Cancer Immunol Immunother (1983) 15:84-86 ancer mmunology mmunotherapy © Springer-Verlag 1983 Immunotherapy of Lewis Lung Carcinoma with Hydrosoluble Peptidoglycan Monomer (PGM) G. Sara 1, T. Giraldi 1, J. Tomagid2, and I. Hrgak 2 1 Istituto di Farmacologia e Farmacognosia, Universita' di Trieste, via A. Valerio 32, 1-34100 Trieste, Italy 2 'Rudjer Boskovic' Institute, JU-41000 Zagreb, Bijenicka 54, Yugoslavia Summary. The water-soluble peptidoglycan monomer (PGM) isolated from the culture fluid of Brevibacterium divaricatum, which has immunostimulating activity, has been examined for its antitumor effects in C57BL mice bearing Lewis lung carcinoma. The formation of spontaneous lung metastases from SC tumor implants is significantly inhibited. The growth of SC primary tumors, including advanced ones, is also significantly inhibited, though to a less pronounced extent than the growth of metastases. The effects on metastases are evident with all treatment schedules used, whereas those on SC primary tumors are treatment schedule-dependent. The treatment with PGM was found to be therapeutically useful when combined with surgical removal of IM implants; in conditions where a single post-operative treatment was ineffective, combined post-opera- tive and immediately pre-operative administration of PGM significantly increased (by 40%) the survival time of treated animals over that of controls undergoing surgery only. Introduction The development of primary malignant tumors and of their metastases leads to depression of the immunological response of the host, attributable to suppressor cells or factors which abrogate the general and tumor-directed reactions [4, 9, 13]. In view of this, the use of substances capable of restoring the immune reactivity of the host might reduce the proliferation of the tumor in both locations. Immunotherapeutic approaches for the eradication of metastases with or without removal of primary tumor by irradiation or surgery have been widely studied with several immunostimulating agents in mice bearing Lewis lung carcinoma. The results obtained indicate that the restoration of the tumor-depressed functions of the immuno- logical system of the host by means of administration of BCG, C. parvum, glucan and pyran copolymer leads to a reduction of metastasis formation dependent on the dose and on the timing-schedule employed [10, 17, 19, 21]. However, the use of whole bacterial cell wails, as in the case of BCG and C. parvum, or of cell wall components, such as MDP and its analogs, is inconvenient in various ways. Immunostimulating activity is critically dependent on the dosage; at active dosages various side-effects are severe, though recently apyrogenic MDP derivatives active when given as adjuvant therapy have been described [3, 15], and administration as suspension in oil or after incorporation into liposomes is required [1, 5, 14]. Reprint requests should be addressed to G. Sava We therefore thought it worthwhile to test a new immunostimulating compound, the peptidoglycan mono- mer GlnNAc-MurNAc-L-Ala-B-iso-Gln-meso-diaminopimelic acid-D-Ala-D-Ala (PGM), in mice bearing Lewis lung carci- noma. PGM is a hydrosoluble substance originating from the cell wall of Brevibacterium divaricatum [11, 12], and is devoid of toxic effects for the host in the range of doses having immunostimnlating activity [6]. The differential effects of PGM on primary tumor growth and on the formation of spontaneous lung metastases have thus been examined. Furthermore, the effects of PGM on the survival time of mice undergoing surgical eradication of primary tumor have also been determined and are reported below. Materials and Methods Peptidoglycan Monomer (PGM). PGM was prepared by lysozyme digestion of uncrosslinked peptidoglycan chains isolated from culture fluids of penicillin-treated Brevibacte- rium divaricatum NRRL-2311, as described in detail elsewhere [11, 12]. PGM was tested by the limulus amebocyte lysate (LAL) test (Pyrostat kit: Millipore, New Jersey, USA), and the samples used in the present study were found to contain 0.015 ng endotoxin per mg PGM. Animal Treatment. C57BL female mice weighing 18-20 g, purchased from Charles River (Calco, Como, Italy), were used. PGM was freshly dissolved in 0.9% NaC1 and was administered IV in volumes of 0.05 ml per 10 g animal weight: control animals received only the solvent. Tumor Transplantation and Evaluation. Lewis lung carcinoma was transplanted by SC injection, in the axillary region of syngeneic C57BL mice, of 25-100 mm 3 of tumor fragments aseptically prepared from donors similarly inoculated 2 weeks before. Primary tumor weight was determined by caliper measurements, taking tumor density equal to 1, as the volume of the rotation ellipsoid having the short and long axes equal to A and B, respectively: Tumor weight = st/6 x A 2 x B. (Eq. 1) The number of spontaneous lung metastases was determined at sacrifice by examining the surface of the lungs with a low-power stereo microscope. The weight of metastases was determined as the sum of their individual weights calculated according to Eq. 1.