Abstract B54: Dissecting immune cell heterogeneity in human cancer by single-cell RNA-sequencing

Checkpoint blockade therapies that aim to reactivate anti-tumor immune responses have revolutionized cancer treatment, resulting in durable responses in a significant proportion of patients with advanced disease. Nevertheless, many patients fail to reach long-term clinical benefit, and therefore, a better insight into the mechanisms underlying response to immunotherapy is required to enhance its success rates. Recent findings suggest that immune cell infiltrates in cancer can be highly heterogeneous, potentially contributing to differential therapy outcomes. However, immune cell states have thus far been studied mainly in patients with end-stage melanoma and diverse treatment histories, whereas patients with early-stage metastatic disease are generally treatment naïve, allowing assessment of naturally evolved T cell functionality and immune cell composition. To characterize the heterogeneity in the tumor immune environment within and between patients, we profiled the tumor microenvironment of six stage III melanoma patients by single-cell RNA sequencing, generating an unbiased map of the expression signatures of immune cells as well as tumor and stromal cells. In parallel, we also derived the T cell receptor sequences for single T cells, and used them to determine intra- and inter-tumoral T cell clonality and infer the functionality of clonally expanded T cell populations in the tumor. A series of different cell types and cellular states that have been described previously in end-stage melanoma was also observed in this early-stage metastatic disease, including cytotoxic, exhausted, and regulatory T cells, as well as various myeloid subsets. Notably, despite identical disease stage and treatment background, the composition of the immune cell populations differed considerably between patients. In addition, while analysis of T cell states showed high variability between and within patients, clonally expanded T cells identified within patients predominantly adapted similar cell profiles. Currently we are assessing whether the occurrence of distinct T cell states can be coupled to other aspects of the immune infiltrate. In addition to describing T cell heterogeneity, our data suggest the existence of novel immune cell subsets within the tumor microenvironment. Our findings demonstrate that, using our single-cell RNA-sequencing approach, we can determine the immunological make-up in melanoma during early stages of metastatic disease, in an unbiased manner. Collectively, our approach should be helpful in determining the mechanisms underlying the development and effectiveness of tumor-specific immune responses in metastatic melanoma. Citation Format: Ido Yofe, Hanjie Li, Anne van der Leun, Lubling Yaniv, Assaf Weiner, Alexander van Akkooi, Amos Tanay, Ton Schumacher, Ido Amit. Dissecting immune cell heterogeneity in human cancer by single-cell RNA-sequencing [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B54.