Abstract 3474: Breast cancer control and increased TIL with intratumoral TLR2 agonist and IFNγ

Abstract Background: High densities of tumor-infiltrating lymphocytes (TILs) in triple-negative breast cancer (TNBC) are associated with better outcomes and response to anti-PD-1 checkpoint blockade therapy. CXCL10 is a critical chemoattractant for activated T-cells and is lowly expressed in breast cancers, thus, approaches to increase CXCL10 may boost TIL, and enhance TNBC control. We have previously found that a TLR2 agonist (FSL-1) plus IFN-gamma enhances the production of CXCL10 by human melanoma cells. We hypothesized that intratumoral therapy with these agents would enhance CXCL10 production, TIL, and tumor control in TNBC. Methods: Chemokine production and TNBC cell viability were evaluated in vitro after treatment of TNBC cell lines with PBS, FSL-1, IFNγ, or FSL-1+IFNγ. In vivo, changes in TIL, tumor outgrowth, and murine survival were evaluated after intratumoral therapy of E0771 tumors grown in the mammary fat pad of C57BL/6 mice. Results: CXCL10 production by 12 human and 3 murine TNBC cell lines was evaluated by flow cytometry and ELISA assay. FSL-1+IFNγ stimulation synergistically enhanced CXCL10 production by human and murine TNBC cell lines when compared to IFNγ, FSL-1, or PBS alone (p≤0.03). Luminex assay revealed that CXCL10 was the most abundant chemokine produced by murine TNBC cells in response to FSL-1+IFNγ (1185 pg/ml), while human TNBC cells dominantly produced CXCL10 (36498 pg/ml) and CXCL9 (31480 pg/ml) and produced low levels of CXCL11, (341 pg/ml) CXCL12 (8 pg/ml), and CCL5 (391 pg/ml). Intratumoral therapy with FSL-1+IFNγ treatment increased overall survival when compared to those receiving PBS, FSL-1, or IFNγ alone (58 vs 34 days P<0.0001). FSL-1+IFNγ induced visible tumor necrosis and induced sustained full tumor regression in 2 of 11 mice evaluated for long-term survival. Gene signatures associated with T-cell infiltrates and T-cell activation were elevated in FSL-1+IFNγ treated tumors evaluated by Nanostring analysis. CD8+ T-cell infiltrates, evaluated by multiparameter immunofluorescence histology (mIFH), were increased in FSL-1+IFNγ treated tumors (117 cells/mm2) compared to those treated with PBS, FSL-1, IFNγ (52, 29, 33 cells/mm2 respectively, p≤0.02). Additionally, in flow cytometric assays, FSL-1+IFNγ treated tumors had increased percentages of CD8+ T-cells (27% p≤0.001) relative to control groups treated with PBS 12%, FSL-1 8%, and IFNγ 5%. Conclusions: FSL-1+IFNγ treatment increases TIL, murine survival, and tumor growth control, and promotes the complete regression of a subset of tumors. Thus, further studies are warranted to identify the mechanisms that modulate response to this treatment to improve treatment efficacy and to evaluate whether FSL-1+IFNγ can drive immunogenic cell death of TNBC and impart a systemic benefit for tumor control. Citation Format: Ileana Soto Mauldin, Elaheh Najafianashrafi, Kelly T. Smith, Marya E. Dunlap-Brown, Chengli Shen. Breast cancer control and increased TIL with intratumoral TLR2 agonist and IFNγ [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 3474.