Abstract 916: Combined focal radiation and immune checkpoint inhibition enhances anti-tumor responses over single agent treatment in a murine mammary carcinoma model
Authors: Yarzabek Brogan , Lapinski Philip , Draper David , Wise Scott , Franklin Maryland 
Published: September 2020

Abstract
While immunologically “cold” tumor models, such as the murine 4T1 mammary carcinoma model, are generally resistant to treatment by immune checkpoint inhibition, we have seen improved treatment response in this model by combining antibody therapy with focal radiation.
4T1-luc (4T1) mammary carcinoma cells were orthotopically implanted into BALB/c mice and treated with anti-mCTLA-4 or isotype control antibody, focal radiation (SARRP; Xstrahl), or a combination of the anti-mCTLA4 and radiation. Tumors were analyzed by flow cytometry, using panels that examined lymphoid and myeloid lineages, production of some cytokines, effector/memory phenotypes of T cells, and a calculation of cells per gram of tumor (absolute counts).
Analysis of T cells with a traditional gating method revealed that, while we do not see a change in absolute counts of CD8+ T cells within the tumor following combination treatment, we see a shift in the phenotype of these cells when compared to single treatments. An increase in the percentage of CD8+ T effector memory cells with a corresponding decrease in CD8+ T central memory cells in the double treatment group showed shifts toward a more activated phenotype. The percentage of CD8+ T cells expressing Granzyme B was also increased following combination treatment. NK T cells showed an increase in the percentage of cells that expressed Interferon-γ with the combined treatment. This suggests that the increased activation seen in the CD8+ T cells is not specific to only that cell type and may be occurring more broadly within the tumor microenvironment. B cell and regulatory T cell absolute counts were decreased, and the tumors shrank in size with the combined treatment as compared to single agent treatments.
These data suggest that combining anti-mCTLA4 and focal radiation can convert the 4T1 tumor microenvironment to an immunogenic state more like that of a “warm” tumor model. Reduction in tumor size, and in numbers of regulatory T cells and B cells, correlated with increased percentages of CD8+ T cells and NK T cells that had a phenotype consistent with improved anti-tumor activity. The radiation treatment appeared to prime the cells to allow the checkpoint inhibition treatment to have an effect like that seen in “warm” tumors.
With complex flow cytometry panels, like utilized in this study, there is an element of investigator bias inherent in traditional gating. tSNE dimensionality reduction analysis combined with a clustering algorithm is a method of flow cytometry analysis that reduces this bias. It also allows for more complex analysis of high parameter panels by examining all parameters at once and examining complex shifts in phenotypes between treatment groups. We are currently investigating use of this analysis method to further explore the effects of the combination treatment on the 4T1 tumor model and data will be presented.
Citation Format: Brogan Yarzabek, Philip Lapinski, David Draper, Scott Wise, Maryland Franklin. Combined focal radiation and immune checkpoint inhibition enhances anti-tumor responses over single agent treatment in a murine mammary carcinoma model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 916.