Relaxin Expression From Tumor-Targeting Adenoviruses and Its Intratumoral Spread, Apoptosis Induction, and Efficacy

Relaxin Expression From Tumor-Targeting Adenoviruses and Its Intratumoral Spread, Apoptosis Induction, and Efficacy Joo-Hang Kim, Young-Sook Lee, Hoguen Kim, Jing-Hua Huang, A-Rum Yoon, Chae-Ok Yun 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-ΔE1BRLX) adenoviruses by inserting a relaxin gene into the E3 adenoviral region. Controls were parental adenoviruses (dllacZ and Ad-ΔE1B) 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-ΔE1B-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-ΔE1B-RLX, and 50%, 95% CI = 15.7% to 84.3%, for C33A tumor-bearing mice treated with Ad-ΔE1B). Infection with Ad-ΔE1B-RLX substantially decreased the collagen content of tumor tissue but not of adjacent normal tissue, compared with noninfected tissues. Intratumoral injection of Ad-ΔE1B-RLX inhibited the formation of lung metastases in mice (PBS = 268 mg of metastatic tumor per mouse and Ad-ΔE1B-RLX = 10 mg; difference = 258 mg, 95% CI = 94 to 426; P = .003, Mann–Whitney test). Systemic treatment with Ad-ΔE1B-RLX completely inhibited the growth of Hep1 hepatocellular carcinomas (PBS = 20.2 mg of tumor per mouse and Ad-ΔE1B-RLX = 0 mg; difference = 20.2 mg, 95% CI = 3.7 to 36.7; P = .004, Mann–Whitney test). Conclusion: Extracellular matrix degradation by relaxin expressed by adenoviruses increased viral distribution and tumor penetration, inhibited tumor growth and metastasis, and increased survival of mice. [J Natl Cancer Inst 2006;98:1482–93] 1482 ARTICLES Although the selective replication and spread of oncolytic adenoviruses within cancer cells and tissues has had some success as an anticancer treatment, its promise has been limited because of the uneven penetration and distribution of these viruses in tumor tissues. To date, ONYX-015, an oncolytic adenovirus in which the E1B 55-kDa gene has been deleted, has been administered to more than 300 cancer patients with various tumor types (1,2) but has had only limited success (i.e., local tumor regression rates of 0%–14%). Sauthoff et al. (3) demonstrated that high levels of adenoviruses persisted in xenograft tumors for at least 8 weeks after intratumoral injection of wild-type adenoviruses, but the viral distribution pattern in the tumor tissue was uneven and patchy. Harrison et al. (4) detected a high level of viruses in persistent viable tumors up to 100 days after the initial viral injection. Thus, despite long-term viral persistence in tumors, the limited spread of virus to cells throughout the tumor could explain the low response rates observed. Connective tissue and the extracellular matrix appear to play a role in inhibiting viral spread in tumors. Kuriyama et al. (5) demonstrated that treatment of human U87, U251, or SF767 glioblastoma multiforme–derived brain tumor xenografts with collagenase, dispase, or trypsin, before the intratumoral injection of adenovirus enhanced virus-mediated gene transduction, and Maillard et al. (6) reported that elastase pretreatment before delivery of adenoviral vectors into rabbit iliac arteries increased viral transduction efficiency. Relaxin is a peptide hormone that is structurally related to insulin and insulin-like growth factors (7). Treatment of human lung fibroblasts and bleomycin-induced mouse lung fibrosis (i.e., the common end stage of many pneumopathies) tumors with relaxin decreases the synthesis and secretion of interstitial collagens and increases the expression of matrix metalloproteinase and procollagenase (8). Relaxin is a potent inhibitor of collagen expression when collagen is overexpressed, but it does not markedly alter basal levels of collagen expression, in contrast to other collagen-modulatory cytokines, such as interferon gamma (9). To further explore the barrier role of the extracellular Affiliations of authors: Brain Korea 21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center (JHK, YSL, JHH, ARY, COY) and Department of Pathology (HK), Yonsei University College of Medicine, Seoul, Korea. Correspondence to: Chae-Ok Yun, PhD, Yonsei Cancer Center, Yonsei University College of Medicine, 134 Shinchon-Dong, Seodaemun-Gu, Seoul, Korea (e-mail: ). See “Notes” following “References.” DOI: 10.1093/jnci/djj397 © 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/2.0/uk/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Journal of the National Cancer Institute, Vol. 98, No. 20, October 18, 2006 matrix and connective tissue in inhibiting viral spread and penetration within tumor masses, we determined whether the expression of relaxin by adenoviruses increases their spread in tumor tissues. MATERIALS AND METHODS Cell Lines and Cell Culture The human embryonic kidney cell line 293 that expresses the adenoviral E1 region, the brain cancer cell lines U343 and U87MG, the cervical cancer cell line C33A, the liver cancer cell line Hep3B, and the non–small lung cancer cell line A549 were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Murine B16BL6 melanoma cells (a metastatic variant of B16 melanoma cells) were obtained from Dr Y. S. Park (Yonsei University, Wonju, South Korea). All cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco BRL), 2 mM l-glutamine, penicillin (100 (...truncated)