Abstract 6679: Immunophenotypic and transcriptome analyses of CT26 and 4T1 murine tumor models following anti-CTLA-4 treatment
Authors: Barnes Sheri R. , Draper David , Church Sarah E. , Franklin Maryland Rosenfeld 
Published: September 2020

Model selection for evaluation of immuno-oncology agents is often aided by both response to therapy and changes in immune cell subsets following treatment. To identify underlying factors that influence differences in therapeutic response, CT26 colorectal and 4T1-Luc (4T1) breast tumor models, which are immunologically warm and cold, respectively, were treated with anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4). Flow cytometry and transcriptome analysis were used to describe treatment-related changes in immune cell infiltrate, TIL activation status, cytokine/granzyme B production, and overall changes in gene expression. Response to anti-CTLA-4 therapy was distinct between these models. Tumor growth inhibition was observed in CT26 (74%), but not 4T1. In CT26, CD8+ and NK T cell infiltration increased, while M2 macrophages decreased. In 4T1 tumors, only increased NKT cell infiltration was noted after treatment, and no demonstrable changes were observed in any other cell type or activation marker. Several pharmacodynamic changes in immune cell activation were observed in CT26. Notably, CT26-derived CD4+, CD8+, and NK T cell subsets had increased IFNγ production (70%, 25%, and 17%, respectively, versus control). An increase in polyfunctional T cells producing both IFNγ and TNFα was also noted for CT26 and was correlated with slower tumor growth. Granzyme B was reduced and CD107a was increased in CT26-derived NK T cells, suggesting functionally active T cells. This was also supported by upregulation of genes in the cytotoxicity, antigen presentation, and NFkappaB pathways.

Following CT26 treatment with anti-CTLA-4, transcriptome analysis with the nCounter® Mouse PanCancer IO 360 Panel (NanoString) showed an 8-10 fold induction of several T cell-related genes as well as an 8-14 fold induction of several chemokines and their receptors. These data complement the flow cytometry data and support stimulation of T cell activation and recruitment to CT26 tumors following anti-CTLA-4 treatment. In contrast, analysis of 4T1 tumors indicated only a 2 to 3-fold induction of CCL4, CCL11, and Granzyme A after treatment. Interestingly, genes in the matrix remodeling pathway were upregulated in 4T1 tumors upon anti-CTLA-4 treatment.

In summary, CT26 tumors were responsive to anti-CTLA-4 treatment, showing increased polyfunctional T cell recruitment, NK T cell activity, and reduced M2 macrophage infiltration, as well as marked upregulation of several genes involved in T cell recruitment and activation in tumor. In contrast, 4T1 tumors did not display any remarkable response to therapy, changes to TIL infiltrates, and only limited changes to gene expression with treatment. Collectively, these results suggest tumor profiles following treatment can provide insight into potential targets of interest and are important for preclinical model selection.

Citation Format: Sheri R. Barnes, David Draper, Sarah E. Church, Maryland Rosenfeld Franklin. Immunophenotypic and transcriptome analyses of CT26 and 4T1 murine tumor models following anti-CTLA-4 treatment [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 6679.