Abstract B055: Macrophage and T reg expression of PD-L1 in the tumor draining lymph node and not the primary tumor is associated with anti-PD-1 resistance in a model of metastatic triple negative breast cancer

Abstract Introduction: The main cause of breast cancer mortality is metastasis. Triple negative breast cancer (TNBC) is an aggressive subtype with high rates of metastasis to the regional tumor draining lymph nodes (TDLN) and visceral organs, which is driven in part by immune escape. Recently, the addition of pembrolizumab (anti-PD-1) to neo-adjuvant chemotherapy gained FDA approval for both the management of metastatic and early stage TNBC. However, only about 20% of patients benefit and responses are completely independent of PD-L1 tumor expression, indicating a need to better elucidate the complexities the TNBC immune microenvironment. A major hurdle in identifying the mechanisms of immune escape and anti-PD-1 resistance in TNBC, is the lack of clinically representative mouse models of metastatic progression. We hypothesized that the serial re-implantation of metastatic lesions from a relatively slow growing syngeneic mouse model of TNBC would faithfully recapitulate the immune microenvironment of primary, lymphatic and distant metastatic disease. Methods: We previously reported the establishment of a syngeneic basal TNBC mouse model KPB25Luv (KLuv) that has comparable mutational burden, genomic profile, B-T cell cooperativity and IgG signature to human TNBC. The KLuv model develops both primary and TDLN tumors over several months, with rare liver metastases. KPB25Luv tumors were maintained in vivo and liver metastases were harvested and re-transplanted orthotopically over 1 year to develop the KLuv_LVMMR tumor line. Confocal microscopy, immunohistochemistry, bulk and single cell RNseq characterize and contrast the tumor microenvironments of each site. Longitudinal flow cytometry and TCR and BCR sequencing assess changes in adaptive lymphocyte dynamics. Findings are validated with corresponding patient data. Results: We report the development of the KLuv_LVMMR tumor line which is innately metastatic and reliably develops primary tumors, TDLN and liver metastases over 10 weeks. Gene expression signatures were comparable to human tumor tissues, including a significant decrease in immune activity (p=0.02) in metastases relative to primary tumors. This was associated with a reduction in MHCI and PD-L1 expression in metastatic lesions (p < 0.05). As metastatic tumors progress, CD8+ PD1+ T-cells increase 20-fold, with only a 2.5-fold increase in cytotoxic activity and IFN-gamma production, while T regulatory cells increase by 10-fold. No significant change was seen in CD4+ T cells or B220+ B cells. Confocal microscopy and single cell RNAseq reveal that PD-L1 expression localizes to macrophages and T-regs in the tumor draining lymph node rather than primary or metastatic tumor cells. Anti-PD-1 response is lost. Conclusions: The KLuv_LVMMR tumor line faithfully models the TNBC metastatic cascade, and immune microenvironment. This model represents the large population of anti-PD-1 resistant patients and has potential utility in identifying mechanisms of immune escape that can be harnessed into novel therapeutics for patients with advanced TNBC. Citation Format: Monika J Quackenbush, Asona Lui, Daniel P Hollern. Macrophage and T reg expression of PD-L1 in the tumor draining lymph node and not the primary tumor is associated with anti-PD-1 resistance in a model of metastatic triple negative breast cancer [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B055.