Journal of Leukocyte Biology Volume 56, July 1994 41 Role of macrophages in the immunotherapy of Lewis lung peritoneal carcinomatosis Paul K. Wallace and Page S. Morahan Department of Microbiology and Immunology, Medical Abstract: The underlying cellular mechanisms for the antitumor effects of biological response modifiers (BRMs) have not been clearly resolved. We have investigated this issue in the Lewis lung (3LL) peritoneal carcinomatosis model in which treatment with the BRM MVE-2 slows tumor growth and enhances survival. MVE-2 is a potent inducer of cytotoxic macrophages (ms); however, the in vivo tumoricidal properties of these ms remain to be firmly established. To directly establish that ms were at least in part responsible for the in vivo efficacy of MVE-2, a novel method of obtaining highly enriched m4 suspen- sions was developed which gave high purity, satisfactory yield, and excellent viability without affecting antitumor activity. Using the 3LL peritoneal carcinomatosis model and adoptive transfer techniques, we directly demon- strate that the majority of antitumor activity was as- sociated with the adherent cell fraction enriched for ms. Histological observations supported this conclusion, in- dicating that MVE-2 treatment initially activated cells associated with nonspecific immunity, retarding tumor growth in the ascites long enough for a multifaceted im- mune response to develop J. Leukoc Biol. 56: 41-51; 1994. Key Words: macmphage . biological response nwdzfiers . MVE-2- adoptive immunotherapy . flow cytometry - Lewis lung carcinoma INTRODUCTION Considerable attention has been focused on the use of bio- logical response modifiers (BRMs) as an adjunct to the con- ventional treatment of cancer and for the prevention of metastatic disease [1, 2]. Because of their pleiotropic effects on the immune system, the cellular mechanisms for their antitumor affects have been difficult to resolve. Mechanisms that have been actively considered include augmentation of both cell-mediated and nonspecific immunity. We have been addressing the role of macrophages (ms) in nonspecific im- munity to neoplasia using the BRM MVE-2, a potent aug- mentor of natural killer (NK) cell activity, inducer of cyto- toxic ms, and stimulator of specific immunity [3, 4]. MVE-2-activated ms have clearly been shown to be cyto- toxic in vitro to a variety oftransformed, but not normal, cell lines [5-7]. Their in vivo tumoricidal properties, however, remain to be firmly established. MVE-2 is a low-molecular-weight fraction (15,500) of the maleic anhydride-vinyl ether polyanion, derived from its broad-molecular-weight parent compound, pyran copolymer [8]. The drug has a wide variety of effects on the immune system, including modification of the reticuloendothelial sys- tern, interferon (IFN) production, immunoadjuvant activity, antiviral activity, and antitumor activity [3, 4]. It is paren- terally active against a variety of spontaneous, virus- induced, and carcinogen-induced tumors [3, 9] and has been evaluated in several clinical trials [10-12]. College of Pennsylvania, Philadelphia MVE-2 is a potent m4 activator and several lines of cvi- dence, including both histological [13] and in vitro studies, strongly suggest that these cells are principally responsible for its antitumor activity [6, 7, 9]. However, other mecha- nisms, besides enhancement of m4 antitumor activity, have been proposed to contribute to the effects mediated by MVE-2, including augmentation ofNK cell activity [14, 15]. Elevated levels of NK activity have been reported following MVE-2 therapy [16], which may inhibit tumor growth and contribute to the antimetastatic properties induced by MVE-2. MVE-2 is also an IFN inducer [17], but its anti- tumor activity does not appear to correlate directly with either IFN induction or IFN levels [3, 18]. Moreover, pre- treatment ofanimals with antibodies to IFN has been shown not to alter the antitumor activity of MVE-2, although aug- mentation of NK activity was blocked by this antibody [19]. The cellular mechanisms through which the antitumor effects of MVE-2 are exerted can be directly assessed in the Lewis lung (3LL) peritoneal carcinomatosis model. The sen- sitivity of the 3LL to MVE-2, the accessibility of the peritoneal cavity, and the presence of relevant effector cells (NK cells and mcs) make this a suitable model in which to establish directly the in vivo cellular mechanisms. The most direct experimental approaches include adoptive transfer of ms or whole body m4 depletion techniques. The m deple- tion experiments have been discouraging because of the in- ability to eliminate totally resident and bone marrow-derived ms, particularly following immunotherapy [20, 21]. Adop- tive transfer methods have heretofore been inconclusive be- cause of the difficulty in obtaining relatively pure and viable suspensions of MVE-2-activated m4s free of NK or other effector cells. Using a novel method to purify peritoneal ms, we demonstrated directly that activated ms are at least one of the effector cells responsible for the immunotherapeutic efficacy of MVE-2 in vivo. MATERIALS AND METHODS Mice Male and female, 6-10-week-old C57BL/6N mice were sup- plied through the Animal Genetics and Production Branch of the National Cancer Institute (Charles River Breeding Laboratories, Kingston, NY). All animals were maintained in an American Association for Accreditation of Laboratory Abbreviations: BRM, biological response modifier; m, macrophage; NK cells, natural killer cells; MVE-2, maleic anhydride divinyl ether copolymer; IFN, interferon; 3LL, Lewis lung carcinoma; HBSS, Hanks’ buffered saline solution; TG, thioglycollate broth; EIJFA, disodium ethylenediaminetetraacetic acid; MST, median survival time. Reprint requests: Paul K. Wallace, Department of Microbiology, HB 7556, Dartmouth Medical School, I Medical Center Drive, Lebanon, NH 03756. Received November 19, 1993; accepted March 8, 1994.