Abstract 2630: Identifying most promising combination therapies for immune suppressive breast cancer using a combined in vitro/in vivo approach
Authors: Silberzahn Hannah , Bleisch Mattias , Löhr Anne , Knauff Volker , Lashuk Kanstantsin , Schueler Julia B. 
Published: March 2024

Abstract
Combining epigenetic therapy with immunotherapy is a promising strategy to address the complexities of cancer biology. However, it's important to note that the field is still evolving, and more research is needed to understand the optimal combinations, timing, and patient populations that will benefit the most from these approaches. We evaluated a combination of two different checkpoint inhibitor therapies (anti-CTLA4 and anti-PD1) with two different hypomethylating agents (5-azacytidine, AZA and Decitabine, DEC) in vitro and in vivo using a mouse breast cancer cell line 4T1 known to be resistant towards checkpoint inhibition (CPi). Beside tumor cell killing and tumor volume measurement, cytokine expression and immune cell phenotyping was determined. In a co-culture assay with autologous T cells and 4T1 cells, antitumor efficacy weas increased in all combination arms using AZA but not in combination with DEC. The combination of AZA with anti-CTLA4 was significantly better than the respective monotherapies (oneway ANOVA, p&lt; 0.001). This effect was observed as well in the combination with anti-PD1 and AZA, however it was less pronounced (oneway ANOVA, p&lt; 0.01). Of note, AZA restored the CTLA4 expression on CD8+ T cells in vitro, which was reduced by co-culture with tumor cells alone. In the follow-up in vivo experiment we focus on the combination of both CPi with AZA on different treatment schedules in the combination arms. The combination of anti-CTLA4 and AZA in a staggered treatment schedule displayed significant efficacy compared to control (oneway ANOVA, p&lt; 0.05). The implantation of 4T1 reduced the number of T cells in bone marrow (BM), peripheral blood (PB) and spleen of the mice, confirming the immune-suppressive nature of the breast cancer line. This effect was restored under treatment with AZA in monotherapy and combination with CPi. Interestingly AZA reduced the number of immunosuppressive MDSC in BM, spleen and in tumor tissue. Vice versa NK cells were increased in BM, spleen and tumor tissue under treatment with AZA. AZA reduced the number of circulation tumor cells in PB and increased as already shown in vitro the percentage of CD8/CTLA4++ in PB. The PCA analysis of 36 mouse cytokines in vivo displayed two main clusters separated mainly by the presence of absence of AZA in the treatment arms. The combination arms which displayed the highest efficacy showed an increase in CXCL1, G-CSF, IL-6, LIF, M-CSF, MCP-1 (CCL2) and MCP-3 (CCL7) and a decrease of IFN-gamma, CXCL2. This is in line with the more inflammatory pattern of the tumor infiltrating lymphocytes in those groups. In summary, we have strong evidence that AZA influences the immune landscape of breast cancer towards a more inflammatory subtype. The combination with CPi was superior to the monotherapy. In addition, our data indicate that the treatment schedule of the combination might have a crucial influence on efficacy.
Citation Format: Hannah Silberzahn, Mattias Bleisch, Anne Löhr, Volker Knauff, Kanstantsin Lashuk, Julia B. Schueler. Identifying most promising combination therapies for immune suppressive breast cancer using a combined in vitro/in vivo approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2630.