Abstract LB-351: Tumor infiltrating T cells: complete workflows allow faster and improved flow cytometric analysis of syngeneic mouse tumors
Authors: Richter Anne , Siemer Ramona , Steinbrueck Carolin , Herber Manuela , Agorku David , Brauner Janina , Hardt Olaf , Dose Christian , Evaristo Cesar 
Published: August 2018

Abstract
Immunotherapy approaches that engage T cells to attack tumors have proven clinical efficacy and tremendous potential in multiple cancers. Syngeneic mouse tumor models represent the gold standard to develop and analyze effects of immunotherapy, as they possess a fully competent immune repertoire. However, the phenotypic and functional analysis of tumor-infiltrating leukocytes (TILs) is technically challenging and labor intensive. The number of tumor infiltrating T cells can be very low and small subpopulations might escape analysis as they get lost in the background noise. Importantly, tumor infiltrating T cells are embedded in a cellular environment where antigen is abundant and surrounding cells express highly immunomodulatory molecules, such that unbiased cell-intrinsic functional characterization is hindered. When working with large cohort sizes, even immunophenotyping of TILs by flow cytometry is usually time consuming and data processing can be laborious. Therefore, it is fundamental to use effective innovative tools to streamline the workflow and to generate reliable data.
We established complete workflows combining tissue storage, dissociation, T cell isolation and flow cytometric phenotyping. These workflows were validated in four different syngeneic mouse tumor models (B16-F10, B16-OVA, 4T1 and CT26.WT). Tissues were either processed immediately or stored in a solution that was shown to maintain cell viability and phenotype up to 48h after collection (Tissue Storage Solution™). Tumor dissociation was automated and optimized for epitope preservation using a tissue dissociator (gentleMACS™ Octo). Phenotypic analysis revealed that optimal enzymatic dissociation was essential for analysis of critical tumor-specific sub-populations, such as PD1hiTim3+Lag3+CD39+CD8+ T cells present in B16-F10 tumors. We developed new T cell-specific enrichment reagents for manual and (semi-) automated magnetic cell sorting, based on MACS® Technology, which enrich for rare tumor infiltrating T cells by up to 500-fold, while maintaining activation status and phenotype. Importantly, we compared labeling of TILs using conventional hybridoma-derived antibodies with recombinant antibodies engineered to eliminate Fc receptor-mediated background (REAfinity™). Use of REAfinity antibodies significantly diminished non-specific labeling of cells present in the tumor microenvironment. Finally, flow cytometric analysis was performed using an automated analyzer (MACSQuant X™). This instrument decreased hands-on as well as total acquisition time by facilitating fast and fully automated sample processing, including sample mixing and absolute cell counting.
In conclusion, we have optimized workflows that include standardized processing of tumor samples, newly developed tools for (semi-) automated magnetic isolation of tumor infiltrating T cells and automated flow cytometric analysis. These workflows greatly reduce experimental time and allow the performance of more complex experimental setups. We believe the use of these innovative tools and workflows can significantly increase the quality of the data obtained in immuno-oncology and immunotherapy research.
Citation Format: Anne Richter, Ramona Siemer, Carolin Steinbrueck, Manuela Herber, David Agorku, Janina Brauner, Olaf Hardt, Christian Dose, Cesar Evaristo. Tumor infiltrating T cells: complete workflows allow faster and improved flow cytometric analysis of syngeneic mouse tumors [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 LB-351.