[CANCER RESEARCH 53, 5067-5070, November 1, 1993] Advances in Brief Protective and Curative Potential of Vaccination with Interleukin-2- Gene-transfected Cells from a Spontaneous Mouse Mammary Adenocarcinoma1 Federica Cavallo, Francesco Di Fierro, Mirella Giovarelli, Alberto Gulino, Alessandra Vacca, Antonella Stoppacciaro, Marco Forni, Andrea Modesti, and Guido Forni2 CNR-lmmunogenetics and Histocompatibilily Center at the Institute of Microbiology, University of Turin, Via Sanlena 9, Turin [F. C., F. D-P., M. G., G. F./; Department of Experimental Medicine, University of L'Aquila, f>7100, Collemaggio ¡A.G.j; Department of Biomedicine, University La Sapienza, Viale R. Elena 324, 00161. Rome [A. S., A. V.j; Regina Margherita Children!çHospital, Piazza Polonia 40, 10126 Turin [M. F./; and Institute of Experimental and Social Medicine, University of Chieti, Via Ventini, 66013, Chieti, Italy ¡A.M.J Abstract The potential of interleukin 2-gene-transfected tumor cells to prevent tumor growth and cure established tumors was evaluated using cells from a spontaneous, invasive, and metastasizing mouse mammary adenocarci- noma. Tumor cells engineered to secrete interleukin 2 initially trigger a local inflammatory reaction that leads to inhibition of established parental adenocarcinomas, as well as an antigenically unrelated fibrosarcoma. The ensuing systemic immunity selectively inhibits subsequent parental cell challenges and cures established parental adenocarcinomas and their lung métastases,although less effectively as the neoplastic mass increases. Mul tiple injections of interleukin 2-gene-transfected tumor cells may thus be considered a new form of vaccination in the management of minimal residual disease and incipient métastases. Introduction Several data have shown that the local presence of cytokines in a tumor growth area triggers a potent antitumor reaction in both experi mental models (1-3) and clinical trials (4). IL-23 appears of particular interest due to its central regulatory role in the immune response. It is a progression factor for T-helper and T-cytotoxic lymphocytes, which participates in B-lymphocyte activation, boosts natural killer cells, generates lymphokine activated killer activity (for a review see Ref. 5), and triggers nonspecific cytotoxicity in macrophages (6) and neu- trophils (7). Studies with IL-2 that is repeatedly injected at the tumor site or around tumor-draining lymph nodes (1,2), as well as with IL-2 directly released by IL-2 gene transduced tumor cells (for reviews see Refs. 2 and 3), have shown that it provides the signals for the efficient induction of both a nonspecific and a specific antitumor immune response. The nonspecific, delayed type of hypersensitivity reaction first elicited involves a knotty cross-talk between granulocytes, mac rophages, fibroblasts, and CD4+ T-lymphocytes that often results in the induction of a tumor-specific, CDS4 T-lymphocyte mediated im mune memory (1-3). These data suggest that the local availability of IL-2 increases tumor immunogenicity and stress its use as a component of new tumor vaccines. Phase I clinical trials based on the injection of IL-2 gene transduced tumor cells are underway (8, 9), while the ability of these cells to cure established tumors has not been extensively explored in mouse models. Moreover, the biological meaning of the experimental Received 7/29/93; accepted 9/20/93. 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 This work was supported by grants from the Italian Association for Cancer Research (AIRC), the ISS Italy-USA Program on Cancer Therapy and AIDS, and PF CNR (Con siglio Nazionale Ricerche)-ACRO. 2 To whom requests for reprints should be addressed. 1 The abbreviations used are: IL. interleukin; TS/A-pc, TS/A parental cells; Mit-C. mitornycin-C treated. data is difficult to determine, since proper staging of the established tumors is not reported (10-12). To obtain a realistic assessment of the potential of IL-2 gene trans duced tumor cells to vaccinate mice before tumor challenge and cure those with established tumors, we used an expression vector to intro duce the complementary DNA coding for mouse IL-2 into the cells of a spontaneous, invasive, and metastatizing mouse mammary adeno- carcinoma (TS/A) that arose in a female of a mouse strain of low tumor incidence (1). Materials and Methods In Vitro Cultures. Cell cultures were performed with sterile, disposable flasks and plates from Nunc. Roskildc, Denmark, at 37°Cin a humidified 5% CO2 atmosphere, using RPMI 1640 supplemented with 10% fetal calf scrum, 100 units/ml penicillin. 100 units/ml streptomycin, 50 fig/ml gcntamycin. and 2.5 X 10~5 M 2ß-mercapto-ethanol (Flow Laboratories, Opera, Italy). Tumors. TS/A-pc derive from the eighth in vitro passage of a tumor cell line established from the first in vivo transplant of a moderately differentiated mammary adenocarcinoma which arose spontaneously in a BALB/cnAnCr female mouse. TS/A-pc express major histocompatibility complex class I, but not class II, molecules and do not stimulate a syngeneic antitumor response in vitro nor in vivo (1, 13). Four X IO4 are about the minimal 100% TS/A-pc tumor-inducing doses in mice of the BALB/cnAnCr (BALB/c mice from Charles River Laboratories, Calco, Italy) of its origin used in these experi ments. When necessary, TS/A-pc were treated with 60 /ig/ml of Mit-C (Sigma, St. Louis, MO)/1 X IO7 cells/ml for 30 min at 37°C(13). CE-2 is a methyl- cholanthrene induced sarcoma of BALB/c mice that does not cross-react an tigenically with TS/A-pc (13). Two X IO* cells are the minimal 100% CE-2 tumor inducing dose. In Vivo Evaluation of Tumor Growth. Seven-weck-old female BALB/c were challenged (day 0) s.c. in the left flank or i.v. with the lethal dose of TS/A-pc or CE-2 cells. For 120 days, mice were inspected twice weekly and s.c. neoplastic masses were measured with calipers in the two perpendicular diameters in a fashion blind to the group in which they had been treated. Mice with a tumor >4 mm mean diameter were classed as tumor bearers. Mice were killed for humane reasons when the tumor exceeded 10 mm mean diameter. Following i.v. challenge, mice were killed when moribund. Care of mice was in accordance with the European community and Italian guidelines. IL-2 Gene Transfection and IL-2 Titration. TS/A-pc were transfectcd by electroporation with the linearized BCMGneo (neomycin resistance gene) (TS/A-neo cells) or BCMGneo-IL-2 plasmids (14), cloned, and cultured in selective medium as previously described (13). The IL-2 produced by 1 X IO5 transfected cells cultured for 48 h in 1 ml of medium was evaluated as the ability to support the growth of the murine T-cell line CTLL. The IL-2 tiler was calibrated against the Biological Response Modifiers Program Reference re agent human IL-2 (lot ISDP-841) (13). Morphological Analysis. For histological evaluation, groups of 4 mice were killed 1, 7, and 14 days after challenge. Tissues were fixed, embedded in paraffin, sectioned at 4 mm. and stained with hcmatoxylin-eosin. Métastases were counted in lungs injected with 2 ml of 15% black India ink. 5067 on August 1, 2021. © 1993 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from