency and viral spread throughout the tumor mass. Infection with Ad-deltaE1B-RLX, compared with parental virus, elicited greater viral persistence and spread, leading to increased survival (e.g., 100%, 95% confidence interval [CI] = 63.1% to 100%, for C33A tumor-bearing mice treated with Ad-deltaE1B-RLX, and 50%, 95% CI = 15.7% to 84.3%, for C33A tumor-bearing mice treated with Ad-deltaE1B). Infection with Ad-deltaE1B-RLX substantially decreased the collagen content of tumor tissue but not of adjacent normal tissue, compared with noninfected tissues. Intratumoral injection of Ad-deltaE1B-RLX inhibited the formation of lung metastases in mice (PBS = 268 mg of metastatic tumor per mouse and Ad-deltaE1B-RLX = 10 mg; difference = 258 mg, 95% CI = 94 to 426; P = .003, Mann-Whitney test). Systemic treatment with Ad-deltaE1B-RLX completely inhibited the growth of Hep1 hepatocellular carcinomas (PBS = 20.2 mg of tumor per mouse and Ad-deltaE1B-RLX = 0 mg; difference = 20.2 mg, 95% CI = 3.7 to 36.7; P = .004, Mann-Whitney test).


CONCLUSIONS
Extracellular matrix degradation by relaxin expressed by adenoviruses increased viral distribution and tumor penetration, inhibited tumor growth and metastasis, and increased survival of mice.
Kim
Joo-Hang
JH
Brain Korea 21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemun-Gu, Seoul, Korea.
Lee
Young-Sook
YS
Kim
Hoguen
H
Huang
Jing-Hua
JH
Yoon
A-Rum
AR
Yun
Chae-Ok
CO
D000256:Adenoviridae; D000818:Animals; D017209:Apoptosis; D003238:Connective Tissue / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000378:metabolism; D005434:Flow Cytometry; D015967:Gene Expression Regulation, Viral; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D051379:Mice; D008819:Mice, Nude; D009369:Neoplasms / Q000235:genetics / Q000628:therapy*; D012065:Relaxin / Q000235:genetics / Q000378:metabolism*; D018874:Spheroids, Cellular; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous
D012065:Relaxin