Abstract 6171: Flt3 ligand and CD40 agonist-based therapy in breast cancer promotes myeloid cell CXCL9 secretion and CD8 T cell recruitment but is not dependent on CXCL9 for efficacy
Authors: Preteesh L. Mylabathula, Anas Alkhani, James Zhu, Skanda Hebbale, Riyasat Ali, Vivek Mathesh, Isaac Chan, Tao Wang, Sangeetha M. Reddy
Journal: Cancer Research (2025)

Abstract Background: Breast cancers have defects in antigen presentation, limiting T cell-mediated immunity. Flt3 ligand (Flt3L) is a growth factor that supports the differentiation of DC1 antigen-presenting dendritic cells (DCs), which are deficient in breast tumors, while CD40 agonist (CD40a) activates dendritic cells and also macrophages. We previously demonstrated that the combination of Flt3L, CD40a, and chemotherapy synergizes to improve outcomes in murine breast cancer models. Methods: C57BL/6 mice (6-8 weeks old) were orthotopically injected with E0771, AT3, and 4T1 breast cancer cells in the mammary fat pad. Tumors were allowed to grow to ∼50 mm3 before mice were randomized into treatment groups. Mice received triplet therapy consisting of pegylated liposomal doxorubicin (PLD) administered via tail vein, followed by Flt3L intraperitoneally (IP) for 5 consecutive days, and CD40a IP one week later. Control groups included monotherapy and doublet therapy. Tumor growth and survival were monitored. Tumors and tumor draining lymph nodes were harvested from E0771 tumor bearing mice for single-cell RNA sequencing (scRNAseq) and spectral flow cytometry. To study if CXCL9 secretion and CD8 T cells mediate effects of therapy, CXCL9 chemokine and CD8 T-cells were systemically depleted with anti-CXCL9 and anti-CD8 antibodies administered IP every 4 and 2 days respectively during treatment. Results: Triplet therapy led to improved tumor control compared to no treatment, monotherapy, and doublet controls (p&amp;lt;0.0001). Treatment led to significant increase in CD8 T cell recruiting chemokine CXCL9 expression from all intra-tumoral cDC populations including, cDC1, cDC2, and mregDCs (p&amp;lt;0.01). Additionally, there was a significant increase in an inflammatory macrophage population characterized by high CXCL9 expression. Increased tumor infiltration with CD8 T cells was observed with triplet therapy via both scRNAseq and flow cytometry (p&amp;lt;0.01), with an increase in stem-like and effector memory phenotype (p&amp;lt;0.01) and a decrease in exhaustion (p&amp;lt;0.01). While depletion of CD8 T cells significantly reduced treatment effect, efficacy was maintained in setting of CXCL9 depletion. Conclusion: Our findings demonstrate that the combination of Flt3L, CD40 agonist, and chemotherapy significantly enhances tumor control in murine breast cancer models through increased recruitment of CD8 T cells which have more stem-like and effector memory phenotype. While significant increase in CXCL9 was observed, the efficacy of treatment was independent of CXCL9, suggesting importance of factors improving CD8 T cell quality and not just quantity. A clinical trial with this combination in metastatic triple negative breast cancer patients is open and recruiting (NCT05029999). Citation Format: Preteesh L. Mylabathula, Anas Alkhani, James Zhu, Skanda Hebbale, Riyasat Ali, Vivek Mathesh, Isaac Chan, Tao Wang, Sangeetha M. Reddy. Flt3 ligand and CD40 agonist-based therapy in breast cancer promotes myeloid cell CXCL9 secretion and CD8 T cell recruitment but is not dependent on CXCL9 for efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6171.