Checkpoint inhibitor (CPI)-based immunotherapy such as PD-1/PD-L1 blockade has shown an unprecedented success for the treatment of a variety of malignancies; however, meaningful clinical activity is seen only in a subset of breast cancer patients. This is due, at least in part, to poor T-cell infiltration into the tumor prior to CPI therapy, which is negatively correlated with the treatment response (non-T cell-inflamed tumor). Effective priming and trafficking of tumor-specific T cells to the tumor microenvironment (TME) can theoretically overcome current limitations of immunotherapy for non-T cell-inflamed tumors such as breast cancer.

Accumulating evidence indicates that tumor-residing Batf3-dependent CD103+ dendritic cells (DCs) play critical roles in not only priming of tumor-specific CD8+ T cells, but also trafficking of CD8+ T cells to the TME, thus enhancing responses to PD-1/PD-L1 blockade. In the present study, we hypothesized that mobilization and activation of cross-presenting CD103+ DCs in the TME would increase CD8+ T cell infiltrate to the tumor, and facilitate effective regression of breast cancer. To this end, we utilized two triple-negative syngeneic mammary cancer models, 4T1 on a H-2d background and AT3 derived from the PyMT-MMTV model on a H-2b background, and evaluated antitumor efficacy of a combinatorial therapeutic regimen comprised of intratumoral administration of Fms-like tyrosine kinase 3 receptor ligand (Flt3L) to mobilize CD103+ DCs to the TME, local radiotherapy (RT) to induce immunogenic cell death and maturation of DCs, and peritumoral administration of a toll-like receptor 3 (TLR3) agonist to activate DCs.

We found intratumoral administration of Flt3L increased the number of CD103+ DCs in the tumor, blood and lymph nodes, and local RT induced upregulation of TLR3, CD40 and CD86 on the tumor-residing CD103+ DCs. Following intratumoral administration of Flt3L, local RT, and in situ delivery of a TLR3 agonist significantly increased CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs), mediated effective regression of large established tumors, and significantly improved survival in multiple mouse tumor models. Phenotypic analysis of circulating T cells revealed that the combinatorial regimen increased the frequency of effector memory and central memory T cell subsets with upregulation of PD-1 and C-X3-C motif chemokine receptor 1 (CX3CR1), suggesting effective activation and differentiation of T cells. Interestingly, we found the frequency of macrophages significantly decreased in the tumor after combinatorial treatment, indicating remodeling of both the myeloid and lymphoid compartments in the TME. Furthermore, increased PD-1 expression on CD4+ and CD8+ TILs, and PD-L1 expression in the TME suggest that antitumor efficacy of the combinatorial regimen can be additionally enhanced by PD-1/PD-L1 blockade.

Taken together, combinatorial in situ immunomodulation with intratumoral Flt3L administration, RT and, peritumoral administration of a TLR3 agonist can convert non-T cell-inflamed tumors to T-cell inflamed tumors, remodel an immunosuppressive TME, and potentially overcome primary resistance to PD-1/PD-L1 blockade for triple-negative breast cancer.

Acknowledgement: This work was supported by Uehara Memorial Foundation (T.O.), NIH/NCI K08CA197966 (F.I.) and P30CA016056 involving the use of Roswell Park Comprehensive Cancer Center’s Flow Cytometry Core.

Citation Format: Takaaki Oba, Tibor Keler, Henry C Marsh, Scott Abrams, Fumito Ito. Conversion of non-T cell-inflamed triple-negative breast cancer by in situ immunomodulation of induced intratumoral cross-presenting dendritic cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-03.