Abstract A005: Utility of an oncogene-driven spontaneous triple-negative breast cancer model for immunotherapeutic testing

One in eight women will develop breast cancer in their lifetime and it is the second leading cause of cancer-related deaths among women. Breast cancer is a heterogeneous disease with multiple subtypes that have distinct patient outcomes, biomarkers, and treatment options. Triple negative breast cancer (TNBC) is an aggressive form of breast cancer that has limited treatment options. It recently received FDA approval for treatment with anti-PD1 immunotherapy, though predictors of patient success remain unclear. A major hurdle to studying TNBC is that there is a profound lack of clinically relevant animal models. Most studies investigating TNBC utilize limited murine cell lines, which do not capture the intricacies of immune evasion and eventual escape of the tumor as well as the heterogeneity of human breast cancer. Our lab has developed a spontaneous model of TNBC which utilizes CRIPSR guides targeting three commonly mutated genes in TNBC: TP53, PTEN, and BRCA1. This model, upon intraductal injection of an adenovirus expressing Cre (Ad-Cre-GFP), induces expression of Cas9, GFP, Luciferase, and the three CRISPR guide RNAs in the mammary epithelium. This allows for a more natural disease progression where the tumor develops initially in the duct before expanding into the surrounding mammary gland and gives rise to heterogenous, trackable tumors that have developed in a fully immunocompetent host. Each tumor has a unique mutation profile across TP53, PTEN, and BRCA1 driving the tumor progression and microenvironment. These tumors have a long latency period, so we have also used them to generate a library of over twenty TNBC-like cell lines that are compatible with C57BL/6 mice. We have characterized these tumors in terms of hormone receptor and cytokeratin staining via IHC, tumor-infiltrating lymphocytes via flow cytometry, gene expression profile via RNAseq, and more. We have found our cell lines to have unique mutational profiles. We have also found these tumors to be immunologically diverse; some have large proportions of myeloid infiltrates, while others skew largely lymphoid, and some remain essentially immune excluded. By having a diverse library of TNBC cell lines compatible with C57BL/6 mice, we can capture the heterogeneity of human breast cancer in our studies, both by gene expression and by immune involvement. Our library of cell lines also includes tumors that have arisen in diet-induced obese mice, which offers novel insights into how the environment of a developing tumor affects immune infiltration, immunogenicity, and mutational burden. Overall, we have developed both a spontaneous model of TNBC in C57BL/6 mice and a highly diverse repertoire of TNBC implantable cell lines, which will allow us to better model human breast cancer in mice. Moving forward, we will be able to characterize the responsiveness of these diverse tumor lines to immunotherapy and use them to better understand predictors if immunotherapy efficacy in patients.

Duke Scholars

Author Erika J Crosby Surgery, Surgical Sciences