Abstract 586: CD4+and CD8+T cells influence 4T1.2luc mammary tumor growth and survival
Authors: Xu Yitong , Duan Shizhao , Turbitt William J. , Mastro Andrea M. , Rogers Connie J. 
Published: August 2019

Abstract
The 4T1.2 syngeneic murine breast cancer model is an aggressive model of stage IV triple-negative breast cancer. Despite its significant strength, the 4T1.2 model is poorly immunogenic with few defined tumor antigens. The firefly luciferase gene, luc2, has been transfected into various tumor cell lines as a reporter for bioluminescence imaging. However, studies have shown that the luciferase (Luc) may serve as a tumor antigen and induce CD8+ cytotoxic T cell responses. The goal of the current study was to determine if luciferase expression in 4T1.2 tumor cells would enhance the immunogenicity of the tumor, and explore the role of immune effector cells in the parental 4T1.2 and luciferase-transfected 4T1.2luc tumor model. In vitro proliferation of the 4T1.2 and 4T1.2luc cells was assessed using the MTS cell proliferation assay. For in vivo experiments, female BALB/c mice were randomized into 2 groups and orthotopically injected with 5x104 4T1.2 or 4T1.2luc cells, respectively. Tumor growth was assessed until day 35 post tumor implantation. Each group was further randomized into 5 subgroups and received i.p. injections of PBS, rat IgG2b mAb, anti-CD4 mAb, anti-CD8 mAb, or anti-asialo GM1. Mice were sacrificed between day 28-35 post tumor implantation, and lung metastases were analyzed for each group. To further evaluate the immunogenicity of luciferase in the 4T1.2luc model, a separate cohort of mice were orthotopically injected with 5x104 4T1.2luc cells and sacrificed at day 35 post tumor implantation. Splenocytes were harvested and cultured in the presence of 1 or 10 µg/ml peptides representing MHC I-restricted Luc epitopes for 5 days, and cytotoxicity of the effector cells was assessed by the chromium-release assay. In vitro proliferation rates were not significantly different between 4T1.2 and 4T1.2luc tumor cells. However, 4T1.2luc tumor-bearing mice showed a significant reduction in primary tumor growth compared to 4T1.2 tumor-bearing mice. 4T1.2 primary tumor growth was not altered by the depletion of any immune cell type, while 4T1.2luc primary tumor growth was significantly increased by the depletion of CD8+ T cells, and reduced by the depletion of NK cells. Depletion of CD4+ T cells, CD8+ T cells or NK cells in 4T1.2luc tumor-bearing mice resulted in a significant reduction in median survival. Splenocytes from 4T1.2luc tumor bearing mice demonstrated Luc-specific cytotoxicity upon ex vivo restimulation with Luc peptides, and a stronger response was observed when cells were restimulated with 1 µg/ml compared to 10 µg/ml Luc peptides. These findings suggest that luciferase may serve as a tumor antigen in the 4T1.2luc tumor model, and host immune components (CD4+ and CD8+ T cells) may play a role in controlling 4T1.2luc primary tumor growth and survival. The stronger immunogenicity of 4T1.2luc conferred by luciferase will enable future immunotherapeutic research that ultimately may benefit breast cancer patients.
Citation Format: Yitong Xu, Shizhao Duan, William J. Turbitt, Andrea M. Mastro, Connie J. Rogers. CD4+and CD8+T cells influence 4T1.2luc mammary tumor growth and survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 586.