[CANCER RESEARCH 60, 3013–3018, June 1, 2000] CD59 Expressed on a Tumor Cell Surface Modulates Decay-accelerating Factor Expression and Enhances Tumor Growth in a Rat Model of Human Neuroblastoma 1 Shaohua Chen, Theresa Caragine, Nai-Kong V. Cheung, and Stephen Tomlinson 2 Department of Pathology, New York University School of Medicine, New York, New York 10016 [S. C., T. C., S. T.], and Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [N-K. V. C.] ABSTRACT It has been hypothesized that complement inhibitors expressed on the surface of tumor cells prevent effective immune-mediated clearance. Whereas there are in vitro data to support this hypothesis, the species- selective activity of complement inhibitors has been a hindrance to inves- tigating the role of membrane-bound complement inhibitors in rodent models of human cancer. The CD59-positive LAN-1 human neuroblas- toma cell line was significantly more sensitive to lysis by rat complement than by human complement, illustrating the species selectivity of endog- enously expressed complement inhibitors. Transfection of LAN-1 cells with rat CD59, an inhibitor of the terminal cytolytic membrane attack complex, effectively protected the cells from lysis by rat complement in vitro. When LAN-1 cells stably expressing rat CD59 were inoculated into immune-deficient rats, the onset of tumor growth and the rate of tumor growth were significantly enhanced compared with those of control- transfected LAN-1 cells. These data show directly that the expression of a complement inhibitor on a tumor cell promotes tumor growth. Flow cytometric analysis revealed that the endogenous expression of decay- accelerating factor (DAF), an inhibitor of complement activation, was up-regulated on the surface of cells after in vivo growth. Of further interest, higher levels of DAF were present on CD59-transfected cells than on control-transfected cells derived from tumors. Increased DAF expres- sion correlated with decreased complement deposition on the tumor cell surface. These results show that expression of complement inhibitors on a tumor cell has functional consequences with regard to complement dep- osition in vivo and indicate that CD59 can indirectly effect complement activation and C3 deposition in vivo via a link between CD59 and DAF expression. INTRODUCTION Normal cells are protected from inappropriate complement attack by membrane-bound complement-inhibitory proteins that either pre- vent complement activation or block the formation of the terminal cytolytic MAC. 3 Tumor cells also express complement-inhibitory proteins, sometimes at elevated levels, and provide tumor cells with protection from complement-mediated injury. Blocking the function of complement inhibitors expressed on the surface of tumor cells may allow effective immune-mediated clearance of some tumors and im- prove prospects for immunotherapy using complement-activating an- titumor antibodies. Complement effector mechanisms that may be involved in host response to tumor cells include the activation and amplification of an inflammatory response, recruitment of immune effector cells, promotion and enhancement of cell-mediated lysis, and direct complement-mediated cytolysis. The major inhibitors of com- plement activation on human cells are DAF and MCP. These proteins regulate complement enzymatic complexes that are involved in the amplification of the cascade and the resulting generation of C3/C4 opsonizing fragments and physiologically active C3a and C5a pep- tides. Formation of the cytolytic and proinflammatory MAC on host cell membranes is inhibited by CD59, a glycosylphosphatidyl inositol- linked glycoprotein that binds to C8 and C9 in the assembling com- plex. Complement inhibitors have been found on nearly all primary tumors and cancer cell lines that have been examined, and some studies indicate that complement-inhibitory proteins are up-regulated on tumor cells. DAF and the serum complement inhibitor factor H or related proteins have been identified as tumor-associated antigens (1, 2), and the overexpression of DAF confers a poor prognosis in colorectal cancer patients (2). In vitro studies have shown that com- plement inhibitors expressed on tumor cells can inhibit both comple- ment opsonization and direct cytolysis by the MAC (for recent re- views of immune evasion and complement resistance of tumor cells, see Refs. 3 and 4). However, there is little information regarding how tumor-expressed complement inhibitors relate to complement deposi- tion in situ, and the in vivo relevance of complement effector mech- anisms and the importance of tumor-expressed complement inhibitors in controlling tumor growth remain largely unexplored. One reason for this is that complement inhibitor proteins (particularly CD59) are species selective, and human complement inhibitors are less effective against rat and mouse complement (5, 6). Thus, endogenous comple- ment inhibitors expressed on the surface of human tumor cells will not provide the cells with effective protection from complement in rodent models of human cancer. Indeed, the species-selective activity of membrane complement-inhibitory proteins may be a basis for obser- vations that complement-activating mAbs effective at causing regres- sion of human tumors in rodents have, in most cases, proven ineffec- tive in clinical trials. When investigating the role of complement-inhibitory proteins in immune evasion of tumor cells in vivo, it is therefore relevant to study rodent complement inhibitors in rodent models of cancer. The ubiq- uitous and high level of expression of membrane complement inhib- itors on normal tissues has not allowed for the targeted blocking of complement inhibitors (using current technologies) on tumor cells in syngeneic rodent models of cancer. In the studies described here, we investigated the effect of heterologously expressed rat CD59 on the growth of a human neuroblastoma cell line in nude rats. The neuro- blastoma cell line endogenously expressed CD59, but we have pre- viously determined on a quantitative basis that human CD59 is sev- eralfold less effective at inhibiting rat complement compared to human complement (6). The data show for the first time in vivo that the complement inhibitor CD59 expressed on a tumor cell surface significantly promotes tumor growth. We also show that growth in vivo resulted in the up-regulation of DAF on the tumor cell surface and that the level of DAF expression was further up-regulated by the expression of functional CD59. Received 2/14/00; accepted 4/17/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grant AI 34451 and Department of the Army Grants DAMD17- 97-1-7273 and DAMD12-99-1-9325. 2 To whom requests for reprints should be addressed, at Department of Microbiology & Immunology, Medical University of South Carolina, BSB 201, 173 Ashley Avenue, Charleston, SC 29425, E-mail: tomlinss@musc.edu. 3 The abbreviations used are: MAC, membrane attack complex; DAF, decay-acceler- ating factor; MCP, membrane cofactor protein; mAb, monoclonal antibody. 3013 Research. on November 20, 2015. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from