Nonuniform T-Cell Infiltration Induced By Pd-1 Checkpoint Blockade, Ex Vivo, Predicts Distinct Clinical Response

Background: The presence and activity of lymphocytes within the tumor is critical for clinical response to cancer immunotherapy, such as immune checkpoint blockade. Tumors with poor T-cell inflamed phenotypes, often referred to as a ‘cold9 tumor, is associated with modest clinical response. High baseline infiltration of effector T-cell lymphocytes is considered ‘hot9, and patients are predicted to respond more favorably to treatment. However, patient-to-patient response and durability remains highly variable. There is an urgent gap in available methods to study lymphocyte infiltration, trafficking and spatial heterogeneity induced by different cancer immunotherapies in individual patients. Moreover, there is a poor correlation between therapy-induced lymphocyte infiltration with clinical response, which could be shaped using personalized approaches to therapy. Methods: Here, we used CANscript TM , an ex-vivo human tumor model that recapitulates and preserves the native, patient-autologous tumor microenvironment, including autologous patient-derived peripheral blood mononucleated cells (PBMC). Utilizing tissue from breast cancer patients classified as either ‘cold9 or ‘hot9, we studied lymphocyte infiltration under pressure of a-PD-1 immune checkpoint blockade (pembrolizumab) over a 72h time course. Using fluorescent labelling and flow cytometric analysis we characterized infiltrating lymphocytes, studying the role of T-cell repertoires under different environmental and immunotherapy pressures. We coupled these analyses with multiplex immunohistochemistry (CD3 + , CD4 + , CD8 + , pan-cytokeratin, DAPI) to map spatial heterogeneity of tumor cells and lymphocytes before and after treatment, ex-vivo. Results: We determined that immune checkpoint blockade induced unique patterns of migration and infiltration of effector T-cells (T eff ) and T-regulatory (T reg ) cells in ‘hot9 vs ‘cold9 tumors. Furthermore, we determined that, in some instances, ‘cold9 tumors can be driven towards a ‘hot9 phenotype characterized by trafficking of active immune lymphocytes following treatment, which corresponded to differential ratio of T eff to T reg compared to baseline. Concluding remarks: Taken together, these data demonstrate the utility of CANscript TM as a platform to characterize response to immunotherapy in a spatial context, providing insight into the migratory patterns of immune cell subsets at the individual patient level. Such an advance in our preclinical methods to study immuno-modulators may help guide treatment decisions for clinicians while simultaneously functioning as a platform to study and discover mechanisms of clinical efficacy for emerging drug combinations. Citation Format: Munisha Smalley, Basavaraja Shanthappa, Hans Gertje, Mark Lawson, Baraneedharan Ulaganathan, Allen Thayakumar, D.C. Doval, Anurag Mehta, S. P. Somashekhar, Padhma Radhakrishnan, Pradip Majumder, Aaron J. Goldman. Nonuniform T-cell infiltration induced by PD-1 checkpoint blockade, ex vivo, predicts distinct clinical response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 705.